WO2001016373A2 - Techniques d'analyse de la translocation des proteines de l'espace intermembranaire mitochondrial - Google Patents

Techniques d'analyse de la translocation des proteines de l'espace intermembranaire mitochondrial Download PDF

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WO2001016373A2
WO2001016373A2 PCT/US2000/023638 US0023638W WO0116373A2 WO 2001016373 A2 WO2001016373 A2 WO 2001016373A2 US 0023638 W US0023638 W US 0023638W WO 0116373 A2 WO0116373 A2 WO 0116373A2
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fusion polypeptide
intermembrane space
mitochondrial
cell
bcl
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WO2001016373A3 (fr
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Anne N. Murphy
Sandra Eileen Wiley
Alexander Y. Andreyev
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Mitokor
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5035—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on sub-cellular localization
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • the invention relates generally to methods for detecting the translocation of mitochondrial intermembrane space proteins. More specifically, the invention relates to compositions and screening methods for use in identifying agents that alter the translocation of epitope tagged mitochondrial intermembrane space proteins, which occurs for example as a result of altered outer mitochondrial membrane permeability, in response to various agents including, for example, apoptogens.
  • Mitochondria are the main energy source in cells of higher organisms, and provide direct and indirect biochemical regulation of a wide array of cellular respiratory, oxidative and metabolic processes. Such processes include electron transport chain (ETC) activity, which drives oxidative phosphorylation to produce metabolic energy in the form of adenosine triphosphate (ATP), and which controls mitochondrial regulation of intracellular and intramitochondrial calcium homeostasis.
  • ETC electron transport chain
  • ATP adenosine triphosphate
  • Mitochondrial ultrastructural characterization reveals the presence of an outer mitochondrial membrane that serves as an interface between the organelle and the cytosol, a highly folded inner mitochondrial membrane that appears to form attachments to the outer membrane at multiple sites, and an intermembrane space between the two mitochondrial membranes.
  • the subcompartment within the inner mitochondrial membrane is commonly referred to as the mitochondrial matrix.
  • the cristae originally postulated to occur as infoldings of the inner mitochondrial membrane, have recently been characterized using three-dimensional electron tomography as also including tube-like conduits that may form networks, and that can be connected to the inner membrane and/or the intermembrane space by open, circular (30 nm diameter) junctions (Perkins et al., 1997, Journal of Structural Biology 119:260-212).
  • the inner mitochondrial membrane While the outer membrane is freely permeable to ionic and non-ionic solutes having molecular weights less than about ten kilodaltons, the inner mitochondrial membrane exhibits selective and regulated permeability for many small molecules, including certain cations, and is impermeable to large (> ⁇ 10 kDa) molecules.
  • Altered or defective mitochondrial activity may result in catastrophic mitochondrial collapse that has been termed "permeability transition” (PT) or “mitochondrial permeability transition” (MPT). This collapse can also be less catastrophic, causing MPT within local domains of an individual mitochondrion.
  • PT permeability transition
  • MPT mitochondrial permeability transition
  • proper ETC respiratory activity requires maintenance of an electrochemical potential ( ⁇ m) in the inner mitochondrial membrane by a coupled chemiosmotic mechanism.
  • Altered or defective mitochondrial activity may dissipate this membrane potential, thereby preventing ATP biosynthesis and halting the production of a vital biochemical energy source.
  • mitochondrial intermembrane space proteins such as cytochrome c may be transported or may leak out of the mitochondria after permeability transition intitiating the genetically programmed cell suicide sequence known as apoptosis or programmed cell death (PCD).
  • PCD programmed cell death
  • mitochondria (or, at least, mitochondrial components) participate in apoptosis (Newmeyer et al., 1994, Cell 79:353-364; Liu et al., 1996, Cell 86:141-151).
  • Apoptosis is apparently also required for, inter alia, normal development of the nervous system and proper functioning of the immune system.
  • some disease states are thought to be associated with either insufficient ⁇ e.g., cancer, autoimmune diseases) or excessive ⁇ e.g., stroke damage, AD- associated neurodegeneration) levels of apoptosis.
