WO2005099745A2

WO2005099745A2 - Gcp-170 fusion proteins and uses thereof

Info

Publication number: WO2005099745A2
Application number: PCT/US2005/012839
Authority: WO
Inventors: Elizabeth Sztul
Original assignee: Uab Research Foundation
Priority date: 2004-04-13
Filing date: 2005-04-13
Publication date: 2005-10-27
Also published as: WO2005099745A3

Abstract

The present invention relates to chimeric or fusion proteins comprising an amino acid sequence encoding a fluorescent protein and an amino acid sequence encoding a GCP170 protein or fragment thereof. The invention also provides the use of these fusion proteins to identify agents that modulate protein aggregation in cells.

Description

GCP-170 FUSION PROTEINS AND USES THEREOF This application claims priority to U.S. Provisional Application Serial No. 60/561 ,634 filed April 13 , 2004 which is hereby incorporated in its entirety by this reference. FIELD OF THE INVENTION The present invention relates to chimeric or fusion proteins comprising an amino acid sequence encoding a fluorescent protein and an amino acid sequence encoding a GCP170 protein or fragment thereof. These fusion proteins can be to identify agents that modulate protein aggregation in cells. BACKGROUND Newly synthesized proteins must be properly folded and modified to function correctly. Eukaryotic cells have developed extensive folding machineries to ensure the fidelity of protein processing. Nevertheless, misfolding can occur due to mutations within a protein, outside stresses, or the over-expression of proteins. Misfolded proteins often expose their hydrophobic domains, which leads to nonproductive protein associations and results in aggregation. Aggregated proteins tend to coalesce and form large deposits termed inclusion bodies, Russell bodies, or aggresomes, depending on their composition and location. Formation of such inclusions underlies a number of aggresomal diseases, including Alzheimer's disease, Parkinson's disease, familial amyotrophic lateral sclerosis, and the poly-glutamine (poly-Q) (reviewed in (Zoghbi and Orr, 2000; Garcia-Mata et al, 2002)). The biological processes leading to protein aggregation have been actively investigated (for review: (Kopito, 2000; Garcia-Mata et al., 2002; Goldberg, 2003; Selkoe, 2003)). Aggregation of proteins most likely occurs cotranslationally, while nascent peptide chains are synthesized on polyribosomes. If the nascent peptides cannot fold correctly, they will aggregate to form aggresomal particles. Small aggresomal particles form throughout the cell, and are quickly transported towards the microtubule (MT)-organizing center (MTOC), where they coalesce to form aggresomes (Johnston et al., 1998; Garcia-Mata et al., 1999). Aggresome formation is blocked by drugs that depolymerize microtubules, and by the expression of p50/dynamitin, suggesting that a dynein-based transport along microtubules is required for aggresome formation (Johnston et al., 1998; Garcia-Mata et al., 1999). Cytoplasmic aggresomes are enriched in molecular chaperones (including Hsc70, Hdjl and Hdj2, and chaperonin TCP), and in proteasomal subunits (Wojcik et al., 1996; Wigley et al., 1999). The active recruitment of re-folding and degradative machineries suggests that the formation of aggresomes is a dynamic process that cells use to cope with misfolded proteins. The preferential localization of aggresomes to the peri-centriolar region in mammalian cells suggests that the cytoplasmic milieu contains regions specialized to sequester and clear misfolded proteins. In addition to cytoplasmic aggresomes, nuclear inclusions are often found in patients with Huntington's disease (HD) or spinocerebellar ataxias (SCAs) (DiFiglia et al., 1997; Perez et al., 1998; Chai et al., 2001; Waelter et al., 2001; Yamada et al., 2001). HD and SCAs are neurodegenerative diseases caused by expanded poly-Q repeats in huntingtin and ataxins, respectively. The mutant proteins are aggregation-prone and form both cytoplasmic and intranuclear inclusions. In vitro studies with purified disease-causing proteins show that aggregation is based on a nucleated polymerization reaction, suggesting that self- aggregation of poly-Q may occur when the protein concentration reaches a critical level (Scherzinger et al., 1999; Chen et al., 2002). The formation of nuclear inclusions depends on the length of poly-Q repeats and on as yet unidentified factors in the host cells. Studies in cell culture systems and in transgenic mice show that the nuclear inclusions recruit molecular chaperones, ubiquitin and proteasomal subunits (Cummings et al., 1998; Chai et al., 1999b; Kim et al., 2002). The association of the degradative machineries suggests that nuclear inclusions may be involved in the proteolytic clearing of poly-Q aggregated substrates. Such nuclear inclusions may be analogous to cytoplasmic aggresomes, suggesting that the nucleus may also contain specialized sites to compartmentalize and clear misfolded proteins. The link between poly-Q content and the ability to form nuclear aggregates has led to the suggestion that the formation of nuclear aggregates may involve poly-Q-dependent mechanisms. Aggresome deposition is of particular interest due to the appearance of similar inclusions in a variety of human deposition diseases. These diseases include, but are not limited to Parkinson's disease, Huntington's disease, Alzheimer's disease, prion diseases, various ataxias and amyloidoses. One of the key challeneges to the pharmaceutical industry is to develop drugs that eliminate or reduce protein aggregation. Multiple mechanisms can be utilized to achieve this goal, among them the prevention of protein aggregation or the promotion of degradation of aggregated proteins. Therefore, methods identifying molecules that reduce, prevent or eliminate aggresome formation are necessary. SUMMARY OF THE INVENTION The present invention provides fusion polypeptides comprising a fluorescent proteiri and a Golgi protein GCP170 or a fragment of GCP-170. The present invention also provides cells comprising cytoplasmic and nuclear aggregates formed by chimeric polypeptides comprising a fluorescent protein and a Golgi protein GCP170 or a fragment of GCP-170. These chimeras can be utilized to identify agents that prevent protein aggregation or promote degradation of aggresomes or protein aggregates. These chimeric proteins can also be utilized to identify antiviral agents, agents that reduce cellular toxicity and agents that inhibit the recruitment of transcription factors to protein aggregates. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows GFP 170* deposits within the cytoplasm and the nucleus. A) A schematic diagram of full length GCP170 (1-1530), GFP-GCP170 and GFP170*(566- 1375). The full length GCP170 contains an amino-terminal head domain followed by a coiled-coil stalk of 6-coiled coils (shaded boxes). GFP170* contains GFP fused to an internal segment (amino acid 566 to 1375 of GCP170). B) COS-7 cells were transfected with a GFP-tagged full-length GCP170 or GFP-250 construct. After 48 h, cells were processed for indirect immunofluorescence using antibody against the Golgi marker protein, giantin. In a cell expressing low levels of GFP-GCP170 (lower left), most of the molecules are targeted to the Golgi region, and co-localize with giantin (arrow). In addition, small peripheral aggregates are visible (arrowheads). In a cell expressing high levels of GFP- GCP170 (upper right), the GFP-GCP170 forms large aggregates that surround the Golgi (double arrow). The GFP170* aggregates appear "ribbon-like" and are distinct from the spherical aggregates formed by GFP-250 (insert). C) COS-7 cells were transfected with GFP170*. After 48 h, cells were processed for indirect immunofluorescence using antibody against giantin. In a cell expressing low levels of GFP 170* (lower left), most of the molecules are targeted to the Golgi region, as demonstrated by co-localization with giantin (arrow). In a cell expressing high levels of GFP 170* (upper right), GFP 170* forms large aggregates that surround the Golgi (double arrow). In addition, GFP 170* is detected in numerous nuclear foci (double arrowhead). D) COS-7 cells were transfected with GFP170*. After 24 to 48 h, cells were processed for epifluorescence. Images of COS-7 cells containing different numbers of nuclear aggregates were selected. The average size of

GFP 170* nuclear foci was determined using IPLab software, and plotted as a function of number of foci per nucleus. E) COS-7 cells were transfected with GFP170*. After 24 h, cells were processed for immunofluorescence using anti-laminA antibody. Spherical GFP 170* foci are enclosed within the lamin A-defined space. Figure 2 shows the ultrastructure of GFP 170* aggregates. Non-transfected COS-7 cells (a) or COS-7 cells transfected with GFP 170* (b-i) were processed for transmission electron microscopy (b-f), fluorescence (g), or immunogold labeling (h, i). a) Non- transfected COS-7 cell lacks aggregates, b) Transfected COS-7 cell contains cytoplasmic aggregates (arrows) and nuclear aggregates (arrowheads), c, d) Cytoplasmic aggregates are ribbon-like, and are surrounded by mitochondria, e) Nuclear aggregates are either spherical or ovoid, f) Nuclear aggregates contain internal electron-lucent spaces (arrowheads), g)

Internal GFP 170* substructure within the nuclear aggregates is also visible by fluorescence (arrowheads), h, i) The content of the cytoplasmic and nuclear aggregates was confirmed by immuno-gold labeling with anti-GFP antibodies followed by secondary antibodies conjugated to 12-nm gold particles (arrowheads). N: nucleus. Figure 3 shows the recruitment of molecular chaperones to GFP 170* aggregates.

COS-7 cells were transfected with GFP 170*. After 48 h, cells were processed for indirect immunofluorescence using antibodies against Hsc70 (A), Hsp70 (B) or Hdj2 (C). Chaperones are recruited to GFP 170* aggregates. Figure 4 shows the involvement of microtubules in the formation of GFP 170* aggregates and the recruitment of vimentin and proteasomal components to GFP 170* aggregates. A) COS-7 cells were transfected with GFP 170*. After 8 h, cells were left untreated (-noc) or supplemented with 1.5 μM nocodazole (+noc), and cultured for additional 25 h. Cells were processed for indirect immunofluorecence using anti β-tubulin antibody. The nuclei were stained with Hoechst 33258. Nocodazole treatment leads to disruption of the microtubule cytoskeleton. In cells treated with nocodazole, cytosolic and nuclear aggregates of GFP 170* are smaller and more dispersed. B-D) COS-7 cells were transfected with GFP 170*, GFP-250 or ΔF508-CFTR. After 48 h, cells were processed for indirect immunofluorescence using antibodies against vimentin (B), α-subunit of proteasome (C), or ubiquitin (D). Vimentin envelopes the cytosolic GFP 170* and GFP-250 aggregates (B and insert). Proteasomal subunits are recruited to cytoplasmic and nuclear GFP 170* aggregates (C). Ubiquitin is recruited to GFP 170* and ΔF508-CFTR aggregates (D and insert). Figure 5 shows the degradation and solubility of GFP170*. COS7 cells were transfected with GFP170* or GFP-250. A) 24 h after transfection, cells were pulse labeled with ³⁵S-methionine for 1 hr and chased for indicated times. Cells were lysed and the lysates analyzed by SDS-PAGE and autoradiography. A representative autoradiogram is shown. The density of GFP 170* and GFP250 bands was quantified with ImmageQuant software. Results from three independent experiments are presented in the graph. B) 48 h after transfection, cells were lysed using indicated buffers. The supernatant (S) and the pellet (P) fractions from each lysis were analyzed by SDS-PAGE, followed by immunoblotting with antibody against either GFP or β-tubulin. Most of GFP 170* is present in the insoluble fraction. Figure 6 shows FRAP and FLIP analysis of GFP 170* aggregates. COS-7 cells were transfected with GFP 170* (A and D) or Q82-GFP (B and E). A-C) FRAP analysis of GFP 170* and Q82-GFP aggregates. A) A defined cytosolic (rectangle, arrowhead) or nuclear (circle, arrowhead) GFP 170* aggregate was photobleached and the recovery of fluorescence into that region was monitored. B) A nuclear Q82-GFP aggregate (circle, arrowhead) was photobleached and the recovery of fluorescence into that region was monitored. C) The relative fluorescence intensity (RFI) was determined for each time after bleaching and is represented as the average analysis of 3 to 5 cells. Error bars represent standard deviations of FRAP results from different cells. D-F) FLIP analysis of GFP 170* and Q82-GFP aggregates. D) A defined cytosolic region (circle, arrowhead) of a cell containing GFP 170* aggregates was repeatedly bleached and the loss of fluorescence from nuclear and cytosolic GFP 170* aggregates was monitored. E) A defined region (circle, arrowhead) of a cell containing Q82-GFP aggregates was repeatedly bleached and the loss of fluorescence from the Q82-GFP aggregates was monitored. F) Relative fluorescence intensity (RFI) was determined for each time point and is represented as the average analysis of 3 to 5 cells. Error bars represent standard deviations of FLIP results from different cells. Figure 7 shows the association of nuclear GFP 170* and G3 aggregates with PML bodies. COS cells were transfected with GFP170* (A-C) or G3-FLAG (D). After 48 h, cells were processed for immunofluorescence using anti-PML antibody (A-D), anti-Sc35 antibody (A) or anti-FLAG antibody (D). A) A non-transfected COS-7 cell (lower left) contains numerous PML bodies in the nucleus (arrowheads). In a cell with nuclear GFP 170* aggregates, PML is concentrated on the surface of GFP 170* aggregates (arrows). In contrast, the distribution of nuclear speckles marked by Sc35 is not changed in the presence of GFP 170* aggregates. B) A panel of COS-7 cells containing GFP 170* nuclear aggregates of different sizes. Small GFP 170* aggregates usually co-localize with, or are adjacent to individual PML bodies (first row, arrowheads). Larger GFP 170* aggregates contain PML bodies on their surface (arrows). C) An enlarged image shows the preferential association of nuclear GFP 170* aggregates with PML bodies. D) G3 nuclear inclusions also associate with PML bodies. Nuclei are visualized with DAPI staining. Figure 8 shows the dynamic movements of GFP 170* aggregates. COS-7 cells were transfected with GFP170*. After 20 h, two cells expressing low levels of GFP170* were imaged every five min for additional 12 h. A) A panel of images at the time points indicated. B) Tracing of the movement of selected GFP170* aggregates. Arrows indicate the directions of the movement and dots indicate fusion events between aggregates. Figure 9 shows fusions and rearrangements of nuclear GFP170* aggregates. COS-7 cells were transfected with GFP170*. After 48 h, cells were subject to time-lapse imaging, with images acquired every 20 sec for 2 h. A) A panel of images at time points indicated. Arrows indicate fusion between cytosolic aggregates. Arrowheads indicate fusion between nuclear aggregates. B) A panel of images at 1 min intervals. Fusion of two nuclear aggregates is accompanied by extensive rearrangements of internal structures. An arrow shows an inclusion containing one fluorescent-lucent internal structure. Arrowheads indicate multiple internal structures within an inclusion. Double arrowheads mark two aggregates that undergo fusion and rapid internal reorganization. Figure 10 shows a comparison between cytoplasmic and nuclear aggregates of GFP 170* and Q80-GFP. COS-7 cells were transfected with GFP 170*, GFP-GCP170FL or Q80-GFP contstruct. 48 hours after transfection, cells were fixed and processed for fluorescence (A, B), electron microscopy (C, D), or immunogold labeling (E, F). (A and B), Light microscopic analysis of GFP 170*, GFP-GCP170FL and Q80-GFP aggregates. The nuclei are stained with Hoechst 33258. Arrows point to cytoplasmic aggregates. Arrowheads point to nuclear inclusions. (C and D), Ultrastructure of the GFP 170* and the Q80-GFP aggregates. The cytoplasmic GFP 170* aggregates (arrows in C) have irregular shapes and are frequently surrounded by mitochondria. Nuclear aggregates (arrowheads in C and D) range in size from 0.5 to 3 μm, and are spherical or ovoid. (E and F), Cytoplasmic (E) and nuclear (F) inclusions of GFP 170* are labeled with anti-GFP antibodies conjugated to gold particles. N, Nucleus. Figure 11 shows that GFP 170* and Q80-GFP form cytoplasmic and nuclear aggregates in neuronal cells. Mouse primary cortical neurons were transfected with GFP170* (A) or Q80-GFP (B). 48 hours after transfection, cells were fixed and the nuclei were stained with Hoechst 33258. Arrows point to cytoplasmic aggregates. Arrowheads point to nuclear inclusions. Figure 12 shows that GFP 170* aggregates recruit chaperones and proteasomes. COS-7 cells were transfected with GFP170*. 48 h after transfection, cells were processed for immuno-fluorescence using antibodies against Hsp70 (A), Hdj2 (B), or the proteasomal α-subunit (C). Recruitment to cytosolic and/or nuclear aggregates is evident. (D), COS-7 cells were transfected with GFP 170*, and were lysed 48 h later in RIPA buffer containing 0.1% SDS. The soluble and insoluble fractions were separated by centrifugation. Equivalent amounts of total cell lysate (T), the soluble (S) and the insoluble pellet (P) fractions were resolved by SDS-PAGE and immunoblotted with antibodies against GFP or β-tubulin. Most of GFP 170* is present in the insoluble fraction. Figure 13 shows that nuclear GFP 170* aggregates recruit PML protein and SUMO-

1. COS-7 cells were transfected with GFP 170*. 48 h after transfection, cells were processed for indirect immunofluorescence using antibodies to PML and SUMO-1. PML distribution is normal in cells expressing low levels of GFP170* (A). PML distribution is altered in cells expressing high levels of GFP 170* (B). PML structures are surrounding the large GFP 170* aggregates. Arrowheads point to PMLs in the control cells. Arrows point to PMLs in a nucleus containing GFP 170* aggregates. (C), SUMO-1 co-localizes with nuclear GFP 170* aggregates. (D), COS-7 cells were mock transfected (control lanes) or transfected with GFP170* (GFP170* lanes) for 48 h. Cells were lysed and the lysates were processed by SDS-PAGE and immunoblotting with anti-GFP antibody (anti-GFP panel). A GFP170* band (~124kD) is present only in the transfected cells. The same membrane was stripped and reprobed with anti-SUMO antibody (anti-SUMO panel). The GFP170* band is recognized. An additional band (~98-kD) is recognized in control and in transfected cells, and may represent PML. , Figure 14 shows that nuclear GFP 170* aggregates recruit transcriptional regulators. COS-7 cells were transfected with GFP170*. 48 h after transfection, cells were processed for indirect immunofluorescence using antibodies to CBP (A), p53 (B) and SC35 (C).