  • At least one recognized apoptotic mechanism therefore involves the release of cytochrome c from the mitochondrial intermembrane space to the cytosol, a process refened to as translocation of the mitochondrial intermembrane space protein ⁇ e.g., Single et al., 1998 Cell Death Diff. 5:1001).
  • Improved understanding of the regulation of mitochondrial outer membrane permeability is therefore desirable in order to diagnose and/or screen for agents useful for treating conditions associated with altered ⁇ e.g., increased or decreased) apoptosis.
  • cytochrome c translocation from the mitochondrial intermembrane space is not well understood, and may be specific to cytochrome c or may, alternatively, be accompanied by translocation of one or more additional intermembrane space proteins (Single et al., 1998).
  • translocation models including (i) a model whereby specific channels for cytochrome c release are opened by pro-apoptotic Bcl-2 family members, (ii) a model wherein MPT underlies cytochrome c translocation and (iii) a model wherein mitochondrial outer membrane swelling and rupture permit nonspecific translocation of cytochrome c and other intermembrane space proteins such as adenylate kinase (see, e.g., Kohler et al., 1999 FEBS Lett. 447:10-12, and references cited therein).
  • Technical problems related to the reliable detection of these and other intermembrane space proteins have prevented further determination of mitochondrial events during translocation.
  • modifications to intermembrane space proteins that dramatically increase their molecular mass may disrupt their ability to translocate if mitochondrial outer membrane permeability is a function of solute size.
  • the high degree of immunological crossreactivity between the mitochondrial and cytosolic isoforms of adenylate kinase (AK) may preclude reliable determination of translocated mitochondrial AK isoforms in the cytosol.
  • agents that alter such translocation may be beneficial, and assays to specifically detect such agents are needed.
  • the present invention fulfills these needs and further provides other related advantages.
  • the present invention is directed to compositions and methods for detecting mitochondrial intermembrane space protein translocation, including screening assays for agents that alter such translocation. Accordingly, it is an aspect of the invention to provide methods of detecting mitochondrial intermembrane space protein translocation, comprising contacting a sample comprising a cell (which may optionally be permeabilized) containing a mitochondrion, or an isolated mitochondrion, or an apoptosis reconstitution system, with an agent known or suspected to induce the translocation of one or more mitochondrial intermembrane space proteins, under conditions and for a time sufficient to induce mitochondrial intermembrane space protein translocation, the mitochondrion comprising at least one mitochondrial intermembrane space protein fusion polypeptide, wherein the fusion polypeptide comprises at least one mitochondrial intermembrane space protein domain and at least one affinity domain; and contacting the sample with a detectable ligand that specifically binds to the affinity domain under conditions and
  • an agent that alters mitochondrial intermembrane space protein translocation is provided, and the object of the invention is to provide a method of identifying a compound that alters ⁇ e.g., enhances or inhibits) the activity of the agent that alters mitochondrial intermembrane space protein translocation.
  • Such a method comprises contacting a sample comprising a mitochondrion with, sequentially in either order or simultaneously, (i) an agent that alters mitochondrial intermembrane space protein translocation and (ii) a candidate compound suspected of being able to alter the activity of the agent, under conditions and for a time sufficient to induce mitochondrial intermembrane space protein translocation, the mitochondrion comprising at least one mitochondrial intermembrane space protein fusion polypeptide, wherein the fusion polypeptide comprises at least one mitochondrial intermembrane space protein domain and at least one affinity domain; contacting the sample with a detectable ligand that specifically binds to said affinity domain under conditions and for a time sufficient for the detectable ligand to bind to the fusion polypeptide in extramitochondrial spaces in order to form detectable ligand:fusion polypeptide complexes, and detecting the ligand:fusion polypeptide complexes; and comparing the level of extramitochondrial ligand:fusion polypeptid
  • the mitochondrion is contained in a cell, which is a neural cell in certain embodiments, and in certain embodiments the cell is a neuroblastoma cell. In certain further embodiments the neuroblastoma cell is a SH-SY5Y cell. Regardless of cell type, the cell may be permeabilized by the addition of permeabilizing agents such as digitonin, streptolysin O, Staphylococcus aureus -toxin ( ⁇ - hemolysin), saponin (all available from Sigma Chemical Co., St.
  • permeabilizing agents such as digitonin, streptolysin O, Staphylococcus aureus -toxin ( ⁇ - hemolysin), saponin (all available from Sigma Chemical Co., St.
  • the permeabilized cell is depleted of cytosol.
  • the mitochondrion is provided substantially isolated from other cellular components. In embodiments wherein isolated or purified mitochondria are used, release of a mitochondrial intermembrane space protein from mitochondria into their media is taken as a measure of protein "translocation" even though, to be precise, only dissociation from mitochondria actually occurs, as the cellular compartment(s) to which the proteins would translocate in intact cells are absent.
  • the mitochondrial intermembrane space protein is adenylate kinase-2, cytochrome c or sulfide oxidase, and in particularly preferred embodiments the mitochondrial intermembrane space protein is adenylate kinase-2.
  • the affinity domain is a hemagglutinin epitope tag, a FLAG® epitope tag, an XPRESSTM epitope tag, a myc epitope tag or a polyhistidine epitope tag and in particularly preferred embodiments the affinity domain comprises a hemagglutinin epitope tag.
  • the detectable ligand comprises an antibody, and in certain further embodiments the antibody comprises a monoclonal antibody while in certain other further embodiments the antibody comprises a polyclonal antibody. In still other further embodiments the antibody comprises a single chain antibody, and in other embodiments the antibody comprises a fusion protein.
  • translocation of a mitochondrial intermembrane space protein occurs with some degree of activation of the mitochondrial permeability transition (MPT) "pore" and/or biochemical events in apoptotic pathways.
  • MPT mitochondrial permeability transition
  • protein translocation is observed in the absence of MPT; in these embodiments, the translocation of mitochondrial intermembrane space proteins can be detected and assayed in isolation from confounding signals that might result from MPT and/or apoptosis.
  • translocation of mitochondrial intermembrane space proteins may not result in MPT and/or apoptosis per se, other pathological consequences may ensue.
  • the translocation of cytochrome c from mitochondria under conditions wherein MPT and/or apoptosis are not seen to occur may be assayed according to the methods of the invention.
  • the cell comprises a polypeptide that is a Bcl-2 family member, and in certain further embodiments the cell comprises a nucleic acid expression construct comprising a promoter operably linked to a polynucleotide encoding a Bcl-2 family member.
  • Bcl-2 family members include, by way of example and not limitation, Bcl-2, Bcl-X L , Bcl-w, Mcl-1, Al, NR-13, BHRF1, LMW5-HL, ORF16, Ks-Bcl-2, E1B-19K (U.S. Patent No. 5,858,678) and Ced-9 (Adams et al., Science 281:1322-1326, 1998).
  • the Bcl-2 family member polypeptide may be modified, for example by way of mutation, to contain one or more altered or substituted amino acid residues (see, for example, U.S. Patent No. 5,856,171).
  • the agent of the methods is an apoptogen, such as a pro-oxidant or a calcium ionophore, and in certain further embodiments the pro-oxidant is hydrogen peroxide, tert-butylhydroperoxide or per oxy nitrite.
  • the calcium ionophore is ionomycin.
  • the apoptogen is an appropriate amount of atractyloside, bongkrekic acid, thapsigargin, glutamate, N-methyl-D-aspartic acid, carbachol, A23187or ionomycin.
  • such apoptogens may further include Ca , pro-apoptotic factors such as Bax (J ⁇ rgensmeier et al., Proc. Natl. Acad. Sci. U.S.A. 95:4991-5002, 1998; U.S. Patent No. 5,837,838), mutants of Bax (U.S. Patent No. 5,856,445) and Bid, and the like.
  • pro-apoptotic factors such as Bax (J ⁇ rgensmeier et al., Proc. Natl. Acad. Sci. U.S.A. 95:4991-5002, 1998; U.S. Patent No. 5,837,838), mutants of Bax (U.S. Patent No. 5,856,445) and Bid, and the like.