Levels of CBP and p53 are increased in cells expressing GFP 170*, and both proteins are recruited to nuclear inclusions of GFP 170*. The levels and distribution of SC35 appears similar in non-transfected cells and in cells containing GFP 170* aggregates. Figure 15 shows that Expression of GFP 170* represses transcription and is cytotoxic. (A), COS7 cells were co-transfected with the p21-Luc vector expressing firefly luciferase and either a control plasmid, GFP170* or Q80-GFP.48 h after transfection, cell lysates were made and luciferase activity was measured. The activity in each sample was calculated as arbitrary units per milligram protein and normalized to that in the control sample. The p53 transcriptional activity is inhibited by GFP 170* or Q80-GFP. (B), COS-7 cells were transfected with GFP 170* or ρEGFP-C2 (GFP alone). 32 h after transfection, cells were incubated with 30 μM BrdU, followed by immunofluorescent staining with anti- BrdU monoclonal antibody. Nuclei were stained with Hoechst 33258. GFP170*-containing cells do not incorporate BrdU. (C), COS-7 cells were mock transfected (control), or transfected with GFP170* or Q80-GFP. 48 h after transfection, cells were incubated with a viability indicator dye and sorted by FACS. The number of dead cells was counted and is plotted as % of total cells. Expression of GFP170* or Q80-GFP results in cell death. DETAILED DESCRIPTION OF THE INVENTION The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Example included therein. Before the present compounds and methods are disclosed and described, it is to be understood that this invention is not limited to specific proteins or specific methods. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an aggregate" includes two or more aggregates, reference to "a nucleic" includes two or more such nucleic acids, and the like. Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. The present invention provides a fusion protein or fusion polypeptide comprising a GCP-170 protein or a fragment thereof, and a fluorescent protein. These fusion polypeptides are also referred to herein as chimeric GCP-170 polypeptides or fusion GCP-170 polypeptides. GCP-170 is also known as golgin 160 and is localized to the Golgi complex. This protein was characterized by Misumi et al. (Molecular Characterization of GCP170, a 170kDa Protein Associated with the Cytoplasmic Face of the Golgi Membrane" J. Biol. Chem. 272(38):23851-23858 (1997)). The amino acid sequence of GCP-170 and the nucleic acid encoding GCP- 170 are set forth herein as SEQ ID NO: 1 and SEQ ID NO: 2 respectively.

Nucleotides 270-4862 of SEQ ID NO:2 encode GCP-170 or SEQ ID NO: 1. This sequence can also be found in Misumi et al. and this reference is hereby incorporated in its entirety by this reference. The nucleic acid sequence encoding GCP-170 and the amino acid sequence of GCP-170 can also be found under GenBank Accession No. D63997. All of the information provided under GenBank Accession No. D63997 including GCP-170 nucleic acid sequences and GCP-170 amino acid sequences is incorporated herein by this reference. The polypeptides of this invention include a fusion polypeptide comprising a GCP-170 protein, or a fragment thereof, and a fluorescent protein. These polypeptides are also called GCP-170 fusion polypeptides or GCP-170 fusion proteins. The GCP-170 polypeptides of the present invention that are linked or fused to a fluorescent protein to produce a GCP-170 fusion polypeptide can be full- length GCP-170 or fragments thereof. For example, the GCP-170 polypeptide linked to a fluorescent protein can be a full-length GCP-170 comprising amino acids 1-1530 of GCP-170, a fragment of GCP-170 comprising amino acids 1-1374, a fragment of GCP-170 comprising amino acids 1-566 of GCP-170, a fragment of GCP-170 comprising amino acids 566-1530, a fragment of GCP-170 comprising amino acids 1374-1530 of GCP-170, a fragment of GCP-170 comprising amino acids 566-1375 of GCP-170 or any other fragment of GCP-170 that when linked to a fluorescent protein to form a fusion polypeptide of the present invention results in protein aggregation or aggresome formation in cells. One of skill can readily obtain a fragment of GCP-170 and link it to a fluorescent protein in order to determine the protein aggregating ability of the GCP-170 fragment. Based on the guidance provided herein and the Examples, one of skill in the art can assess the protein aggregating ability of any GCP-170 fragment as well as the location of the protein aggregates formed by a polypeptide comprising a GCP-170 fragment. Further examples of these polypeptides include, but are not limited to, a polypeptide comprising the amino acid sequence of a green fluorescent protein (GFP) fused to amino acids 1-1530 of SEQ ID NO: 1 (GCP-170) which forms aggregates in the cytoplasm. Also provided is a polypeptide comprising the amino acid sequence of a GFP fused to amino acids 566-1530 of SEQ ID NO: 1 (GCP-170) which forms aggregates in the cytoplasm of cells. Also provided is a polypeptide comprising the amino acid sequence of a GFP fused to amino acids 566-1375 of SEQ ID ON: 1 (GCP-170) which forms aggregates in both the cytoplasm and the nucleus of cells. The present invention also contemplates fusion proteins wherein the amino acid sequence of the GCP-170 polypeptide fused to the fluorescent protein is not the full-length amino acid sequence of GC170. The fusion GCP-170 polypeptides of the present invention comprise a fluorescent protein, examples of which include, but are not limited to, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP). Other examples include the green fluorescent protein from Aequorea coerelescens (AcGFP), DsRedExpress, and red coral fluorescent proteins (for example, AmCyan, ZsGreen, ZsYellow, AsRed2, DsRed2, and HcRedl). The fluorescent protein can optionally be linked or fused to a GCP-170 protein or a fragment thereof by a polypeptide linker. The fluorescent protein can be linked or fused to the N-terminus of the GCP-170 protein or fragment thereof. The fluorescent protein can also be linked or fused to the C-terminus of the GCP-170 protein or fragment thereof. Also, one of skill in the art can link the GCP-170 protein or fragment thereof to the C-terminus of the fluorescent protein or to the N- terminus of the fluorescent protein. Numerous vectors that comprise a nucleic acid encoding a fluorescent protein are available from commercial sources, for example, from Clontech (Palo Alto, CA, USA). These Clontech vectors include, but are not limited to C-terminal fluorescent protein vectors such as a pAcGFP-Nl vector, a pAmCyanl-Nl vector, apAsRed2-Nl vector, a pDSRed-Express-Nl vector, a pDsRed-Monomer-Nl vector, a pHcRedl-Nl/1 vector, a pZsGreenl-Nl vector, or a pZsYellowl-Nl vector. C-terminal fluorescent protein vectors are also available and they include, but are not limited to, a pEGFP-C2 vector, a pAcGFP-Cl vector, a pAmCyanl-Cl vector, apAsRed2-Cl vector, a pDSRed-Express-Cl vector, a pDsRed-Monomer-Cl vector, a pHcRedl-C 1/1 vector, apZsGreenl-Cl vector, or a pZsYellowl-Cl vector. One of skill in the art can select a vector described herein or any other vector that encodes a fluorescent protein of interest and insert a nucleic acid that encodes a GCP-170 protein or a fragment thereof in operable linkage with the fluorescent protein, to obtain a vector comprising a nucleic acid that encodes a fusion polypeptide comprising the fluorescent protein and the GCP-170 protein or a fragment thereof. This vector can then be utilized to express the fusion polypeptide in cells. With regard to the polypeptides of this invention, as used herein, "isolated" and/or "purified" means a polypeptide which is substantially free from the naturally occurring materials with which the polypeptide is normally associated in nature. Also as used herein, "polypeptide" refers to a molecule comprised of amino acids which correspond to those encoded by a nucleic acid. The polypeptides or fragments thereof of the present invention can be obtained by isolation and purification of the polypeptides from cells where they are produced naturally or by expression of an exogenous nucleic acid encoding a GCP-170 polypeptide. Fragments of the GCP-170 polypeptide can be obtained by chemical synthesis of peptides, by proteolytic cleavage of the polypeptide and by synthesis from nucleic acid encoding the portion of interest. The polypeptide may include conservative substitutions where a naturally occurring amino acid is replaced by one having similar properties. Such conservative substitutions do not alter the function of the polypeptide. Thus, it is understood that, where desired, modifications and changes may be made in the nucleic acid and/or amino acid sequence of the GCP-170 polypeptides and fusion polypeptides of the present invention and still obtain a protein having like or otherwise desirable characteristics. Such changes may occur in natural isolates or may be synthetically introduced using site-specific mutagenesis, the procedures for which, such as mis-match polymerase chain reaction (PCR), are well known in the art. For example, certain amino acids may be substituted for other amino acids in a GCP-170 polypeptide or a fragment thereof, or a fusion polypeptide comprising a GCP-170 polypeptide without appreciable loss of functional activity. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a GCP- 170 amino acid sequence (or, of course, the underlying nucleic acid sequence) and nevertheless obtain a GCP-170 polypeptide with like properties. It is thus contemplated that various changes may be made in the amino acid sequence of the GCP-170 polypeptide (or underlying nucleic acid sequence) without appreciable loss of biological utility or activity and possibly with an increase in such utility or activity. Also provided by the present invention is a GCP-170 fusion polypeptide comprising amino acids 1-1530 of GCP-170, a GCP-170 fusion polypeptide comprising amino acids 1- 1374 of GCP-170, a GCP-170 fusion polypeptide comprising amino acids 1-566 of GCP- 170, a GCP-170 fusion polypeptide comprising amino acids 566-1375 of GCP-170 a GCP- 170 fusion polypeptide comprising amino acids 566-1530 of GCP-170 and a GCP-170 fusion polypeptide comprising amino acids 1374-1530 of GCP-170 with one or more conservative amino acid substitutions. These conservative substitutions are such that a naturally occurring amino acid is replaced by one having similar properties. Such conservative substitutions do not alter the function of the polypeptide. For example, conservative substitutions can be made according to the following table:

TABLE 1: Amino Acid Substitutions

Original Residue Exemplary Substitutions

Arg Lys

Asn Gin TABLE 1: Amino Acid Substitutions

Original Residue Exemplary Substitutions

Asp Glu

Cys Ser

Gin Asn

Glu Asp

Gly Pro

His Gin lie leu; val

Leu ile; val

Lys arg; gin

Met leu; ile

Phe met; leu; tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr trp; phe

Val ile; leu

Thus, it is understood that, where desired, modifications and changes may be made in the nucleic acid encoding the polypeptides of this invention and or amino acid sequence of the polypeptides of the present invention and still obtain a polypeptide having like or otherwise desirable characteristics. Such changes may occur in natural isolates or may be synthetically introduced using site-specific mutagenesis, the procedures for which, such as mis-match polymerase chain reaction (PCR), are well known in the art. For example, certain amino acids may be substituted for other amino acids in a polypeptide without appreciable loss of functional activity. It is thus contemplated that various changes may be made in the amino acid sequence of the polypeptides of the present invention (or underlying nucleic acid sequence) without appreciable loss of biological utility or activity and possibly with an increase in such utility or activity. Nucleic Acids The present invention also provides nucleic acids encoding the polypeptides of the present invention. As used herein, the term "nucleic acid" refers to single or multiple stranded molecules which may be DNA or RNA, or any combination thereof, including modifications to those nucleic acids. The nucleic acid may represent a coding strand or its complement, or any combination thereof. Nucleic acids may be identical in sequence to the sequences which are naturally occurring for any of the moieties discussed herein or may include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence. These nucleic acids can also be modified from their typical structure. Such modifications include, but are not limited to, methylated nucleic acids, the substitution of a non-bridging oxygen on the phosphate residue with either a sulfur (yielding phosphorothioate deoxynucleotides), selenium (yielding phosphorselenoate deoxynucleotides), or methyl groups (yielding methylphosphonate deoxynucleotides), a reduction in the AT content of AT rich regions, or replacement of non-preferred codon usage of the expression system to preferred codon usage of the expression system. The nucleic acid can be directly cloned into an appropriate vector, or if desired, can be modified to facilitate the subsequent cloning steps. Such modification steps are routine, an example of which is the addition of oligonucleotide linkers which contain restriction sites to the termini of the nucleic acid. General methods are set forth in in Sambrook et al. (2001) Molecular Cloning - A Laboratory Manual (3rd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook). Once the nucleic acid sequence is obtained, the sequence encoding the specific amino acids can be modified or changed at any particular amino acid position by techniques well known in the art. For example, PCR primers can be designed which span the amirio acid position or positions and which can substitute any amino acid for another amino acid. Alternatively, one skilled in the art can introduce specific mutations at any point in a particular nucleic acid sequence through techniques for point mutagenesis. General methods are set forth in Smith, M. "In vitro mutagenesis" Ann. Rev. Gen., 19:423-462 (1985) and Zoller, M.J.

"New molecular biology methods for protein engineering" Curr. Opin. Struct. Biol., 1:605-610 (1991), which are incorporated herein in their entirety for the methods. These techniques can be used to alter the coding sequence without altering the amino acid sequence that is encoded. Vectors, Cells, and Methods of Using Also provided is a vector, comprising a nucleic acid of the present invention. The vector can direct the in vivo or in vitro synthesis of any of the polypeptides described herein. The vector is contemplated to have the necessary functional elements that direct and regulate transcription of the inserted nucleic acid. These functional elements include, but are not limited to, a promoter, regions upstream or downstream of the promoter, such as enhancers that may regulate the transcriptional activity of the promoter, an origin of replication, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter, antibiotic resistance genes or other markers which can serve to select for cells containing the vector or the vector containing the insert, RNA splice junctions, a transcription termination region, or any other region which may serve to facilitate the expression of the inserted gene or hybrid gene. (See generally, Sambrook et al.). The vector, for example, can be a plasmid. The vectors can contain genes conferring hygromycin resistance, gentamicin resistance, or other genes or phenotypes suitable for use as selectable markers, or methotrexate resistance for gene amplification. There are numerous other E. coli (Escherichia coli) expression vectors, known to one of ordinary skill in the art, which are useful for the expression of the nucleic acid insert.

Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).

In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences for example, for initiating and completing transcription and translation. If necessary, an amino terminal methionine can be provided by insertion of a Met codon 5' and in-frame with the downstream nucleic acid insert. Also, the carboxy-terminal extension of the nucleic acid insert can be removed using standard oligonucleotide mutagenesis procedures. Also, nucleic acid modifications can be made to promote amino terminal homogeneity. Additionally, yeast expression can be used. The invention provides a nucleic acid encoding a polypeptide of the present invention, wherein the nucleic acid can be expressed by a yeast cell. More specifically, the nucleic acid can be expressed by Pichia pastoris or S. cerevisiae. There are several advantages to yeast expression systems, which include, for example, Saccharomyces cerevisiae and Pichia pastoris. First, evidence exists that proteins produced in a yeast secretion systems exhibit correct disulfide pairing. Second, efficient large scale production can be carried out using yeast expression systems. The Saccharomyces cerevisiae pre-pro-alpha mating factor leader region (encoded by the MFa-1 gene) can be used to direct protein secretion from yeast (Brake, et al.). The leader region of pre-pro-alpha mating factor contains a signal peptide and a pro-segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene: this enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage signal sequence. The nucleic acid coding sequence can be fused in-frame to the pre-pro-alpha mating factor leader region. This construct can be put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter, alcohol oxidase I promoter, a glycolytic promoter, or a promoter for the galactose utilization pathway. The nucleic acid coding sequence is followed by a translation termination codon which is followed by transcription termination signals. Alternatively, the nucleic acid coding sequences can be fused to a second protein coding sequence, such as Sj26 or beta-galactosidase, used to facilitate purification of the fusion protein by affinity chromatography. The insertion of protease cleavage sites to separate the components of the fusion protein is applicable to constructs used for expression in yeast. Efficient post translational glycosylation and expression of recombinant proteins can also be achieved in Baculovirus systems. Mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, and secretion of active protein. Vectors useful for the expression of active proteins in mammalian cells are characterized by insertion of the protein coding sequence between a strong viral promoter and a polyadenylation signal. The vectors can contain genes conferring hygromycin resistance, genticin or G418 resistance, or other genes or phenotypes suitable for use as selectable markers, or methotrexate resistance for gene amplification. The chimeric protein coding sequence can be introduced into a Chinese hamster ovary (CHO) cell line using a methotrexate resistance-encoding vector, or other cell lines using suitable selection markers. Presence of the vector DNA in transformed cells can be confirmed by Southern blot analysis. Production of RNA corresponding to the insert coding sequence can be confirmed by Northern blot analysis. A number of other suitable host cell lines have been developed in the art, and include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc. The vectors containing the nucleic acid segments of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transformation is commonly utilized for prokaryotic cells, whereas calcium phosphate, DEAE dextran, or lipofectin mediated transfection or electroporation may be used for other eukaryotic cellular hosts. Alternative vectors for the expression of genes or nucleic acids in mammalian cells, those similar to those developed for the expression of human gamma-interferon, tissue plasminogen activator, clotting Factor VIII, hepatitis B virus surface antigen, protease Nexinl, and eosinophil major basic protein, can be employed. Further, the vector can include CMV promoter sequences and a polyadenylation signal available for expression of inserted nucleic acids in mammalian cells (such as COS-7). Insect cells also permit the expression of mammalian proteins. Recombinant proteins produced in insect cells with baculovirus vectors undergo post-translational modifications similar to that of wild-type proteins. Briefly, baculovirus vectors useful for the expression of active proteins in insect cells are characterized by insertion of the protein coding sequence downstream of the Autographica calif ornica nuclear polyhedrosis virus

(AcNPV) promoter for the gene encoding polyhedrin, the major occlusion protein. Cultured insect cells such as Spodopterafrugiperda cell lines are transfected with a mixture of viral and plasmid DNAs and the viral progeny are plated. Deletion or insertional inactivation of the polyhedrin gene results in the production of occlusion negative viruses which form plaques that are distinctively different from those of wild-type occlusion positive viruses. These distinctive plaque morphologies allow visual screening for recombinant viruses in which the AcNPV gene has been replaced with a hybrid gene of choice. The invention also provides for the vectors containing the contemplated nucleic acids in a host suitable for expressing the nucleic acids. The host cell can be a prokaryotic cell, including, for example, a bacterial cell. More particularly, the bacterial cell can be an E. coli cell. Alternatively, the cell can be a eukaryotic cell, including, for example, a COS- 7 cells, a Chinese hamster ovary (CHO) cell, a myeloma cell, a Pichia cell, or an insect cell. The coding sequence for any of the polypeptides described herein can be introduced into a Chinese hamster ovary (CHO) cell line, for example, using a methotrexate resistance- encoding vector, or other cell lines using suitable selection markers. Presence of the vector DNA in transformed cells can be confirmed by Southern blot analysis. Production of RNA corresponding to the insert coding sequence can be confirmed by Northern blot analysis. A number of other suitable host cell lines have been developed and include myeloma cell lines, fibroblast cell lines, and a variety of tumor cell lines such as melanoma cell lines. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc. The vectors containing the nucleic acid segments of interest can be transferred into the host cell by well- known methods, which vary depending on the type of cellular host. For example, calcium chloride transformation is commonly utilized for prokaryotic cells, whereas calcium phosphate, DEAE dextran, lipofectin mediated transfection or electroporation or Streptolysin-O-mediated permeabilization may be used for other cellular hosts. The present invention further provides antibodies which specifically bind the GCP- 170 fusion polypeptides of the present invention. The antibodies of the present invention include both polyclonal and monoclonal antibodies. Such antibodies may be murine, rabbit, fully human, chimeric or humanized. These antibodies can also include Fab or F(ab')₂ fragments, as well as single chain antibodies (ScFv) (See, e.g., Harlow and Lane, 1989). The antibodies can be of any isotype IgG, IgA, IgD, IgE and IgM. Such antibodies can be produced by techniques well known in the art which include those described in Kohler et al. (42) or U.S. Patents 5,545,806, 5,569,825 and 5,625,126, incorporated herein by reference. Antibodies that specifically bind the GCP-170 fusion polypeptides of the present invention can be utilized to detect GCP-170 fusion polypeptides in a cell as well as protein aggregates formed by the GCP-170 fusion polypeptides of the present invention. Optionally, the antibody of the invention is labeled with a detectable moiety. For example, the detectable moiety can be selected from the group consisting of a fluorescent moiety, an enzyme-linked moiety, a biotin moiety and a radiolabeled moiety. The antibody can be used in techniques or procedures such as screening, or imaging. The present invention also provides a cell that expresses a GCP-170 fusion polypeptide of the present invention and comprises a protein aggregate or aggresome, wherein the protein aggregate comprises two or more GCP-170 fusion polypeptide molecules of the present invention. As utilized herein "protein aggregate" means that the aggregate comprises fusion polypeptides of the present invention that associate with each other. One or more protein aggregates can occur in the nucleus of the cell, in the cytoplasm of the cell or in both the nucleus and the cytoplasm of the cell. The protein aggregates can comprise other proteins associated with the fusion polypeptides of the present invention. For example, the protein aggregate can comprise molecular chaperones such as, for example, BIP, HSP70, HSP40, and Hdj2. The protein aggregates can also comprise transcription factors such as p53 and CBP as well as proteasomes, for example, proteasomal subcomplexes 20S, 1 IS and 19S. Therefore, in addition to detecting the presence of protein aggregates via fluorescence of the GCP-170 fusion polypeptides and antibodies that bind the GCP-170 fusion polypeptides, the present invention also contemplates the use of antibodies that bind the proteins associated with the fusion polypeptides of the present invention (i.e. molecular chaperones, transcription factors, proteasomes) for the detection of protein aggregates. The present invention also provides a cell that expresses a GCP-170 fusion polypeptide of the present invention wherein the GCP-170 fusion polypeptide comprises a fluorescent protein and the cell expresses a second GCP-170 fusion polypeptide of the present invention wherein the GCP-170 fusion polypeptide comprises a different fluorescent protein. For example, a cell can express a GCP-170 fusion polypeptide of the present invention wherein the GCP-170 fusion polypeptide comprises a green fluorescent protein and this cell can also express a GCP-170 fusion polypeptide of the present invention wherein the GCP-170 fusion polypeptide comprises a red fluorescent protein. Utilizing these cells, one of skill in the art can view the differences between protein aggregates formed by the GCP-170 fusion polypeptide comprising GFP and the protein aggregates formed by the GCP-170 fusion polypeptide comprising red fluorescent protein. This also allows the skilled artisan to engineer cells that comprise protein aggregates in the nucleus and the cytoplasm utilizing two different GCP-170 fusion polypeptides. For example, a cell can express a GCP-170 fusion polypeptide comprising GFP in the nucleus of the cell (such as for example a GCP-170 fusion polypeptide comprising amino acids 566-1375 of GCP- 170) and a GCP-170 fusion polypeptide (such as, for example, a GCP-170 fusion polypeptide comprising amino acids 1-1530 of GCP-170) comprising red fluorescent protein in the cytoplasm of the cell. These examples are not meant to be limiting as one of skill in the art would know how to select appropriate fluorescent proteins to observe differences within a cell. Furthermore, based on the teachings of the present invention, one of skill in the art would know how to make and use a GCP-170 fusion polypeptide that formed protein aggregates in the nucleus of the cell. One of skill in the art would also know how to make and use a GCP-170 fusion polypeptide that formed protein aggregates in the nucleus of the cell.

Screening Methods The present invention provides a method of identifying an agent that reduces protein aggregation comprising: a) contacting a cell containing aggregated fusion polypeptides comprising an amino acid sequence encoding a fluorescent protein and an amino acid sequence encoding a GCP-170 protein with a test agent; b) comparing the cell contacted with the test compound with a cell containing aggregated polypeptides that was not contacted with the test agent; and c) determining the effect of the test agent on protein aggregation, such that if protein aggregation in the cell contacted with the test compound is less than protein aggregation in the cell that was not contacted with the test compound, the test agent is an agent that reduces protein aggregation. Any of the GCP-170 fusion polypeptides set forth herein can be utilized in the methods of the present invention. As mentioned above, the aggregated polypeptide can comprise a full-length GCP-170 polypeptide or a fragment thereof. The fluorescent protein can be, but is not limited to a fluorescent protein selected from the group consisting of green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), green fluorescent protein from Aequorea coerelescens (AcGFP), DsRedExpress, and a red coral fluorescent protein. Also, as mentioned above, more than one GCP-170 fusion polypeptide can be utilized to obtain protein aggregates in a cell. For example, a GCP-170 fusion polypeptide that forms aggregates in the cytoplasm and a GCP-170 fusion polypeptide that forms aggregates in the nucleus can be utilized to obtain cells that form protein aggregates in the nucleus and the cytoplasm of the cell. For example, a GCP-170 fusion polypeptide comprising amino acids 1-1530 of GCP-170 or amino acids 566-1530 of GCP-170 can be expressed in a cell with a GCP-170 fusion polypeptide comprising amino acids 566-1375 to obtain a cell that contains protein aggregates in the nucleus and in the cytoplasm of the cell. The GCP-170 fusion polypeptide comprising amino acids 1-1530 of GCP-170 or amino acids 566-1530 of GCP-170 and the GCP-170 fusion polypeptide comprising amino acids 566-1375 can both be linked to the same type of fluorescent protein or different fluorescent proteins. For example, both the GCP-170 fusion polypeptide comprising amino acids 1-1530 of GCP-170 or amino acids 566-1530 of GCP-170 and the GCP-170 fusion polypeptide comprising amino acids 566-1375 can be linked to a green fluorescent protein. Alternatively, the GCP-170 fusion polypeptide comprising amino acids 1-1530 of GCP-170 or amino acids 566-1530 of GCP-170 can be linked to a green fluorescent protein and the GCP-170 fusion polypeptide comprising amino acids 566-1375 can be linked to a fluorescent protein that is not green fluorescent protein, for example, red fluorescent protein. These examples are not meant to be limiting, as one of skill in the art can utilize any combination of GCP- 170 polypeptides or fragments thereof and fluoresecent polypeptides described herein in the methods of the present invention. The "cells" utilized in the screening methods of this invention include any cell described herein and any cell type that can express a GCP-170 fusion polypeptide of the present invention. The cells can be in vitro, ex vivo and in vivo. Examples include, but are not limited to, eukaryotic cells such as neuronal cells, kidney cell, endothelial cells, pancreatic cells, liver cells, ovary cells, muscle cells, intestinal cells, blood cells, adrenal cells. The amount of protein aggregation in a cell can be measured via fluorescence as described in the Examples set forth herein and via other methods available in the art. The amount of protein aggregation can also be measured via antibody detection as described above. A reduction in aggregation does not have to be complete as this can range from a slight decrease in aggregation to complete elimination of a protein aggregate. For example, one of skill in the art can observe the aggregation of GCP-170 fusion polypeptides in a cell. An amount of fluorescence will be associated with one or more aggregates formed by the GCP-170 fusion polypeptides. Upon administering an agent that reduces protein aggregation, one of skill in the art will know, either visually via microscopy or via quantitative methods, that the amount of fluorescence has decreased in one or more of the aggregates. For example, a reduction in protein aggregation can result in a decrease in the number of protein aggregates and/or a decrease in protein aggregate size. A reduction in protein aggregation can also be characterized by decrease in the density and/or or a change in the morphology of the protein aggregate. For example, if prior to administration of the test agent, the protein aggregate comprising GCP-170 fusion polypeptides is densely packed and after administration of the test agent, the aggregate is less dense or more loosely arranged, this agent causes a reduction in protein aggregation. A reduction in protein aggregation can be due to disassociation or degradation of the GCP-170 fusion polypeptides. In the methods of the present invention, the aggregates observed in the cell can occur in the nucleus or the cytoplasm. Therefore, the screening methods of the present invention can be utilized to identify agents that reduce aggregation in the nucleus and/or the cytoplasm. The methods of the present invention can also utilize a polypeptide that forms aggregates in both the nucleus and the cytoplasm, for example, a GCP 170 fusion polypeptide as described in the Examples which comprises a fluorescent protein fused to amino acids 566-1375 of GCP 170. A cell expressing this fusion protein will form aggregates in both the nucleus and the cytoplasm. Therefore, the present methods can be utilized to identify agents that reduce protein aggregation in both the nucleus and the cytoplasm. The present methods can also be utilized to identify agents that reduce protein aggregation in the nucleus or the cytoplasm. Therefore, if an agent reduces protein aggregation in the nucleus to a greater extent than it reduces protein aggregation in the cytoplasm, this agent preferentially reduces aggregation in the nucleus. Similarly, if an agent reduces protein aggregation in the cytoplasm to a greater extent than it reduces protein aggregation in the nucleus, this agent preferentially reduces aggregation in the cytoplasm. As another example, if an agent reduces protein aggregation in the nucleus and does reduce protein aggregation in the cytoplasm, this agent specifically reduces protein aggregation in the nucleus. As another example, if an agent reduces protein aggregation in the cytoplasm and does reduce protein aggregation in the nucleus, this agent specifically reduces protein aggregation in the cytoplasm. The present invention provides a method of identifying an agent that prevents protein aggregation comprising: a) contacting a cell expressing a polypeptides comprising an amino acid sequence encoding a fluorescent protein and an amino acid sequence encoding a GCP-170 polypeptide with a test agent; b) comparing the cell contacted with the test compound with a cell expressing a polypeptide comprising an amino acid sequence encoding a fluorescent protein and an amino acid sequence encoding a GCP-170 polypeptide that was not contacted with the test agent; and c) determining the effect of the test agent on protein aggregation, such that if protein aggregation in the cell contacted with the test compound occurs to a lesser extent than protein aggregation in the cell that was not contacted with the test compound, the agent is an agent that prevents protein aggregation. As utilized herein, an agent that prevents protein aggregation is an agent that inhibits protein aggregation or recurrence of protein aggregation. In the methods of identifying an agent that prevents protein aggregation, one of skill in the art will know how to administer the test agent before protein aggregation has occurred in a cell expressing a polypeptide comprising an amino acid sequence encoding a fluorescent protein and an amino acid sequence encoding a GCP-170 polypeptide, so that the extent to which the agent prevents protein aggregation can be assessed. For example, the GCP-170 fusion polypeptide of the present invention can be expressed under the control of an inducible promoter such that a test agent can be administered to the cell prior to inducing expression of the GCP-170 polypeptide, thus allowing the test agent to be present in the cell prior to protein aggregation. Alternatively, one of skill in the art can administer a test agent to a cell in which protein aggregates have formed and determine the extent to which new aggregates form and/or the extent to which existing aggregates are prevented from changing in morphology, increasing in size and/or changing in density by the test agent. The test agents or test compounds utilized in these methods include, but are not limited to, antibodies, chemicals, oligonucleotides, antisense compounds, siRNAs, ribozymes, small molecules, drugs and secreted proteins. Test compounds in the form of cDNAs or nucleic acids encoding therapeutic polypeptides can also be tested in the methods of the present invention. These nucleic acids can be administered to a cell via methods standard in the art such as via transfection, lipofection, viral transduction, electroporation and Streptolysin-O-mediated permeabilization. The present invention provides a method of identifying an agent that increases protein aggregation comprising: a) contacting a cell containing aggregated fusion polypeptides comprising an amino acid sequence encoding a fluorescent protein and an amino acid sequence encoding a GCP-170 protein with a test agent; b) comparing the cell contacted with the test compound with a cell containing aggregated polypeptides that was not contacted with the test agent; and c) determining the effect of the test agent on protein aggregation, such that if protein aggregation in the cell contacted with the test compound is greater than protein aggregation in the cell that was not contacted with the test compound, the test agent is an agent that increases protein aggregation. Such a method can be utilized to screen an agent for its effect on protein aggregation, such that if protein aggregation is increased, one of skill in the art would know that this agent could increase protein aggregation in a subject and potentially lead to a disease associated with protein aggregation. For example, drugs administered to subjects for disease indications can be screened utilizing this method to evaluate their effects on protein aggregation. If protein aggregation is increased by a drug, one of skill in the art would know that this drug can increase protein aggregation in a subject, for example in the brain, and lead to increased cellular toxicity potentially resulting in cell death. The present invention also contemplates administering a test agent to a cell in a non human transgenic animal model, such as a drosophila, c.elegans, mouse, rat, rabbit, monkey, guinea pig, zebrafish, pig, sheep or rodent model, expressing a polypeptide comprising an amino acid sequence encoding a fluorescent protein and an amino acid sequence encoding a GCP-170 polypeptide that forms aggregates, such that one of skill in the art can observe the effects of an agent on protein aggregation in vivo. Also, any of the agents identified utilizing the screening methods of the present invention can be administered to a non human transenic animal expressing a polypeptide comprising an amino acid sequence encoding a fluorescent protein and an amino acid sequence encoding a GCP-170 polypeptide that forms aggregates. Furthermore, any of the agents that reduce protein aggregation, identified utilizing the screening methods of the present invention, can be administered to a nonhuman animal model of a disease associated with protein aggregation in order to assess its in vivo effects on pathologies associated with protein aggregation. For example, these agents can be administered to an animal model of alcoholic liver disease, Alexander's disease, Alzheimer's disease, Amyloidosis, familial amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, Prion diseases, spinocerebellar ataxia or Wilson's disease. The agents identified in the screening methods of the present invention can also be used in either in vitro or in vivo assays to determine the effect of the agent on proteins known to aggregate in disease states. For example, the agents can be administered to a cell, either in vitro, ex vivo or in vivo comprising aggregated cytokeratins (alcoholic liver disease); GFAP (Alexander's disease); presenilin, Tau, /5-amyloid(Alzheimer's disease); light chain IgG (Amyloidosis); superoxide dismutase (familial amyotrophic lateral sclerosis); huntingtin (Huntington's disease); alpha-synuclein, ubiquitin carboxyl-terminal hydrolase LI (Parkinson's disease); prion protein (prion disease); P or Q-type voltage- sensitive Ca++ channels, axins (spinocerebellar ataxia); or ATP7B (Wilson's disease) in order to assess the effect of the agent on the aggregated proteins and/or the pathology of the disease. The present invention has also established that the domain of the nucleus where the GCP 170 fusion proteins proteins forms deposits is also the domain in which poly-glutamine expansion disease proteins accumulate, and viruses replicate. Thus, GCP-170 fusion proteins can be used to alter this domain or accumulation of proteins within it to prevent poly-Q protein deposition and viral replication. Therefore, the present invention provides a method of identifying a potential antiviral agent comprising: a) contacting a cell containing aggregated fusion polypeptides comprising an amino acid sequence encoding a fluorescent protein and an amino acid sequence encoding a GCP-170 protein with a test agent; b) comparing the cell contacted with the test compound with a cell containing aggregated polypeptides that was not contacted with the test agent; and c) determining the effect of the test agent on protein aggregation, such that if protein aggregation in the cell contacted with the test compound is less than protein aggregation in the cell that was not contacted with the test compound, the agent is a potential antiviral agent. This method can identify agents that reduce protein aggregation in the nucleus such that protein aggregation in the nucleus of the cell contacted with the test compound is less than protein aggregation in the nucleus of the cell not contacted with the test compound. This method can also identify agents that reduce protein aggregation in the cytoplasm such that cytoplasm of the cell contacted with the test compound is less than protein aggregation in the cytoplasm of the cell not contacted with the test compound. As mentioned above, any of the GCP-170 fusion polypeptides of the present invention can be utilized in this method. In particular, a fusion polypeptide comprising a GCP-170 protein and a fluorescent protein, wherein the GCP-170 protein comprises amino acids 566-1375 of SEQ ID NO: 1 can be utilized. Once an agent is identified that reduces protein aggregation in the nucleus or the cytoplasm of the cell, this agent is a potential antiviral agent. Therefore, one of skill in the art can administer the potential antiviral agent to a cell contacted with a virus and determine its antiviral activity. For example, the skilled artisan can administer an agent that reduces protein aggregation in the nucleus to a cell contacted with a virus, such as a DNA virus, that replicates in the nucleus of the cell. At the time of administering the agent, the cell can already be infected with the virus, the virus and the agent can be administered simultaneously or the cell can be contacted with the virus after administration of the agent to the cell. Such viruses include, but are not limited to, parvoviruses, adenoviruses, herpesviruses (herpesvirus 1-7), papillomaviruses (HPVs), polyoma virus, vaccine virus and hepatitis B. One of skill in the art would be able to assess the effects of the agent on viral activities, such viral replication, genomic integration of viral sequences, translation of mRNA, assembly of viral particles, cell lysis and release of virus from the cells. The skilled artisan can also administer the agent to a non human animal model of viral infection, such as a mouse, rat, rabbit, monkey, guinea pig, zebrafish, pig, sheep or rodent model to determine its antiviral activity. The animal can already be infected with a virus prior to administration of the agent or contacted with the virus after administration of the agent. In another example, the skilled artisan can administer an agent that reduces protein aggregation in the cytoplasm to a cell contacted with virus, such as an RNA virus or a poxvirus, that replicates in the cytoplasm of the cell. Such viruses include, but are not limited to, polio virus, human rhino virus 1 A, foot and mouth disease virus, human astrovirus, sindbis virus, rubella virus, yellow fever virus, reovirus 1, blue tongue virus, human rotavirus, influenza virus A, influenza virus B, influenza virus C, newcastle disease virus, measles virus, mumps virus and respiratory synctial virus, rabies virus, hantavirus, retrovirus (for example, HIV-1, HIV-2), human spuma retrovirus, Marburg virus and Ebola virus. One of skill in the art would be able to assess the effects of the agent on viral activities, such viral replication, genomic integration of viral sequences, translation of mRNA, assembly of viral particles, cell lysis and release of virus from the cells. The skilled artisan can also administer the agent to a non human animal model of viral infection, such as a mouse, rat, rabbit, monkey, guinea pig, zebrafish, pig, sheep or rodent model to determine its antiviral activity. The animal can already be infected with a virus prior to administration of the agent or contacted with the virus after administration of the agent. The present invention shows that aggregates of GCP-170 fusion polypeptides result in inhibited cell growth, ultimately leading to cell death. Cell death can occur via apoptosis, the transcription of genes involved in apoptosis, inappropriate transcription of genes possibly leading to necrosis, or other mechanisms of cell death, or DNA damage. Thus, the present invention provides methods of identifying agents that reduce cell death or cellular toxicity by reducing protein aggregation, for example, reducing aggregation of GCP-170 fusion proteins, in cells. The present invention also provides a method of identifying an agent that reduces the growth inhibitory effects of protein aggregation in a cell comprising: a) contacting a cell containing aggregated fusion polypeptides comprising an amino acid sequence encoding a fluorescent protein and an amino acid sequence encoding a GCP-170 protein with a test agent; b) comparing the cell contacted with the test compound with a cell containing aggregated polypeptides that was not contacted with the test agent; and c) determining the effect of the test agent on protein aggregation, such that if protein aggregation in the cell contacted with the test compound is less than protein aggregation in the cell that was not contacted with the test compound, and cell growth is greater in the cell contacted with the test compound than cell growth in the cell that was not contacted with the test compound the agent is an agent that reduces the growth inhibitory effects of protein aggregation in a cell. In the methods of identifying an agent that reduces the growth inhibitory effects or the cytotoxic effects of protein aggregation in a cell, the reduction of the growth inhibitory effects of protein aggregation does not have to be complete as this reduction can range from a slight reduction in growth inhibition to a complete reduction of growth inhibition such that cell growth is comparable to a control cell that does not express the GCP-170 fusion proteins of the present invention. The agents identified via this method can be utilized to assess in vivo reduction of cytotoxicity, for example, in neurodegenerative animal models, such as Parkinson's and Alzheimer's disease where neuronal damage and cell death is associated with the disease. Thus, one of skill in the art can administer an agent to an animal model of a neurodegenerative disease and assess its effect on cholinergic neurons, dopaminergic neurons, catecholaminergic neurons hippocampal neurons, forebrain neurons and/or motor neurons. The present invention has also identified that GCP-170 fusion proteins can sequester transcription factors such as, for example, CBP and p53. Therefore, the present invention provides a method of identifying an agent that inhibits the association of a transcription factor with protein aggregate comprising: a) contacting a cell containing aggregated fusion polypeptides comprising an amino acid sequence encoding a fluorescent protein and an amino acid sequence encoding a GCP-170 protein with a test agent; b) comparing the cell contacted with the test compound with a cell containing aggregated polypeptides that was not contacted with the test agent; and c) determining the effect of the test agent on the association of the transcription factor with a protein aggregate, such that if the amount of the transcription factor associated with a protein aggregate in the cell contacted with the test compound is less than the amount of transcription factor associated with a protein aggregate in the cell that was not contacted with the test compound, the agent is an agent that inhibits the association of a transcription factor with a protein aggregate. The transcription factor can be, but are not limited to, p53, CBP, LANP, PQBP-1, N-coR, ARA24, mSin3A, TAFII130, ETO/MTG8, pl60/GRIPl, Spl,CtBP, CA150, SC35, or MLF1P. The protein aggregates can also comprise combinations of the above- mentioned transcription factors, such that one of skill in the art would be able to assess the effects of an agent on more than one transcription factor utilizing this method. In this method, the transcription factor if associated with a protein aggregate, will be associated with a protein aggregate in the nucleus of the cell and the amount of the transcription factor associated with a protein aggregate can determined by measuring the transcriptional activation activity of the transcription factor in the cell as described in the Examples and via methods known in the art for assessing transcriptional activity. In this method, if an agent reduces the amount of the transcription factor that is associated with a protein aggregate, transcriptional activity of this factor will increase because it is no longer bound to the aggregate or sequestered as part of the aggregate. The amount of the transcription factor associated with a protein aggregate can also be determined by detecting the transcription factor with an antibody that binds to the transcription factor. Agents that inhibit the association of a transcription factor with a protein aggregate can be utilized in animal models for diseases, such as Huntington's disease that are characterized by altered transcriptional activity of transcription factors. Treatment Methods Those agents or compounds found to reduce protein aggregation can be utilized to treat diseases associated with protein aggregation, such as those mentioned above and throughout this application . One of skill in the art will know that the compounds of the present invention can be administered to a subject in a suitably acceptable pharmaceutical carrier. The subject can be any mammal, preferably human, and can include, but is not limited to mouse, rat, cow, pig, guinea pig, hamster, rabbit, cat, dog, goat, sheep, monkey, horse and chimpanzee. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected agent without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. In addition, one can include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc. The compounds of the present invention can be administered via oral administration, nebulization, inhalation, mucosal administration, intranasal administration, intratracheal administration, intravenous administration, intraperitoneal administration, subcutaneous administration, intracerebral delivery (such as intracerebral injection or by convection enhanced delivery) and intramuscular administration. Dosages of the compositions of the present invention will also depend upon the type and/or severity of the disease and the individual subject's status (e.g., species, weight, disease state, etc.) Dosages will also depend upon the form of the composition being administered and the mode of administration. Such dosages are known in the art or can be determined by one of skill in the art. Furthermore, the dosage can be adjusted according to the typical dosage for the specific disease or condition to be treated. Often a single dose can be sufficient; however, the dose can be repeated if desirable. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, sex and other parameters and can be determined by one of skill in the art according to routine methods (see e.g., Remington's Pharmaceutical Sciences). The individual physician in the event of any complication can also adjust the dosage. EXAMPLES Formation of protein aggregates or aggresomes has been proposed to represent a general cellular response to the presence of misfolded proteins. Aggresomes recruit components of the folding and degradative machineries, suggesting that they represent sites specialized for protein clearance. Aggresomes form at the peri-centriolar region, indicating that sequestration of misfolded proteins occurs in spatially defined cellular site. To test the generality of misfolded protein deposition and determine whether cellular sites other than the cytoplasm can form aggresomes, the effects of overexpressing a protein chimera (GFP- GCP170*) in cells were investigated. GFP-GCP170* accumulates in ribbon-like cytoplasmic aggresomes, and unexpectedly, also deposits in discrete foci within the nucleus.