  • treatment with the agent may result in apoptosis whereas, in other embodiments, subcritical amounts of the apoptogen may promote some degree of intermembrane space protein translocation without inducing apopto
  • the agent of the methods is an agent that can, at sufficiently high concentrations or periods of incubation, cause necrosis.
  • treatment with the agent may result in necrosis whereas, in other embodiments, subcritical amounts of the agent may promote some degree of intermembrane space protein translocation without inducing necrosis.
  • the agent of the methods is an agent that promotes some degree of intermembrane space protein translocation but neither induces apoptosis nor causes necrosis.
  • such agents may induce MPT without inducing apoptosis or causing necrosis.
  • permeabilized cells such agents may include, by way of non-limiting example, appropriate concentrations of MPT inducers such as t-BOOH, atractyloside, phenylarsine oxide, or gangliosides.
  • a candidate compound is present and is being tested for its ability to alter ⁇ e.g., enhance or inhibit in a statistically significant manner) the activity of the agent that alters mitochondrial intermembrane space protein translocation.
  • the agent that alters mitochondrial intermembrane space protein translocation may be a calcium ionophore (when unpermeabilized cells are used) or Ca (when permeabilized cells or isolated mitochondria are used), and the compound that is being tested for its ability to alter the activity of such agents may be a Ca chelator.
  • the invention may thus be used, for example, to evaluate the activity of compounds such as cell-permeant Ca chelators (Tymianski et al, J. Cereb. Blood Flow Metab. 74:911-923, 1994).
  • the invention provides a nucleic acid expression construct comprising a promoter operably linked to a polynucleotide encoding an intermembrane space protein fusion polypeptide, wherein the fusion polypeptide comprises at least one mitochondrial intermembrane space protein domain and at least one affinity domain.
  • a promoter operably linked to a polynucleotide encoding an intermembrane space protein fusion polypeptide
  • the fusion polypeptide comprises at least one mitochondrial intermembrane space protein domain and at least one affinity domain.
  • intermembrane space fusion proteins of the invention may be incorporated into a containing device, such as a 96-well or 386-well microtiter plate, that may be combined with methods and mechanisms for high throughput screening of agents and compounds, according to methods known in the art.
  • Figure 1 shows western immunoblot analysis of transiently transfected 293 T and COS-1 cells expressing HA-tagged AK2 mitochondrial intermembrane space protein fusion polypeptide.
  • Lane 1 molecular weight markers; lane 2, 293 T cells transfected with vector; lanes 3-6, 293 T cells transfected with an expression construct encoding an HA- tagged AK2 mitochondrial intermembrane space protein fusion polypeptide; lane 7, COS-1 cells transfected with vector; lanes 8-9, COS-1 cells transfected with an expression construct encoding an HA-tagged AK2 mitochondrial intermembrane space protein fusion polypeptide; lane 10, COS-1 cells taken through the transfection protocol without the addition of exogenous DNA.
  • the sizes of the molecular weight markers are indicated on the lefthand side of the figure.
  • Figure 2 shows western immunoblot analysis of stable doubly transfected cybrid cells expressing HA-tagged AK2 mitochondrial intermembrane space protein fusion polypeptides and Bcl-2 polypeptides.
  • Panel A probed with antibody to Bcl-2
  • panel B probed with antibody to HA tag.
  • Lane 1 molecular weight markers
  • lanes 2-11 individually isolated potential double transfectants.
  • the sizes of the molecular weight markers are indicated on the lefthand side of each panel.
  • Figure 3 shows western immunoblot analysis of stable SH-SY5Y neuroblastoma cells expressing HA-tagged AK2 mitochondrial intermembrane space protein fusion polypeptides.
  • Lanes A and I SH-SY5Y cells taken through the transfection protocol without the addition of exogenous DNA
  • lanes B-E SH-SY5Y cells transfected with vector (pCDNA3) DNA
  • lanes F-H SH-SY5Y cells transfected with expression constructs encoding HA-tagged AK2 fusion polypeptides.