Antibodies and Reagents Anti-giantin antibody was a gift from Dr. Hans P. Hauri (University of Basel, Switzerland). Anti-Hdj2 polyclonal antibody was a gift from Dr. Douglas Cyr (University of North Carolina at Chapel Hill). Anti-GFP polyclonal antibody (Cat. # Ab290) was purchased from Abeam Limited. Anti-FLAG monoclonal antibody was purchased from Eastman Kodak. Anti- β-tubulin monoclonal antibody was purchased from Sigma Chemical Co. Anti-lamin A (Cat. # sc-6215), anti-SC35 and anti-PML (PG-M3) (Cat. # sc-966) monoclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-Hsc70 (Cat. # SPA-815) and anti-Hsp70 (Cat. #SPA-810) monoclonal antibodies were purchased from StressGen Biotechnologies. Anti-20S proteasome (α-subunit) polyclonal antibody was purchased from Calbiochem-Novabiochem. Anti-human CFTR (c-terminus specific) antibody was purchased from R&D Systems Inc. Rabbit polyclonal antibody to ubiquitin- protein conjugates was from Affiniti Research Products Limited. Oregon green-labeled goat anti-rabbit IgG antibody, Texas red-labeled goat anti-mouse IgG antibody, and Texas red- labeled goat anti-rabbit IgG antibody were from Molecular Probes, Inc. HRP-labeled sheep anti-rabbit IgG antibody and HRP-labeled goat anti-rabbit IgG antibody were from Amersham Pharmacia Biotech. Nocodazole was purchased from Sigma Chemical Co. and used at the indicated concentration. SuperSignal West Pico chemiluminescence substrate was from Pierce Chemical Co. Restriction enzymes and molecular reagents were from

Promega, New England BioLabs, Inc., or QIAGEN. All other chemicals were from Sigma- Aldrich or Fisher Scientific. DNA Constructs To make a chimera of EGFP and GCP-170, a primer containing the Xhol restriction enzyme site was designed in front of the start codon of GCP-170. The 770-base pair PCR fragment containing sequences from the start codon of GCP-170 to the EcoRJ site of FQSY1024 (Misumi et al, 1997) was cloned into the Xhol and EcoRI sites of pEGFP-C2 plasmid (Clontech Laboratories Inc.). A 5558-base pair EcoRI fragment from FQSY1024 was then cloned into the EcoRI sites of the plasmid above to generate an EGFP-tagged full length GCP-170. GFP 170* construct was then generated by removing the Bglll fragment and SacII fragment from the N-terminal and C-terminal end of EGFP-GCP-170, respectively. The resultant construct (GFP 170* construct) expresses an EGFP-tagged GCP- 170 fragment from amino acid 566 to 1375. The G3-FLAG construct has been described previously (Chen et al, 2001). It encodes the putative aggrecan signal sequence (the first 23 N-terminal amino acids of aggrecan), a GAG5 consensus sequence, G3, and C-terminally attached His and FLAG epitopes. GFP-250 and ΛF508-CFTR constructs were described previously (Garcia-Mata et al, 1999). The plasmid expressing Q82-GFP was a gift from Dr. Richard Morimoto (Northwestern University) and was described previously (Kim et al, 2002).

Cell Culture, Transfections and Immunofluorescence Microscopy COS-7 and COS-1 cells were grown in DMEM with glucose and glutamine (Mediatech, Inc.) supplemented with 10% FBS (Life Technologies), 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies). Cells were transfected with the Fugene transfection reagent (Roche), with TransIT polyamine transfection reagents (Minis Corporation), or with Lipofectin (Life Technologies), according to manufacturer protocols. At 18-48 h after transfection, cells were fixed with 3% paraformaldehyde, or in some cases cold methanol, and processed for immunofluorescence microscopy as previously described (Alvarez et al, 1999; Chen et al, 2001).

Electron Microscopy and Immunogold Labeling COS-7 cells were transfected with the GFP 170* construct. At 48 h after transfection, cells were washed with PBS, detached from the plate by trypsinization and collected by centrifugation at 300 X g for 5 min at 4°C. Cells were washed twice with PBS and then fixed for 90 min with 1.5% glutaraldehyde in 0.1 M sodium cacodylate pH 7.4. Cells were washed three times with sodium cacodylate and postfixed with 1% OsO₄ in 0.1 M sodium cacodylate pH 7.4 for 60 min on ice. After washing three times with 0.1 M sodium cacodylate, pH 7.4, cells were dehydrated by incubation with a series of ethanol solutions (30, 50, 70, 90, 95, and 3 X 100%) followed by 2 h incubation in 1:1 Spurr's resin/propylene oxide. After two changes of fresh 100% resin, the cell pellets were transferred to gelatin molds and polymerized in fresh resin overnight at 60°C. Gold epoxy sections (100 nm thick) were generated with a Reichert Ultracut ultramicrotome and collected on 200 mesh copper grids. Grid specimens were stained for 20 min with saturated aqueous uranyl acetate (3.5%) diluted 1:1 with ethanol just before use, followed by staining with lead citrate for 10 min. Stained samples were examined on a JEOL 100CX electron microscope. For immunogold labeling, COS-7 cells expressing GFP 170* were harvested by trypsinization 24 h after transfection. Cells were washed three times with PBS and pre-fixed with 3% formaldehyde and 0.2% glutaraldehyde for 40 min, followed by dehydration with series of graded ethanol at room temperature. Cells were infiltrated and embedded with LR White. After polymerization, sections were cut with an ultramicrotome and collected onto nickel grids. The grids were incubated with anti-GFP primary antibody overnight at 4°C and goat anti-rabbit IgG conjugated to 12-nm gold particles for 1 h at room temperature (Jackson ImmunoResearch Laboratories, Inc.), followed by post-fixation with 2% glutaraldehyde for 5 min at room temperature, and counterstain with 2% uranyl acetate for 5 min at room temperature.

Analysis of Soluble and Insoluble GFP170* COS-7 cells were transfected with GFP170*. At 48 h after transfection, cells were washed and harvested in ice-cold PBS. Equal amounts of cells were lysed for 1 h on ice with 100 μl of either 1% Triton X-100 in PBS or RIPA buffer (50 mM Tris-HCl, pH 8.0, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, and 150 mM NaCl) supplemented with protease inhibitor cocktail and 1.0 mM PMSF. Lysates were sonicated for 5 s with microtip sonicator followed by 15 min centrifugation at 15,000 X g. Pellets were resuspended in 100 μl of 1% SDS in PBS. Equal volumes of each pellet and supernatant were boiled in SDS-PAGE sample buffer and resolved on 8% SDS-PAGE. The gel was transferred to nitrocellulose membrane and processed for Western blotting as previously described (Gao and Sztul, 2001). Analysis of Degradation Rate of GFP170* and GFP250 COS-7 cells were transfected with either GFP 170* or GFP250 construct in a 6- well plate for 24 h. Cells were then washed in PBS and incubated in methionine-free DMEM for 1 h. Cells were labeled with 200 μCi/ml 35S-methionine (NEN) for 60 min. Incorporation was terminated by washing the cells with PBS and chasing with DMEM medium supplemented with 0.2 mM methionine for indicated times. At each time point, cells were lysed with RIPA buffer supplemented with protease inhibitor cocktail and 1.0 mM PMSF. Equal amounts of lysate from each time point were resolved by 10% SDS-PAGE followed by autoradiography using a Phospholmage Screen. The relative radioactive intensity of GFP 170* and GFP250 bands was quantified and compared using ImageQuant software.

Time-Lapse Imaging Study The movement of GFP 170* was analyzed by time-lapse imaging as described before (Garcia-Mata et al., 1999). Briefly, COS-7 cells grown on glass coverslips were transfected with GFP 170* construct. At 20 h after transfection, coverslips were placed onto sealed silicon rubber chambers containing culture medium buffered with 25 mM HEPES, pH 7.5. Images were acquired with an Olympus 1X70 inverted microscope equipped with a 40 X/1.35 NA objective lens and a cooled charge-coupled device camera. IpLab Spectrum software (Signal Analytics) was used to control image acquisition and manipulation.

FRAP and FLIP Analysis FRAP and FLIP analysis of the GFP170* and Q82-GFP was performed as previously described (Kim et al., 2002), using a Leica TCS SP2 confocal microscope with a 63X objective lens. COS-7 cells expressing GFP170* were subject to analysis 24 to 48 h after transfection. COS-7 cells expressing Q82-GFP were analyzed 72 hours after transfection. During the experiment, COS7 cells were kept at 37°C in a glass-bottom dish containing DMEM medium buffered with 25 mM HEPES, pH 7.5. For FRAP analysis, fluorescent regions outlined in figures were photobleached at full laser power with zoom and images were taken at lower laser power (-20%) before bleaching and every 10-30 s after photobleaching. Fluorescent recovery was calculated by comparing the intensity ratio in regions of bleached area before the bleach and after recovery. The postbleach intensities were normalized upward to correct for total loss of fluorescence due to the photobleach by comparing the fluorescent intensities outside the bleached area using IpLab Spectrum software (Signal Analytics). For FLIP analysis, cells were repeatedly bleached in the same defined region and imaged at 1 min intervals. At each time point after photobleaching, fluorescence intensities in the cytosolic and nuclear regions were measured separately using IpLab software, and normalized to those values before bleaching.

GFP170* deposits within cytoplasmic and nuclear aggregates The Golgi Complex protein of molecular weight 170 (GCP 170), also known as golgin- 160, was identified as a human auto-antigen in patients with Sjδgren syndrome (Fritzler et al, 1993). Patient sera reacted with an antigen localized in the Golgi, and subsequent studies led to the cloning of GCP170 (Misumi et al, 1997). GCP170 contains 1530 amino acids, arranged into an N-terminal head domain followed by a long coiled-coil stalk and a short C-terminal tail (Figure 1A). The stalk region is divided into 6 coiled-coil segments. Coiled-coil domains have been shown to mediate protein-protein interactions, and GCP 170 may form a parallel homodimer through the intertwining of its coiled-coil segments (Hicks and Machamer, 2002). GCP 170 is a soluble protein that localizes to the cytoplasmic face of Golgi membranes (Misumi et al, 1997; Hicks and Machamer, 2002). Various GFP-tagged constructs of GCP 170 were generated to study targeting of GCP 170 to the Golgi in vivo. Herein, GFP-tagged wild-type GCP 170 and a construct called GFP 170* that encodes amino acids 566 to 1375 of GCP170 and contains coiled-coils 3, 4 and 5 are described (Figure 1 A). The cellular localization of GFP-tagged GCP170 and GFP170*. GFP-tagged was analyzed. The Golgi localization is shown by extensive overlap of the GFP signal with the Golgi marker, giantin. In addition, dispersed cytoplasmic aggregates are also evident (arrowheads). In an adjacent cell (upper right), perhaps expressing tagged GFP-tagged GCP 170 for a longer period or at a higher level, the protein accumulates in large aggregates surrounding the Golgi complex (double arrow). The formation of cytoplasmic aggregates is consistent with previous biochemical findings that GCP170 is aggregation-prone (Misumi et al, 1997). GFP 170* is also targeted to the Golgi when expressed at low levels (Figure 1C, cell at lower left, arrow). In addition, in an adjacent cell, where GFP 170* is present at high levels, most of the protein localizes to large cytoplasmic aggregates in the juxtanuclear region (double arrow). Examination of cells at different times after transfection with GFP 170* suggests that the cytoplasmic aggregates grow by coalescence (Figure ID). Initially, numerous small (<0.5 μm in diameter) particles are distributed throughout the cell. Subsequently, the peripheral aggregates relocate to the peri-centriolar zone, coalesce, and ultimately form a compact ribbon-like structure adjacent to the Golgi. The GFP 170* cytoplasmic aggregates are morphologically similar to those formed by GFP-tagged wild- type GCP 170. The ribbon-like morphology is distinct from the spherical aggresomes formed by overexpressing CFTR (Johnston et al., 1998), GFP-250 (Garcia-Mata et al., 1999), or poly-Q expanded huntingtin (Waelter et al., 2001). In those cases, a compact spherical structure is formed around the microtubule-organizing center (Figure IB, insert). The ribbon-like morphology is observed when GFP170* is expressed in a number of different cell types (ex: simian COS-7, human HeLa, and mouse MEF), indicating that the structure of aggregates is defined by the nature of the aggregating protein, rather than by the cell type. Unexpectedly, a portion of GFP 170* localizes to discrete punctate foci within the nucleus (Figure 1C, cell at upper right, double arrowhead). A quantitative analysis of 50 randomly chosen transfected cells shows a relationship between the size and the number of the nuclear aggregates (Figure ID). In nuclei containing more than 20 foci, the average size of individual foci is less than 5 /xm2, while nuclei containing less than 10 foci have aggregates bigger than 10 μm2 (Figure ID graph). These results suggest that the larger structures form by coalescence of the small foci. This conclusion is also supported by the shift in the ratios of small, medium and large foci during GFP 170* expression. At 24 hours after GFP 170* transfection, -42% of cells contain small (0.5 μm in diameter) aggregates, -54% contain medium (1-1.5 μm in diameter) aggregates, and -4% contain large (>2 μm in diameter) aggregates. These ratios change 48 hours after GFP 170* transfection, at which time -30% of cells contain small aggregates, -51% contain medium aggregates, and -22% contain large aggregates. The nuclear GFP 170* aggregates are contained in regions of the nucleoplasm enclosed within the nuclear membrane (Figure IE). Lamin A forms a mesh-like matrix on the inner face of the nuclear membrane that delineates the nuclear space (Hozak et al, 1995). A focal plane through the nucleus shows GFP 170* aggregates inside the lamin A- enclosed space. Aggregates are not found in association with the nuclear rim. A direct confirmation of nuclear localization, and morphological characterization of the cytoplasmic and nuclear GFP 170* aggregates are provided by transmission electron microscopy and immunogold labeling (Figure 2). COS-7 cells transfected with GFP 170* contain irregular cytosolic aggregates (Figure 2b, arrows) and varying numbers of nuclear aggregates (Figure 2b, arrowheads). Non-transfected control cells never contain such structures (Figure 2a). The cytosolic aggregates appear ribbon-like and can extend to more than 15 μm in length. They display an uneven distribution of components and appear to have an internal architecture (Figure 2c and d). The cytoplasmic aggregates are often surrounded by mitochondria (Figure 2c), similar to the association of mitochondria with aggresomes formed by CFTR (Johnston et al, 1998) or GFP-250 (Garcia-Mata et al, 1999). The nuclear aggregates are spherical or ovoid, and range in diameter from 0.5 μm to 3 μm. Sometimes they appear as homogenous accumulations of granular material without apparent subdomain structure (Figure 2e). Often, however, they contain internal electron lucent spaces (Figure 2f, arrowheads). The presence of internal substructures within the GFP 170* nuclear aggregates is also observed by fluorescence microscopy (Figure 2g, arrowheads). The deposition of GFP 170* within the morphologically defined cytoplasmic and nuclear aggregates was confirmed by immunogold labeling with anti-GFP antibodies. Gold particles label both cytoplasmic and nuclear aggregates (Figure 2h and i). GFP170* aggregates are cytoplasmic and nuclear aggresomes Cytoplasmic aggregates (formed by either poly-Q proteins or non-poly-Q proteins) and nuclear aggregates (formed by poly-Q proteins) have been described as aggresomes, based on a number of defining characteristics. However, the nature of nuclear aggregates formed by a non-poly-Q protein has not been previously described until the present invention. GFP 170* provides a unique tool to characterize the cytoplasmic and nuclear aggregates within the same cell. Therefore, whether the GFP 170* aggregates have the characteristic features of aggresomes was tested. One of the defining characteristics of aggresomes is the recruitment of molecular chaperones. Chaperones have been detected in association with cytoplasmic aggregates of poly-Q expanded huntingtin (Waelter et al., 2001), and with aggregates of the non-poly-Q proteins CFTR (Johnston et al., 1998) and GFP-250 (Garcia-Mata et al., 1999). Chaperones are also recruited to nuclear aggregates of poly-Q expanded ataxin-3 and huntingtin (Chai et al, 1999a; Waelter et al., 2001). The cytoplasmic and nuclear aggregates containing GFP170* appear to be aggresomes, based on their recruitment of Hsc70, Hsp70 and Hdj2, representatives of the Hsp70 and the Hsp40 families of chaperones, respectively, in transfected cells (Figure 3 A-C). Hsc70 is recruited to the periphery of cytosolic aggregates, but is excluded from the nucleus (Figure 3 A). In contrast, the stress-responsive Hsp70 is significantly induced in cells expressing GFP170*, and localizes to both the nuclear and cytoplasmic aggregates (Figure 3B). Hdj2 also appears to be up regulated in cells expressing GFP 170*, and co-localizes with both cytosolic and nuclear aggregates (Figure 3C). The observations presented herein show distinct roles for these chaperones in the formation of cytoplasmic versus nuclear aggregates. The motor-dependent movement of aggregated particles on microtubules is a hallmark of cytoplasmic aggresome formation (Johnston et al, 1998; Garcia-Mata et al, 1999). However, the importance of MT-mediated transport to nuclear aggresome formation has not been explored. Therefore, the role of MTs in the formation of cytoplasmic and nuclear GFP 170* aggregates was examined. In cells treated with nocodazole during GFP 170* expression, cytosolic aggregates are smaller and dispersed throughout the cell

(Figure 4A). In such cells, peripheral aggregates do not coalesce into a compact perinuclear structure. A similar dispersed phenotype has been reported for GFP-250 aggregates in cells defective in MT- and dynein-mediated transport (Garcia-Mata et al, 1999). Interestingly, the nuclear aggregates are also significantly smaller in nocodazole treated cells (Figure 4A). Large nuclear aggregates are not detected at times when larger aggregates are present in untreated cells. This observation suggests that nuclear aggregates form less efficiently in the absence of MT-dependent traffic. MTs have not been detected within the nucleus, but the delivery of various components to the nucleus has been shown to be MT-dependent (Sodeik et al, 1997). In the absence of MTs, these molecules may become limited within the nucleus and influence the coalescence of GFP 170* foci. The formation of cytoplasmic aggresomes has been correlated with the collapse of the vimentin intermediate filament cytoskeleton (Johnston et al, 1998; Garcia-Mata et al, 1999). Specifically, vimentin has been shown to relocate to form a "cage" around the cytosolic aggresomes of GFP-250 (Figure 4B, insert and (Garcia-Mata et al, 1999)) or CFTR (Johnston et al, 1998). The results presented herein show that vimentin also surrounds the ribbon-like GFP 170* cytoplasmic aggregates (Figure 4B). Another characteristic feature of cytoplasmic and nuclear aggresomes is the recruitment of proteasomes (Johnston et al., 1998; Garcia-Mata et al., 1999; Waelter et al., 2001). Proteasomes are composed of a 20S proteolytic core and two 19S regulatory caps responsible for recognizing and unfolding the substrates. The 20S core is composed of two antechambers, each lined with non-proteolytic /3-subunits, and a central cavity lined with proteolytic jS-subunits (Voges et al., 1999). Antibodies against the α-subunits label the cytoplasmic and the nuclear GFP 170* aggregates (Figure 4C). Proteasomes appear to be recruited to the GFP 170* aggregates. Cytoplasmic and nuclear aggregates have been shown to be positive for ubiquitin (Johnston et al., 1998; Waelter et al., 2001). In agreement with a previous report, anti-ubiquitin antibodies label cytoplasmic aggregates of mutant CFTR (ΔF508-CFTR) (Figure 4D, insert). The same antibodies also label GFP 170* aggregates (Figure 4D). The association of chaperones, proteasomes and ubiquitin with GFP 170* aggresomes is likely to increase the local concentration of molecules necessary for unfolding and degradation of the aggregated protein, and may contribute to its clearance. The degradation rate of GFP 170* was therefore examined. Pulse-chase analyses defined a half-life of -8 hours for GFP 170* (Figure 5 A). This degradation rate is comparable with that of previously described aggresomal proteins such as GFP-250 (Figure 5 A, Garcia-Mata et al., 1999). Proteins deposited within aggresomes are largely detergent-insoluble (Ward et al., 1995; Scherzinger et al., 1997; Garcia-Mata et al., 1999; Kopito, 2000). We examined the solubility of GFP 170* in either Triton X-100 or SDS-containing buffers. Both extraction conditions are sufficient to solubilize 100% of cellular /5-tubulin. In contrast, the majority of GFP 170* is present in the insoluble fraction, with less than 5% in the extractable fraction (Figure 5B). Together, the findings set forth herein indicate that the cytoplasmic and nuclear aggregates of GFP 170* share key characteristics of aggresomes.

Cytoplasmic and nuclear GFP170* aggresomes are dynamic Previous studies of nuclear and cytoplasmic aggregates of poly-Q proteins suggest that some poly-Q expanded proteins (ex: ataxin-1) are dynamic and exchange their components (Stenoien et al, 2002), while others (ex: ataxin-3 and Q82-GFP) are immobile (Chai et al, 2002; Kim et al, 2002). Therefore, FRAP and FLIP were used to explore the dynamics of nuclear and cytoplasmic GFP 170* aggregates. The fluorescence within nuclear aggregates of GFP 170* recovers to -65%, 5 min after photobleaching (Figure 6A, circle and Figure 6C). This result indicate that a large portion of GFP 170* molecules deposited within the aggregates are mobile and exchangeable with soluble molecules in the nucleoplasm. The tι_/2 of fluorescence recovery to steady state levels is -50 sec. The recovery of GFP170* is slower than that of a poly-Q expanded GFP-ataxin-l-84Q (tι_/2 of <2 sec) deposited in nuclear inclusions of similar size (Stenoien et al, 2002). However, GFP 170* recovers significantly faster than the almost immobile poly-Q expanded ataxin-3 (Chai et al, 2002) or Q82-GFP (Kim et al, 2002). To provide a direct comparison between dynamics of GFP 170* and an immobile aggregate, FRAP of Q82-GFP was analyzed. The fluorescence recovery of Q82-GFP in nuclear aggregates is limited to only -12% (Figure 6B, circle and Figure 6C). This is consistent with the observation reported previously (Kim et al, 2002), and validates the experimental parameters utilized herein. The different FRAP dynamics of GFP 170* relative to the more mobile GFP-ataxin-l-84Q or the largely immobile Q82-GFP may reflect differences in biophysical characteristics, stabilization by interacting with distinct nuclear components, or other factors. The dynamics of GFP 170* within cytoplasmic aggregates were also analyzed by FRAP. The fluorescence recovers to -35%, 5 min after photobleaching (Figure 6A, rectangle and Figure 6C). Measuring the initial slope of the recovery indicates a t ₂ of -2 min. This result indicates that a portion of GFP 170* molecules within the cytoplasmic aggregates are mobile and constantly exchange with a soluble pool of GFP 170*. The different kinetics of FRAP between the nuclear and cytosolic GFP 170* aggregates may reflect the different structural properties of aggregates in the cytosol and the nucleus (Figure 2). FLIP was also used to compare the dynamics of GFP 170* molecules within the cytosolic and nuclear aggregates. A cytosolic region of a cell containing cytoplasmic and nuclear aggregates was repeatedly photobleached and the loss of fluorescence from the cytosolic and nuclear aggregates was monitored over time. The fluorescence within the nuclear aggregates decreases to -25% after 5 min of bleaching, and is undetectable after 15 min (Figure 6D circle and Figure 6F). The tj/₂ of FLIP to background level is -3.5 min. This result suggests that GFP 170* molecules are efficiently exported from the nucleus to the cytosol. In contrast, the fluorescence of the cytosolic aggregates only decreases to -65% after 5 min of bleaching, and is still -20% of the initial fluorescence after 15 min of photobleaching (Figure 6D and 6F). In this case, the rate of fluorescence loss is slower than from the nuclear aggregates, with a t of -8 min. This result is consistent with the FRAP data, and both data sets indicate that GFP 170* molecules within cytosolic aggregates are less mobile than those within nuclear aggregates. As expected, only -10% of fluorescence is lost from Q82-GFP aggregates after 5 min photobleaching, and -70% of initial fluorescence still remains after 15 min of photobleaching (Figure 6E circle and Figure 6F). This finding is in agreement with our FRAP results and with previously reported findings (Kim et al, 2002). Nuclear GFP170* aggresomes show a relationship to PML bodies The nucleus is organized into defined territories specialized to perform distinct functions (Spector, 2001). Disruption of nuclear structures, as exemplified by the (tl5;17:q22;q21) translocation that results in a fusion of the PML protein and the retinoic acid receptor alpha (RARo:), leads to acute promyelocytic leukemia (Weis et al, 1994). PML bodies are also called nuclear domain 10 (ND10) bodies or PML oncogenic domains (PODs). The mammalian nucleus contains 10 to 30 PML bodies, which vary in size from 0.2 to 1 μm. They are thought to function in transcriptional regulation, cell cycle progresssion, and apoptosis, based on their content of proteins such as SplOO, PML, Daxx, pRB, CBP and p53, which are involved in these processes (Yasuda et al, 1999; Maul et al, 2000; Zhong et al, 2000). PML bodies have been shown to associate with nuclear aggregates of poly-Q expanded proteins (Dovey et al, 2004). Whether this association is related to the poly-Q track or represents a general response to aggregated protein within the nucleus has not been explored. Therefore, the relationship between PML bodies and GFP 170* aggregates was examined. The nuclear aggregates of GFP 170* show a close relationship with PML bodies (Figure 7). In non-transfected cells, anti-PML antibodies label numerous small nuclear foci (Figure 7A, arrowheads). In a transfected cell containing large GFP170* nuclear foci, the number and the distribution of PML bodies in the nucleus are altered. PML-labeled structures appear to be associated with the GFP170* aggregates (Figure 7A, arrows). In contrast, a significant disruption of nuclear speckles was not observed in the experiments provided herein. The distribution of the SC35 marker for nuclear speckles is not altered by GFP170* expression (Figure 7A), suggesting that GFP170* aggregates do not indiscriminately disrupt nuclear architecture. The association of PMLs with GFP 170* aggregates prompted further exploration of this process. Examination of cells at different times after transfection suggests that the initial deposition of GFP 170* often occurs in or adjacent to PML bodies (Figure 7B; first row, arrowheads). Co-localization is first observed when foci are very small, before PML distribution is altered (Figure 7C). In this representative cell, -82% of GFP 170* aggregates are associated with PML bodies. Quantitation of co-localization in multiple transfected cells indicates that 80-90% of GFP170* puncta are adjacent to or overlap with PML bodies in all examined cells. As the small GFP 170* aggregates merge to form larger structures, PMLs are repositioned on the surface of the larger aggregates (Figure 7B, second and third row). In cells containing only a few large aggregates, PML bodies form beaded necklace-like structures on the surface of the aggregates (fourth row, arrows). Thus, deposition of GFP 170* aggregates induces dramatic changes in the nuclear architecture of PML bodies. Analogous rearrangements are observed in other cell lines (ex: human Hela cells and mouse embryonic fibroblasts), suggesting a commonality of the response. The observed phenotype is distinct from "clumping" of PML bodies during mitotic prophase and metaphase (Everett et al, 1999). It is also morphologically distinct from the increased number of small PML bodies observed during heat shock (Everett et al, 1999). The PML body rearrangements observed in cells expressing GFP 170* are likely to be induced by the aggregates. The association of PML bodies with nuclear aggregates of GFP 170*, in addition to the reported association with nuclear aggregates of poly-Q proteins, suggested that it may be a common nuclear response to nuclear aggregates. Therefore, the relationship between PMLs and nuclear inclusions formed by another non-poly-Q protein, the G3 domain fragment of aggrecan was explored. Aggrecan is an extracellular matrix proteoglycan abundant in cartilage, but an engineered G3 domain fragment has been shown to mistarget to the nucleus and form nuclear foci in transfected cells (Chen et al, 2001). G3 aggregates are also associated with PML bodies (Figure 7D). The association of PMLs with G3 as well as with GFP 170*, in addition to poly-Q proteins, indicates that the association is independent of the primary sequence of the misfolded protein. Cytosolic and nuclear GFP170* aggresomes form by fusion The formation of cytoplasmic aggresomes by the poly-Q expanded androgen receptor occurs by joining of smaller cytoplasmic aggregates (Stenoien et al, 1999). The size variations of GFP 170* aggregates in transfected cells suggested that fusion events may underlie formation of large aggresomes. Time-lapse imaging of live cells was utilized to explore aggresome formation. Two cells with small cytoplasmic and nuclear GFP 170* aggregates were selected and imaged for 12 hours (Figure 8A). Tracking the behavior of individual peripheral particles in the cytoplasm indicates that they move inward and coalesce to form larger peri-nuclear aggregates (tracings in Figure 8B). The movements appear quasi-linear, as would be expected for MT-based transport. The centripetal movement of the particles is not constant over time, and the particles move and then hover in place before moving again. Particle fusions are common and occur preferentially within the peri-nuclear zone. A higher magnification series of time-lapse images clearly shows the fusion of cytoplasmic aggregates (Figure 9A, arrows). Note that spherical aggregates move towards the larger ribbon-like aggregate and fuse with it. Events leading to the formation of nuclear aggregates (either by poly-Q or non-poly- Q proteins) have not been previously described by live imaging. Time-lapse analysis of the nuclear aggregates of GFP 170* illustrates three novel phenomena. First, a gradual increase in the fluorescence intensity of nuclear foci (Figure 9 A) could inidcate that GFP 170* molecules are constantly deposited into pre-existing aggregates. The continuous incorporation could utilize the pool of soluble GFP 170* within the nucleus. Second, small nuclear aggregates move and merge to form larger structures (Figure 9 A, arrowheads). Tracking of the nuclear movements indicates that some particles move directionally, which is inconsistent with unrestricted Brownian movement. In addition, smaller aggregates appear to preferentially fuse with medium to large nuclear aggregates, rather than with other small foci. Third, the nuclear aggregates enclose internal substructures that are extremely dynamic and undergo rapid rearrangements (Figure 9B). In some aggregates, GFP170* surrounds a single substructure (Figure 9B, arrow). In other aggregates, multiple substructures of different size are evident (Figure 9B, arrowhead). The substructures are dynamic and undergo continuous movements within the aggregates. Fusion of two aggregates is paralleled by the reorganization and fusion of the substructures (Figure 9B, double arrowheads). This observation indicates that the components of nuclear aggregates may have preferential affinity for each other and separate by phase partitioning. The present invention shows that a chimeric protein containing a GFP-tagged fragment of GCP-170 (GFP-GCP170*) forms protein aggregates in both cytosolic and nuclear regions of the cell. Extensive literature documents the formation of nuclear inclusions by proteins with expanded poly-Q tracks (reviewed in (Zoghbi and Orr, 2000; Ross, 2002)). This has led to the suggestion that the nuclear aggregation may be regulated by mechanisms specific to poly-Q expansions. The present invention shows GFP 170*, a non-polyQ protein forms nuclear aggresomes. The GFP 170* aggregates appear similar to those formed by poly-Q proteins in a number of criteria: their morphology, the recruitment of chaperones, and the association with proteosomes. These findings show that the formation of nuclear aggresomes is not a poly-Q restricted process, and can represent a common response to the accumulation of misfolded proteins within the nucleus. The process of nuclear aggresome formation was investigated by time-lapse and FRAP imaging of GFP170*. The studies presented herein indicate that the formation of nuclear GFP170* aggresomes is a dynamic process. It is initiated by the deposition of GFP 170* at or adjacent to PML bodies followed by the movement of small aggregates towards larger structures and their coalescence to form still larger aggregates. Unexpectedly, internal substructures that exclude GFP 170* within the nuclear aggregates were observed. The internal substructures undergo extensive rearrangements during fusion of the aggregates. These results show that phase partitioning may underlie the formation of GFP 170* aggregates.

Formation of nuclear aggresomes by GFP170* The GFP 170* protein consisting of the GFP and an internal fragment of the Golgi matrix protein GCP-170 forms aggregates within the cytoplasm and within the nucleus. The nucleation process for aggregation of GFP 170* may begin at the site of translation in the cytoplasm, since high local concentrations of GFP 170* may be produced during the translation of a single mRNA. GFP 170* has 1075 amino acids and a 3225-bp coding region that would be represented in its mRNA. Ribosomes attach every ~80-bp on an mRNA, suggesting that -40 ribosomes could be simultaneously translating a single GFP170* message. As a result, -40 GFP170* molecules may be produced in a localized area, and could serve to seed an aggregation particle. It is unknown whether GFP 170* is transported across the nuclear membrane in a non-aggregated form, similar to the import pathway of most transcription factors, or as an oligomeric particulate, similar to the import pathway utilized by viral particles (Swindle et al, 1999; Tang et al, 2000). Dynamic nature of GFP170* aggresomes FRAP and FLIP analyses of GFP 170* within cytoplasmic and nuclear aggregates shows that GFP170* is largely mobile, and rapidly exchanges in and out of the aggregates. Similar dynamic mobility is exhibited by aggregates of poly-Q expanded ataxin-1 molecules (Stenoien et al, 2002). Both contrast with the behavior of aggregates of poly-Q expanded ataxin-3 that are largely immobile and do not exchange their components (Chai et al,

2002). These results indicate that structures defined morphologically as visible inclusions can be true aggregates (as those formed by ataxin-3), or concentrated depositions of rapidly exchanging components (as those formed by ataxin-1 and GFP 170*). The dynamic behavior of GFP 170* observed in vivo is in contrast with its physical state after cells are disrupted. While the vast majority of GFP170* appears mobile in live cells, the majority of GFP170* is recovered in stable aggregates after cell extraction (compare Figures 5B and 6A). A possible explanation for this apparent contradiction is suggested by the recruitment of chaperones to the GFP170* aggregates. It is possible that in vivo, the continuous action of ATP-dependent molecular chaperones mediates the mobility of GFP 170* molecules. In disrupted cells, falling ATP levels may be insufficient to support chaperone function and instead result in insoluble GFP170* aggregates. This possibility is supported by the finding that the association of Hsp70 with htt-82Q aggregates has the same diffusion coefficient as thermally unfolded substrates, suggesting that Hsp70 associates with the aggregates as part of its functional folding cycle (Kim et al, 2002).

Relationship between nuclear aggresomes and PML bodies A relationship between PML bodies and poly-Q aggregates has been documented previously. PML rearrangements have been described in Purkinje cells of transgenic mice and in transfected COS-1 cells expressing the poly-Q expanded ataxin-1 (Skinner et al, 1997). PML alterations also occur in cells expressing the poly-Q expanded ataxins responsible for the Machado-Joseph disease (Yamada et al, 2002) and SCA7 (Takahashi et al, 2002). These reports suggest that PML bodies may represent sites specialized to recognize and sequester foreign proteins within the nucleus. This is supported by the alterations in PML bodies described during DNA and RNA virus infections (reviewed in (Doucas and Evans, 1996)). In cells infected with Hepatitis delta virus (HDV) (Bell et al, 2000), herpes simplex virus (HSV-1), human cytomegalovirus (HCMV) (Ishov et al, 2002), or human papilloma virus (HPV) (Becker et al, 2004), the virus localizes to enlarged PMLs, and in some cases subsequently disrupts PML bodies (Everett and Maul, 1994). It should be stressed that viruses are highly ordered protein aggregates. The present invention unexpectedly shows that two unrelated proteins, GFP 170* and G3, which are distinct from poly-Q expanded proteins or viral components, also associate with and alter PML bodies. The preferential deposition of GFP 170* at such sites can occur by association with nuclear components present in those sites, or by aggregation-promoting environments in those sites. The results presented herein indicate that PML bodies can represent dedicated domains specialized to handle foreign particulates within the nucleus. The concentration of aggregated material by spatial sequestration, and the concurrent recruitment of unfolding and degradative machineries to such structures can improve the efficiency of clearance. A proteolytic function for PML bodies has been suggested by the increased number of PML bodies in cells treated with a proteasomal inhibitor (Burkham et al, 2001). The association of the protein unfolding and degradative machineries with the nuclear GFP 170* aggregates is consistent with PML bodies participating in proteasomal degradation. Protein clearance within the cytoplasm appears to be consigned to a specialized peri-centriolar proteolytic region (Wojcik and DeMartino, 2003). The association of GFP170* aggregates with PML bodies suggests that like the cytoplasm, the nucleus can contain spatially defined degradative regions. The results presented herein show that the over-expression of GFP170* can amplify these nuclear regions.

Formation of nuclear aggresomes Time-lapse imaging of nuclear aggresomes in live cells documents three novel phenomena. First, GFP 170* is continuously deposited at pre-existing nuclear regions as demonstrated by the increased fluorescence intensity of nuclear GFP 170* foci. The growth of GFP 170* aggregates can be a combined result of direct targeting and selective stabilization of GFP170* molecules at the nuclear foci. The concentration of GFP170* at defined foci suggests that, like the deposition of cytoplasmic aggresomes to the peri- centriolar region, formation of aggregates is spatially restricted within the nucleus. Second, small nuclear aggregates of GFP 170* undergo extensive movements and fusions to form larger aggregates. The movement of small GFP 170* aggregates appears similar to that reported for endogenous nuclear structures. Cajal bodies (CBs) move through the nucleus with similar kinetics, and can fuse and split to modify their size (Platani et al., 2000). PML bodies also can move; although most PML bodies (-63%) show only localized oscillations or are (-25%) immobile, a small percentage (-12%) undergo rapid and more extended movements (Muratani et al., 2002). Nuclear movements are most consistent with passive diffusion within a volume that is limited by a constraint (Platani et al., 2002). A model that is compatible with the available data suggests that nuclear structures move within sub-micron channels formed by the exclusion of nuclear components, rather than by vectorial transport on molecular tracks (Carmo-Fonseca et al., 2002). Actin-dependent myosins may control the diffusible volume since the movement of PML bodies can be blocked by an inhibitor of actin-dependent myosins (Muratani et al., 2002). The driving force that regulate nuclear movements of the various nuclear structures and of GFP 170* aggregates remains to be elucidated. Third, imaging of nuclear GFP 170* aggregates uncovered a novel characteristic of nuclear aggregates: the presence of complex internal structures. These internal subdomains are themselves dynamic and undergo constant movements, fusions and fissions. These observations suggest that the aggregating protein encapsulates nuclear components, and that such encapsulation is not static but undergoes extensive remodeling. Thus, the present invention shows that nuclear aggregates should no longer be considered as static precipitates, but rather, as rapidly reforming entities subject to the coordinated restructuring of their constituents. EXAMPLE 2 Transcriptional Repression and Cell Death Induced by Nuclear Aggregates of Non- polyglutamine Protein Nuclear aggregates of polyglutamine (polyQ)-expanded proteins are associated with a number of neurodegenerative diseases including Huntington's disease (HD) and spinocerebellar ataxias (SCAs). The nuclear deposition of polyQ proteins correlates with rearrangements of nuclear matrix, transcriptional disregulation, and cell death. To explore the requirement for polyQ tracks in educing such cellular responses, whether a non-polyQ protein can deposit as nuclear aggregates and elicit similar responses was examined. As stated above, the present invention provides a protein chimera (GFP 170*) composed of the green fluorescent protein (GFP) fused to an internal fragment of the Golgi Complex Protein (GCP-170) that forms nuclear aggregates analogous to those formed by polyQ proteins. Like the polyQ nuclear aggregates, GFP 170* inclusions recruit molecular chaperones and proteasomal components, alter nuclear structures containing the promyelocytic leukemia protein (PML), recruit transcriptional factors such as CREB-binding protein (CBP) and p53, repress p53 transcriptional activity, and induce cell death. These indicate that nuclear aggregation and transcriptional effects are not unique to polyQ-containing proteins, and represent a general response to misfolded proteins in the nucleus. At least nine neurodegenerative diseases, including Huntington's disease (HD), spinobulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), and spinocerebellar ataxias (SCA) 1, 2, 3, 6, 7, and 17, are caused by a single type of mutation, the expansion of CAG repeats encoding for a polyglutamine (polyQ) track in unrelated proteins (Zoghbi and Orr, 2000). The mutant proteins form protein aggregates or inclusions that are the hallmark of polyQ diseases (Ross, 2002). Unlike other mutant proteins that form cytoplasmic aggregates in diseases such as Alzheimer's and Parkinson's diseases, or amyotrophic lateral sclerosis, polyQ proteins can be deposited as cytoplasmic inclusions as well as nuclear inclusions. The nuclear deposition of polyQ proteins has been correlated with cytotoxicity. Transgenic mice expressing polyQ human huntingtin develop neuronal intranuclear inclusions prior to developing a neurological phenotype (Davies et al, 1997). Similarly, nuclear localization of polyQ proteins is essential to induce cell death in cultured cell and transgenic mouse models (Klement et al., 1998; Katsuno et al, 2002; Takeyama et al., 2002). However, the exact correlation between nuclear polyQ aggregates and pathology remains elusive. PolyQ pathogenesis may be linked to the sequestration and inactivation of proteins essential for cellular functions. Analyses of human postmortem brains, animal models, and cell culture systems have shown that polyQ deposits recruit various cellular components. Invariably, proteins involved in protein folding and degradation, as well as transcriptional regulators are associated with polyQ aggregates (Li and Li, 2004). All the major classes of chaperones including members of the Hsp70 family (Hsc70 and Hsp70) and the Hsp40 family (Hdjl and Hdj2) are recruited. Like chaperones, proteasomes have been shown to be associated with polyQ aggregates (Waelter et al., 2001). Often, polyQ inclusions are ubiquitin positive (DiFiglia et al., 1997). The sequestration of folding/degradative machinery to protein aggregates results in compromised proteasomal degradation (Bence et al, 2001). Nuclear factors also have been shown to interact with polyQ nuclear inclusions (Okazawa, 2003). Nuclear aggregates of ataxin-1 recruit the promyelocytic leukemia protein (PML), a components of nuclear PML bodies (Skinner et al., 1997). Direct association of the CREB binding protein (CBP) with polyQ aggregates has been observed in HD cell culture models, HD transgenic mice and human HD postmortem brain (Nucifora et al., 2001). Spl and p53 also interact with polyQ huntingtin fragments (Steffan et al., 2000; Nucifora et al, 2001). The sequestration of transcription factors by aggregates appears to alter their transcriptional activity. Specifically, polyQ expanded huntingtin and atrophin-1

(responsible for DRPLA) decrease CBP-mediated transcription in transfected primary cortical neurons (Nucifora et al., 2001). Similarly, polyQ expanded huntingtin represses transcription of a p53 reporter construct (Steffan et al., 2000). Genomic screens have shown that CBP-regulated genes, such as eukepholin and Jun, are down regulated in HD transgenic mice and in HD post-mortem brains (Richfield et al., 1995; Luthi-Carter et al., 2000). The inhibition of transcription may be a consequence of direct binding, since CBP and p53 interact directly with polyQ tracks of huntingtin (Steffan et al., 2000; Nucifora et al., 2001) and with the polyQ tracks of androgen receptor that causes SBMA (McCampbell et al., 2000). Transcriptional regulators such as CBP and TATA binding protein (TBP) contain polyQ stretches, suggesting that complementary polyQ-polyQ interactions may mediate the sequestration and the inactivation. The ability of nuclear polyQ aggregates to recruit folding/degradative cellular components, disrupt nuclear architecture, sequester transcriptionally relevant proteins, and alter transcriptional activity of the sequestered factors may be specific to the polyQ content, or may represent a general cellular response to nuclear inclusions. To address this question, it was necessary to examine whether non-polyQ proteins can form nuclear inclusions and elicit similar cellular effects as polyQ aggregates. Here, it is surprisingly shown that a non- polyQ protein (GFP 170*), which contains GFP fused to an internal segment (amino acids 566 to 1375) of the Golgi Complex Protein 170 (GCP170), forms nuclear aggregates analogous to those deposited by polyQ proteins. GCP-170, also known as golgin- 160, is a Golgi localized protein that associates peripherally with the cytoplasmic side of Golgi membranes (Misumi et al., 1997; Hicks and Machamer, 2002). GCP-170 was identified as a Golgi auto-antigen in sera of patients suffering from Sjorgen Syndrome (Fritzler et al., 1993). The cellular function of GCP 170 is currently unknown. The internal fragment of GCP 170 utilized in this invention to generate GFP 170* represents a GCP 170 protein that lacks a polyQ-expanded sequence. The ability of GFP 170* to form nuclear aggregates shows that the formation of nuclear inclusions is not a polyQ-specific process. Furthermore, like deposits of polyQ proteins, the nuclear aggregates of GFP 170* recruit molecular chaperones and proteasomal components, cause a redistribution of PML bodies, and sequester transcription factors such as CBP and p53. In addition, expression of GFP170* represses p53 transcriptional activity and causes cell death. The similarity in cellular responses elicited by polyQ proteins and non-polyQ GFP 170* set forth herein is consistent with the proposition that those responses are common to the presence of any misfolded proteins in the nucleus. The present invention shows that the etiology of diverse polyQ diseases such as HD, SBMA, DRPLA, and ataxias could share a set of common cytopathologies elicited solely by nuclear inclusions (irrespective of the polyQ content of the protein), in addition to specific responses elicited by distinct polyQ proteins. Antibodies and reagents Polyclonal anti-GFP antibody was from Abeam Inc. (Cat. # AB-290). Anti-CBP (A-

22) (Cat. # sc-369) polyclonal antibody and anti-SC35 (Y-16) (Cat. # sc-10251) monoclonal antibody were purchased from Santa Cruz Biotechnology, Inc. Anti-Hsp70 (Cat. # SPA-810) monoclonal antibody was purchased from Stressgen Biotechnologies. Anti-Hdj2 polyclonal antibody was a gift from Dr. Douglas Cyr (University of North Carolina at Chapel Hill). Anti-20S proteasome (α-subunit) polyclonal antibody was purchased from Calbiochem-Novabiochem. Mouse anti-GMP-1 monoclonal antibody (clone 21C7) recognizes the SUMO-1 protein was from Zymed Laboratories Inc. A combined monoclonal anti-p53 antibody, 1801 and 421, was kindly provided by Dr. Xinbin Chen (University of Alabama at Birmingham). Texas red-labeled goat anti-mouse IgG antibody, Texas red-labeled goat anti-rabbit IgG antibody and Hoechst 33258 were from Molecular Probes, Inc. Restriction enzymes and molecular reagents were from Promega, New England BioLabs, Inc., or QIAGEN. All other chemicals were from Sigma-Aldrich or Fisher Scientific.

DNA Constructs To make a chimera of GFP and GCP-170, an Xhol restriction enzyme site was generated in front of the start codon of GCP-170. The 770-base pair PCR fragment containing sequences from the start codon of GCP-170 to the EcoRI site of FQSY1024 (Misumi et al., 1997) was cloned into the Xhol and EcoRI sites of pEGFP-C2 plasmid (Clontech Laboratories Inc.). A 5558-base pair EcoRI fragment from FQSY1024 was then cloned to the EcoRI sites of the plasmid above to generate an EGFP-tagged full length GCP-170 (GFP-GCP170FL). GFP170* construct was then generated by removing the Bglll fragment and SacII fragment from the N-terminal and C-terminal end of GCP- 170, respectively. The resulted construct expresses an EGFP-tagged GCP-170 fragment from amino acid 566 to 1375. The Q80-GFP construct has been described previously (Ding et al., 2002) and was kindly provided by Dr. Qunxing Ding (University of Kentucky). The construct expressing firefly luciferase under the control of the p21 promoter with two p53- responsive elements was provided by Dr. Xinbin Chen and has been described previously (Chinery et al., 1997).

Cell culture, transfections and immunofluorescence microscopy COS-7 cells were grown in DMEM with glucose and glutamine (Mediatech, Inc.) supplemented with 10% FBS (Life Technologies), 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies). Cortical neurons isolated from mouse were cultured in Neurobasal Media (Cat.#21103-049, GIBCO) supplemented with B27 (Cat.#l 7504-010, GIBCO). Cells were transfected with the Fugene transfection reagent (Roche) or with TransIT polyamine transfection reagents (Mirus Corporation), according to manufacturer protocols. 18-48 h after transfection, cells were fixed with 3% paraformaldehyde and processed for immunofluorescence microscopy as previously described (Alvarez et al., 1999). Electron Microscopy and Immuno-gold Labeling Cells were transfected with the GFP 170* construct or the Q80-GFP construct. 48 h after transfection, cells were washed with PBS, detached from the plate by trypsinization and collected by centrifugation at 300 X g for 5 min at 4°C. Cells were washed twice with PBS and then fixed for 90 min with 1.5% glutaraldehyde in 0.1 M sodium cacodylate pH 7.4. Cells were then washed three times with sodium cacodylate and postfixed with 1%

OsO4 in 0.1 M sodium cacodylate pH 7.4 for 60 min on ice. After washing three times with 0.1 M sodium cacodylate, pH 7.4, cells were dehydrated with a series of ethanol solutions (30, 50, 70, 90, 95, and 3 X 100%) followed by 2 h incubation in 1:1 Spurr's resin/propylene oxide. After two changes of fresh 100% resin, the cell pellets were treansferred to gelatin molds and polymerized in fresh resin overnight at 60°C. Gold epoxy sections (100 nm thick) were generated with a Reichert Ultracut ultramicrotome and collected on 200 mesh copper grids. The grid specimens were stained for 20 min with saturated aqueous uranyl acetate (3.5%) diluted 1:1 with ethanol just before use, followed by staining with lead citrate for 10 min. Stained samples were examined on a JEOL 100CX electron microscope. For immunogold electron microscopy, cells expressing GFP 170* were harvested by trypsinization 24 h after transfection. Cells were washed with PBS and pre-fixed with 3% formaldehyde and 0.2% glutaraldehyde for 40 min followed by dehydration with series of graded ethanol at room temperature. The cells were then infiltrated and embedded with LR white. After polymerization, sections were cut with ultramicrotome and collected onto nickel grids. The grids were incubated with anti-GFP primary antibody and goat anti-rabbit IgG conjugated to 6-nm gold particles (Jackson ImmunoResearch Laboratories, Inc.) followed by post-fixation with 2% glutaraldehyde and counterstaining with uranyl acetate. Samples were then examined on a JEOL 100CX electron microscope. Analysis of Soluble and Insoluble GFP170* COS-7 cells were either mock-transfected with PBS or transfected with GFP170* construct. 48 h after transfection, cells were washed and harvested in ice-cold PBS. Cells were then lysed for 1 h on ice with RIPA buffer (50 mM Tris-HCl, pH 8.0, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, and 150 mM NaCl) supplemented with protease inhibitor cocktail and 1.0 mM PMSF. Lysates were sonicated for 5 s with microtip sonicator followed by 15 min centrifugation at 15,000 X g. Pellets were washed 2X with RIPA buffer and resuspended with equal volume of RIPA buffer. Equal volumes of samples from the total cell lysate, supernatant and pellet fractions were boiled in SDS-PAGE sample buffer and resolved on 8% SDS-PAGE. The gel was transferred to nitrocellulose membrane and processed for Western blotting as previously described (Gao and Sztul, 2001).

Measurement of DNA synthesis COS-7 cells were transfected with either GFP 170* or pEGFP-C2 (BD Bioscience).

32 h after transfection, the cells were incubated with 30 μM BrdU for 14 h, followed by immunofluorescent staining with anti-BrdU monoclonal antibody, PRB-1 (Molecular Probes).

Measurement of cell viability by FACS analysis COS-7 cells were mock-transfected or transfected with GFP 170* or Q80-GFP. 48 h after transfection, cells were detached from the plate by trypsinization. Cells were then incubated with a red fluorescent dye, L-23102 for 30 min at room temperature. Live cells exclude the dye and therefore can be separated from dead cells based on their low fluorescence intensity. Cells were then fixed with formaldehyde and washed with PBS followed by FACS analysis. Cells were first gated according to the intensity of green fluorescence. Dead cells in GFP-negative or GFP-positive groups were counted separately.

Luciferase assay COS7 cells in 6- well plates were transfected with 300 ng luciferase expressing vector and 300 ng of pcDNA3.1 vector alone, or vector expressing Q80-GFP or GFP170*. 48 h after transfection, cell lysates were made using the passive lysis buffer in the Dual Luciferase Assay system form Promega according to the manufacturer's instructions. Luciferase activity in the lysate was measured with a Luminometer from Promega. The protein concentrations of the lysates were determined by Bradford analysis and luciferase activity was calculated per milligram of protein and then normalized to the activity in the control sample. GFP170* forms cytoplasmic and nuclear aggregates As stated above, GCP-170 contains 1530 amino acids, arranged into an N-terminal head domain followed by a long stalk regions and a short C-terminal tail. The stalk region consists of 6 coiled-coil domains. The coiled-coil domains of GCP 170 may be responsible for its dimerization (Misumi et al, 1997; Hicks and Machamer, 2002). Coiled-coil domains are known to mediate protein-protein interactions and may enhance the propensity of a protein to aggregate. GCP 170 has been shown to be aggregation-prone in vitro (Misumi et al., 1997). In agreement, GFP-tagged full length GCP-170 protein (GFP-GCP170FL) forms aggregates when over-expressed in COS-7 cells (Figure 10A, insert, arrows). Here, a chimera was generated by fusing in frame an internal segment of the coiled- coil region of GCP170 composed of amino acids 566 to 1375 to the C-terminus of the enhanced green fluorescent protein (GFP). The resulting construct is called GFP170*. GFP 170* does not contain polyQ repeats (Misumi et al., 1997). When transiently expressed in COS-7 cells, GFP 170* deposits as cytoplasmic aggregates in the peri-nuclear region (Figure 10A, arrows). The aggregates appear "ribbon-like" and are significantly more dispersed than the "ball-like" aggregates formed by GFP-250 (Garcia-Mata et al., 1999) or CFTR (Johnston et al., 1998). The GFP 170* aggregates appear concentrated around the nucleus, but in some cases extend into the periphery of the cell. In addition to the cytoplasmic inclusions, GFP170* deposits in spherical foci within the nucleus (Figure 10A, arrowheads). The morphology of cytoplasmic and nuclear GFP170* aggregates was compared to those formed by a model polyQ protein (Q80-GFP) (Figure 10B). Q80-GFP encodes a fusion protein containing an 80-glutamine expansion fused to the amino-terminus of GFP. Q80-GFP has been shown to deposit in characteristic cytoplasmic and nuclear aggregates (Onodera et al, 1997). The cytoplasmic inclusions formed by Q80-GFP are irregular in shape (arrow), while the nuclear aggregates are spherical (arrowheads), and resemble the GFP 170* aggregates. The Q80-GFP cytoplasmic inclusions localize to the peri-centriolar region, but appear more compact than those of GFP 170*. Q80-GFP forms one or two aggregates per nucleus, while GFP 170* forms multiple inclusions per nucleus. The ultrastructure of GFP 170* aggregates was examined by transmission electron microscopy (Figure 10C). The cytoplasmic aggregates (arrows) can extend to more than 15 μm in length. They are often surrounded by mitochondria, similar to the close association of mitochondria with the cytoplasmic aggregates formed by the HDQ83 huntingtin mutant

(Waelter et al., 2001). The nuclear aggregates of GFP170* (arrowheads) are spherical or ovoid, and range from 0.5 μm to 3 μm in diameter. They are similar to the nuclear inclusions formed by the Q80-GFP (Figure 10D). In both cases, the nuclear aggregates appear as homogenous accumulations of granular material, without apparent fibrillar content or subdomain structures. Non-transfected control cells never contain cytoplasmic or nuclear aggregates. The deposition of GFP 170* within the morphologically defined cytoplasmic and nuclear aggregates was confirmed by immunogold labeling with anti-GFP antibodies. Gold particles label the cytoplasmic and nuclear aggregates (Figure 10E and F). The clinically relevant deposits of polyQ proteins occur in neuronal cells (Ross, 2002). To test if GFP170* aggregates also form in neuronal cells, GFP170* was expressed in mouse primary cortical neurons. Q80-GFP was used in analogous transfections to allow direct comparisons. As shown in Figure 11, both GFP170* and Q80-GFP form cytoplasmic and nuclear aggregates in cultured mouse primary cortical neuronal cells. Their morphologies are similar. Like in COS-7 cells, GFP 170* forms multiple nuclear inclusions, while Q80-GFP deposits within a single structure. Similar results were also obtained with PC 12 cells, a rat neuronal cell line. These results indicate that GFP 170* forms aggregates in neuronal cells that are morphologically indistinguishable from those formed in non- neuronal cells. This shows that results obtained in COS-7 cells are applicable to neuronal cells.

GFP170* aggregates recruit chaperones and proteasomal components A characteristic feature of polyQ aggregates that parallels cytopathology is the recruitment of various cellular components. Specifically, polyQ aggregates have been shown to recruit molecular chaperones and proteasomes. Inclusions of polyQ-expanded huntingtin recruit proteasomal subcomplexes 20S, 1 IS and 19S and the chaperones BIP, HSP70 and HSP40 (Waelter et al., 2001). Similarly, polyQ-expanded androgen receptor aggregates recruit HSP70 (Kobayashi et al., 2000). This may facilitate the degradative clearance of the aggregates (Cummings et al., 1998). Therefore, the recruitment of similar components by GFP170* aggregates was examined. Hsp70 and Hdj2, representatives of the Hsp70 and the Hsp40 families of chaperones, respectively, are recruited to the nuclear as well as cytoplasmic GFP 170* deposits (Figure 12A and 3B). In addition to chaperones, proteasomal components are also recruited to the cytoplasmic and nuclear GFP 170* aggregates (Figure 12C). Another important feature of the polyQ aggregates is that they are usually detergent insoluble (Perez et al., 1998; Waelter et al., 2001). The solubility of

GFP 170* aggregates was examined after lysing cells in buffer containing detergent. GFP 170* is largely insoluble in a RIPA buffer containing 0.1% SDS (Figure 12D). The recovery of cytosolic β-tubulin in the soluble fraction provides an internal control for the efficacy of the solubilization.

Nuclear aggregates of GFP170* cause redistribution of PML bodies Nuclear inclusions of polyQ proteins have been shown to recruit nuclear structures containing the promyelocytic leukemia protein (PML bodies) (Skinner et al., 1997). PML bodies are also called nuclear domain 10 (ND10) bodies or PML oncogenic domains (PODs). The mammalian nucleus contains 10 to 30 PML bodies, which vary in size from 0.2 to 1 μm. They are thought to function in transcriptional regulation, cell cycle progresssion, and apoptosis, based on their content of proteins such as SplOO, PML, Daxx, pRB, CBP and p53 that are involved in these processes (Yasuda et al., 1999; Maul et al., 2000; Zhong et al., 2000). Disruption of PML bodies caused by the (tl5;17:q22;q21) translocation that results in a fusion of the PML protein and the retinoic acid receptor alpha (RARα), leads to acute promyelocytic leukemia (Weis et al., 1994). GFP 170* aggregates also recruit PML bodies (Figure 13 A and B). In normal untransfected cells, PML bodies are detected as numerous small nuclear foci (Figure 13 A and 4B, arrowheads). PML bodies with similar morphology are also evident in a cell expressing GFP 170* at low levels (Figure 13 A, arrows). A distinct phenotype is observed in cells expressing high levels of GFP 170* and displaying large nuclear aggregates (Figure 13B). In such cells, PML bodies re-distribute to the surface of the GFP 170* aggregates (Figure 13B, arrows). The results set forth herein indicate that like polyQ proteins, GFP170* causes changes in the nuclear architecture of PML bodies. Recently, it has been shown that the mutant huntingtin, Httexlp, is modified with the small-ubiquitin-related modifier (SUMO) (Steffan et al., 2004). Like ubiquitin, SUMO is ligated to lysine residues of a variety of proteins involved in multiple cellular pathways (Verger et al., 2003). For example, the PML protein in the PML bodies is sumoylated (Duprez et al., 1999). Therefore, the relationship between GFP170* aggregates and SUMO- 1 was testetd. SUMO-1 appears diffusely distributed in the nuclei of control cells (Figure 13C, arrows). SUMO-1 appears to be recruited to the GFP 170* nuclear aggregates, but not the cytoplasmic GFP170* aggregates in cells expressing GFP170* (Figure 13C, arrows). The insert shows extensive co-localization of GFP 170* and SUMO-1 in the nuclear aggregates. To explore whether GFP 170*, like Httexlp, is sumoylated, immunoblotting experiments were performed. GFP 170* is detected as a ~124-kD band in transfected cells (Figure 13D, anti-GFP panel), and this protein is also detected by anti-SUMO- 1 antibodies (anti-SUMO panel). A major sumoylated ~98-kD band detected in non-transfected and in transfected cells corresponds in molecular weight to sumoylated PML (Muller and Dejean, 1999).

Nuclear aggregates of GFP170* recruits transcription factors Nuclear inclusions of polyQ proteins have been shown to recruit transcriptional regulators. Specifically, CBP, the coactivator for CREB-mediated transcription, redistributes to huntingtin polyQ (Htt-N63-148Q) protein aggregates (Nucifora et al.,

2001). Similarly, the tumor supressor p53 interacts with aggregates of a pathogenic amino- terminal region of huntingtin, httexlp (Steffan et al., 2000; Suhr et al., 2001). Therefore, CBP and p53 were tested for their ability to relocate in response to GFP 170* aggregates that do not contain polyQ tracks. CBP is diffusely distributed in the nucleus of control cells (Figure 14A, arrowhead), but redistributes to the GFP170* nuclear aggregates in GFP170* transfected cells (arrows). The overall level of CBP is increased in transfected cells, suggesting that transcription of CBP-responsive genes might be altered. The levels of p53 are significantly increased in cells containing GFP 170* aggregates, since p53 is barely visible in non-transfected cells (Figure 14B). Like CBP, p53 is recruited to nuclear inclusions of GFP170* (Figure 14B). In addition to transcriptional factors, the mRNA splicing factor, SC-35, which normally localizes in the nucleus as nuclear speckles (Fu and Maniatis, 1990), relocates to nuclear inclusions formed by a truncated form of ataxin-3 (HA-Q78) (Chai et al., 2001). This response appears uncommon, and SC-35 is not recruited to aggregates formed by the full-length ataxin-3 (myc-ataxin-3-Q84) (Chai et al., 2001). The relationship between nuclear aggregates of GFP170* and nuclear speckles was examined. GFP170* aggregates do not significantly influence the distribution of nuclear speckles marked by SC35 (Figure 14C).

Expression of GFP170* alters function of transcription factors and is cytotoxic The nuclear deposition of polyQ proteins has been linked to alterations in transcription (Okazawa, 2003). For example, a mutant form of huntingtin, httexlp that sequesters p53 in inclusions, represses transcription of the p53-regulated proteins, p21WAFl/CIPl (Steffan et al., 2000). Since p53 is also sequestered by nuclear GFP170* inclusions, the effect of this sequestration on p53 transcriptional activity was tested. The activity of p53 was analyzed by measuring transcription from a reporter construct composed of firefly luciferase fused to p21 promoter with two p53 -responsive elements (p21-Luc) (Chinery et al., 1997). Analogous experiments were performed with Q80-GFP to allow direct comparisons. COS-7 cells were co-transfected with p21-Luc and either GFP 170*, Q80-GFP or a plasmid control. 48 h after transfection, luciferase activity in cell lysates was measured. COS-7 cells tranfected with the control plasmid have wild type p53 activity, which is consistent with the results described previously (Ray et al., 1997). The luciferase activity in cells co-transfected with the GFP 170* and the p21-Luc constructs is reduced to 30% of that in control cells co-transfected with control plasmid and the p21-Luc construct (Figure 15 A). This value is comparable to the luciferase activity in COS-7 cells expressing Q80-GFP, in which luciferase is reduced to 10% of control cells (Figure 15A). These results indicate that p53 transcriptional activity can be repressed by GFP170* to a level similar to that caused by a polyQ protein. Expression of polyQ proteins such as mutant huntingtin and atrophin-1 in cultured cells, or in animal disease models leads to cellular toxicity (Nucifora et al., 2001). Therefore, the effects of expressing GFP 170* on cell cycle regulation and cell viability were analyzed. First the effects of GFP 170* on DNA synthesis were examined by measuring the incorporation of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) in COS-7 cells expressing GFP 170*. BrdU incorporation during the S phase of mitosis is an indirect measure of cell proliferation. BrdU is incorporated in COS-7 cells expressing the GFP control plasmid (Figure 15B). In contrast, BrdU is not incorporated in cells with GFP 170* aggregates (Figure 15B; arrows), suggesting a defect in DNA synthesis or a cell cycle arrest. The effect of GFP 170* expression was further analyzed by measuring the viability of cells expressing GFP 170* by fluorescent-associated cell sorting (FACS) analysis. Control cells (mock transfected) have a death rate of - 2.5%, probably due to transfection and experimental damage (Figure 15C). In contrast, -21% of cells containing GFP170* aggregates die 48 hours after transfection. This number is similar to the -17% of dead cells containing Q80-GFP aggregates 48 hours after transfection. The percentage of dead cells expressing GFP170* or Q80-GFP is similar to that expressing the HD83Q huntingtin mutant (Waelter et al., 2001). Neurodegenerative disorders, including HD, SBMA, DRPLA, and SCAs 1, 2, 3, 6, 7, and 17, are characterized by the formation of cytoplasmic and/or nuclear inclusions of polyQ proteins. The nuclear aggregates recruit molecular chaperones, ubiquitin, and proteasome proteins (Davies et al., 1997; Paulson et al., 1997; Cummings et al., 1998), and cause significant alterations in the nuclear matrix-associated structures containing PML (Skinner et al., 1997). In addition, transcription factors such as TAF (TATA-binding protein-associated factor), CREB (cAMP-responsive element-binding protein), and CBP (CREB-binding protein) are recruited to inclusions of polyQ proteins in vitro and in vivo (Shimohata et al., 2000; Nucifora et al., 2001). This recruitment influences transcriptional regulation (McCampbell et al., 2000; Shimohata et al., 2000; Steffan et al., 2000; Nucifora et al., 2001; Suhr et al., 2001; Dunah et al., 2002; Obrietan and Hoyt, 2004), and has been correlated with cytopathology. The cellular responses to the polyQ proteins appear directly linked to the polyQ tracks, because the wild type proteins without extended polyQ tracks do not form nuclear inclusions and do not elicit cellular defects (Orr, 2001; Ross, 2002; Michalik and Van Broeckhoven, 2003). Some transcriptional factors, exemplified by CBP, contain poly-Q stretches, leading to the proposal that the recruitment and direct interactions with transcriptional regulators may be mediated through the polyQ tracks. Until the present invention, the issue of whether or not proteins lacking polyQ tracks can form nuclear inclusions, alter nuclear structure, and induce transcriptional responses analogous to those caused by polyQ proteins had not been addressed. The present invention shows that nuclear aggregates are formed by GFP 170*, a GFP-tagged fragment of the Golgi protein GCP 170 that lacks a polyQ tract. Like aggregates of polyQ proteins, the nuclear aggregates of GFP 170* recruit molecular chaperones and proteasomal components, and cause redistribution of PML bodies (Davies et al., 1997; Skinner et al., 1997; Schilling et al., 1999; Waelter et al., 2001; Ross, 2002). It is therefore unlikely that the recruitment of these proteins is directly mediated through the polyQ tracks. Rather, it involves mechanisms that recognize any misfolded protein as part of the cellular responses to either re-fold or clear aggregated proteins. PML bodies have been proposed to be depots (Maul et al., 2000; Negorev and Maul, 2001), and the findings set forth herein confirm that they associate with aggregated proteins in the nucleus. Like aggregates of polyQ proteins, the nuclear aggregates of GFP 170* recruit CBP and ρ53 (McCampbell et al., 2000; Steffan et al., 2000; Suhr et al., 2001). It is likely that binding of chaperones and proteasomes or sumoylation provides the link with CBP recruitment. The interaction of polyQ proteins with p53 can be direct or mediated through other cofactors (Steffan et al., 2000). Irrespective of the actual mechanisms, the results set forth herein indicate that the cellular models used to examine the role of polyQ inclusions in pathogenesis reveal general (rather than polyQ-specific) cellular responses to the accumulation of misfolded protein in the nucleus. Significantly, like the expression of polyQ proteins, the expression of GFP 170* represses p53 transcriptional activity and causes cell death. Cell death can be due to the alteration of p53 activity since it has been documented that p53 may induce apoptosis through transcriptional repression (Oren, 2003). Aggregation of non-polyQ proteins other than GFP 170* has been linked to human diseases. For example, expression of mutant forms of aggregation-prone proteins such as superoxide dismutase (Durham et al., 1997; Bruijn et al., 1998), α-synuclein (Masliah et al., 2000), glial fibrillary acidic protein (GFAP) (Messing et al., 1998), or /3-amyloid (Harper and Lansbury, 1997; Selkoe, 2003) in transgenic mice-models of human disease results in the formation of large aggregates in selected neurons and neurodegeneration of the same neurons that mimic the pathology of FALS, Parkinson's disease, Alexander's disease or Alzheimer's disease, respectively. However, in all known cases on non-polyQ proteins, the aggregates are either cytoplasmic or extracellular. Surprisingly, GFP 170* represents the only non-polyQ protein that deposits in cytoplasmic as well as nuclear aggregates. These findings indicate that nuclear aggregation can be caused by general features of misfolded proteins, rather than the presence of polyQ tracks. Aggregate-induced nuclear alterations and transcriptional disregulation could represent a general cellular response to the accumulation of any (polyQ or non-polyQ) aggregated protein in the nucleus, thus indicating that the mechanisms for the neuropathology of polyQ neurodegenerative diseases can include common (polyQ-independent) events in addition to polyQ-specific responses. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

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Claims

What is claimed is:

1. A fusion polypeptide comprising a GCP-170 protein or a fragment thereof, and a fluorescent protein.

2. The fusion polypeptide of claim 1, wherein the GCP-170 protein comprises amino acids 566-1375 of SEQ ID NO: 1.

3. The fusion polypeptide of claim 1 , wherein the GCP- 170 protein comprises amino acids 566-1530 of SEQ ID NO: 1.

4. The fusion polypeptide of claim 1, wherein the GCP-170 protein comprises amino acids 1-1530 of SEQ ID NO: 1.

5. The fusion polypeptide of any one of claims 1, 2, 3 or 4, wherein the fluorescent protein is selected from the group consisting of green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), green fluorescent protein from Aequorea coerelescens (AcGFP), DsRedExpress, and a red coral fluorescent protein.

6. An isolated nucleic acid encoding the fusion polypeptide of claim 1.

7. An isolated nucleic acid encoding the fusion polypeptide of claim 2.

8. An isolated nucleic acid encoding the fusion polypeptide of claim 3.

9. An isolated nucleic acid encoding the fusion polypeptide of claim 4.

10. A vector comprising the nucleic acid of any of claims 1 -9.

11. A cell comprising the vector of claim 10.

12. A cell that expresses the fusion polypeptide of claim 1, wherein the cell comprises a protein aggregate of the fusion polypeptide.

13. The cell of claim 12, wherein the aggregate is located in the nucleus of the cell.

14. The cell of claim 12, wherein the aggregate is located in the cytoplasm of the cell.

15. A cell that expresses the fusion polypeptide of claim 2, wherein the cell comprises an aggregate of the fusion polypeptide.

16. The cell of claim 15, wherein the aggregate is located in the nucleus of the cell.

17. The cell of claim 15, wherein the aggregate is located in the cytoplasm of the cell.

18. A cell that expresses the fusion polypeptide of claim 3, wherein the cell comprises an aggregate of the fusion polypeptide.

19. The cell of claim 18, wherein the aggregate is located in the cytoplasm of the cell.

20. A cell that expresses the fusion polypeptide of claim 4, wherein the cell comprises an aggregate of the fusion polypeptide.

21. The cell of claim 20, wherein the aggregate is located in the cytoplasm of the cell.

22. A method of identifying an agent that reduces protein aggregation comprising: a) contacting a cell containing aggregated fusion polypeptides comprising an amino acid sequence encoding a fluorescent protein and an amino acid sequence encoding a GCP-170 protein with a test agent; b) comparing the cell contacted with the test compound with a cell containing aggregated polypeptides that was not contacted with the test agent; and c) determining the effect of the test agent on protein aggregation, such that if protein aggregation in the cell contacted with the test compound is less than protein aggregation in the cell that was not contacted with the test compound, the test agent is an agent that reduces protein aggregation.

23. The method of claim 22, wherein protein aggregation occurs in the nucleus.

24. The method of claim 22, wherein protein aggregation occurs in the cytoplasm.

25. The method of claim 22, wherein protein aggregation occurs in the nucleus and the cytoplasm.

26. The method of claim 22, wherein the test agent reduces protein aggregation in the nucleus.

27. The method of claim 22, wherein the test agent reduces protein aggregation in the cytoplasm.

28. The method of claim 22, wherein the test agent reduces protein aggregation in the nucleus and the cytoplasm.

29. The method of claim 22, wherein the test agent reduces protein aggregation in the nucleus to a greater extent than it reduces protein aggregation in the cytoplasm.

30. The method of claim 22, wherein the test agent reduces protein aggregation in the cytoplasm to a greater extent than it reduces protein aggregation in the nucleus.

31. The method of claim 22, wherein the fusion polypeptide is a fusion polypeptide comprising a GCP-170 protein and a fluorescent protein, wherein the GCP-170 protein comprises amino acids 566-1375 of SEQ ID NO: 1.

32. The method of claim 31 , wherein the test agent reduces protein aggregation in the nucleus and the cytoplasm.

33. The method of claim 31 , wherein the test agent reduces protein aggregation in the nucleus to a greater extent than it reduces protein aggregation in the cytoplasm.

34. The method of claim 31 , wherein the test agent reduces protein aggregation in the cytoplasm to a greater extent than it reduces reduce protein aggregation in the nucleus.

35. A method of identifying a potential antiviral agent comprising: a) contacting a cell containing aggregated fusion polypeptides comprising an amino acid sequence encoding a fluorescent protein and an amino acid sequence encoding a GCP-170 protein with a test agent; b) comparing the cell contacted with the test compound with a cell containing aggregated polypeptides that was not contacted with the test agent; and c) determining the effect of the test agent on protein aggregation, such that if protein aggregation in the cell contacted with the test compound is less than protein aggregation in the cell that was not contacted with the test compound, the agent is a potential antiviral agent.

36. The method of claim 35, wherein protein aggregation in the nucleus of the cell contacted with the test compound is less than protein aggregation in the nucleus of the cell not contacted with the test compound.

37. The method of claim 35, protein aggregation in the cytoplasm of the cell contacted with the test compound is less than protein aggregation in the cytoplasm of the cell not contacted with the test compound.

38. The method of claim 35, wherein the fusion polypeptide is a fusion polypeptide comprising a GCP-170 protein and a fluorescent protein, wherein the GCP-170 protein comprises amino acids 566-1375 of SEQ ID NO: 1.

39. The method of claim 35, further comprising administering the potential antiviral agent to a cell contacted with a virus and determining its antiviral activity.

40. A method of identifying an agent that reduces the growth inhibitory effects of protein aggregation in a cell comprising: a) contacting a cell containing aggregated fusion polypeptides comprising an amino acid sequence encoding a fluorescent protein and an amino acid sequence encoding a GCP-170 protein with a test agent; b) comparing the cell contacted with the test compound with a cell containing aggregated polypeptides that was not contacted with the test agent; and c) determining the effect of the test agent on protein aggregation, such that if protein aggregation in the cell contacted with the test compound is less than protein aggregation in the cell that was not contacted with the test compound, and cell growth is greater in the cell contacted with the test compound than cell growth in the cell that was not contacted with the test compound the agent is an agent that reduces the growth inhibitory effects of protein aggregation in a cell..

41. The method of claim 40, wherein protein aggregation occurs in the nucleus.

42. The method of claim 40, wherein protein aggregation occurs in the cytoplasm.

43. The method of claim 40, wherein the fusion polypeptide is a fusion polypeptide comprising a GCP-170 protein and a fluorescent protein, wherein the GCP-170 protein comprises amino acids 566-1375 of SEQ ID NO: l.

44. The method of claim 40, wherein protein aggregation occurs in the nucleus and the cytoplasm.

45. The method of claim 43, wherein the test agent reduces protein aggregation in the nucleus.

46. The method of claim 43, wherein the test agent reduces protein aggregation in the cytoplasm.

47. The method of claim 43, wherein the test agent reduces protein aggregation in the nucleus and the cytoplasm.

48. The method of claim 43, wherein the test agent reduces protein aggregation in the nucleus to a greater extent than it reduces protein aggregation in the cytoplasm.

49. The method of claim 43, wherein the test agent reduces protein aggregation in the cytoplasm to a greater extent than it reduces protein aggregation in the nucleus.

50. The method of claim 40, wherein the cell is a neuronal cell.

51. A method of identifying an agent that inhibits the association of a transcription factor with protein aggregate comprising: a) contacting a cell containing aggregated fusion polypeptides comprising an amino acid sequence encoding a fluorescent protein and an amino acid sequence encoding a GCP-170 protein with a test agent; b) comparing the cell contacted with the test compound with a cell containing aggregated polypeptides that was not contacted with the test agent; and c) determining the effect of the test agent on the association of the transcription factor with a protein aggregate, such that if the amount of the transcription factor associated with a protein aggregate in the cell contacted with the test compound is less than the amount of transcription factor associated with a protein aggregate in the cell that was not contacted with the test compound, the agent is an agent that inhibits the association of a transcription factor with a protein aggregate.

52. The method of claim 51 , wherein the transcription factor is associated with a protein aggregate in the nucleus of the cell.

53. The method of claim 51 , wherein the amount of the transcription factor associated with a protein aggregate is determined by measuring the transcription activation activity of the transcription factor in the cell contacted.

54. The method of claim 51 , wherein the fusion polypeptide is a fusion polypeptide comprising a GCP-170 protein and a fluorescent protein, wherein the GCP-170 protein comprises amino acids 566-1375 of SEQ ID NO: 1.