  • Figure 4 shows tracings derived from mitochondria in a multiparameter chamber. The three panels on the left show results under the "non-MPT" conditions described herein, whereas the three panels on the right show results where ATP and Mg have not been added.
  • Figure 5 is a Western immunoblot analysis of cells expressing an HA-tagged derivative of adenylate kinase 2 that were treated under "non-MPT" conditions.
  • Figure 6 shows light scattering tracings derived from mitochondria in a multiparameter chamber (left), and the corresponding Western analysis (right), for cells that have been treated with carboxyatractyloside (CAtr), Bongkrekic Acid (BkA) and alamethacin (Aim).
  • Figure 7 shows Ca electrode tracings derived from mitochondria in a multiparameter chamber (left), and the corresponding Western analysis (right), for cells that have been treated with carboxyatractyloside (CAtr), Bongkrekic Acid (BkA) and alamethacin (Aim).
  • Bcl-2 cells overpressing Bcl-2; "puro,” control cells comprising a puromycin resistance gene.
  • Figure 8 is a Western immunoblot analysis of cells expressing an HA-tagged derivative of adenylate kinase 2 that were exposed to etoposide.
  • Figure 9 is a Western immunoblot analysis of cells expressing an HA-tagged derivative of adenylate kinase 2 that were exposed to thapsigargin.
  • Figure 10 is a Western immunoblot analysis of cells expressing an HA-tagged derivative of adenylate kinase 2 that were exposed to staurosporine.
  • Figure 11 shows calcium and mitochondrial membrane potential measurements in digitonin-permeabilized and cytosol depleted cells.
  • Figure 12 shows absormance measurements in digitonin-permeabilized and cytosol depleted cells.
  • Figure 13 shows Western immunoblot analysis of cytochrome c in digitonin- permeabilized and cytosol depleted cells.
  • the present invention provides compositions and methods for use in detecting mitochondrial intermembrane space protein translocation, and for identifying agents that alter such translocation.
  • the invention is directed in part to detecting mitochondrial intermembrane space protein translocation in a biological sample comprising a cell having a mitochondrion.
  • the cell comprises a host cell transfected with, and expressing, a recombinant nucleic acid expression construct encoding an intermembrane space protein fusion polypeptide comprising an affinity domain as provided herein, for example, a monoclonal antibody defined peptide epitope tag.
  • the cell comprises a host cell transfected with, and expressing or overexpressing, a recombinant nucleic acid expression construct encoding a protein or polypeptide that is a member of the Bcl-2 family, for example, Bcl-2, Bid, Bad or Bax (see, e.g., Reed, Cell 91:559, 1997; Adams et al., Science 281:1322-1326, 1998).
  • the method of the invention comprises, in part, contacting the biological sample with an agent known or suspected to induce the translocation of one or more mitochondrial intermembrane space proteins, under conditions and for a time sufficient to induce mitochondrial intermembrane space protein translocation, and contacting the sample with a detectable ligand as provided herein in order to detect extramitochondrial intermembrane space protein fusion polypeptide, i.e., fusion protein that has translocated.
  • any mitochondrial intermembrane space protein may be selected for use in a MISP fusion polypeptide as provided herein, as long as the MISP is a protein or polypeptide that is naturally present in a mitochondrial intermembrane space of any subject or biological source as described herein.
  • MISPs include cytochrome c (Green et al., 1998 Science 281 :1309; Liu et al., 1996 Cell 86: 147-157; Kluck et al., 1997 Science 275: 1132-36), pro-caspases 2, 3, and 9 (Mancini et al., 1995 J. Cell Biol. 140, 1485-95; Susin et al, 1999 J. Exp.
  • apoptosis inducing factor (Susin et al., 1999 Nature 397: 441-45), adenylate kinase (AK; e.g., Single et al, 1998 Cell Death Diff. 5:1001), nucleoside diphosphate kinase, nucleoside monophosphate kinase (Tzagoloff, Chapter 2 in Mitochondria, Plenum Press, New York, NY, 1982, p. 28), and sulfide oxidase ⁇ e.g., Newmeyer presentation, 1998 Society of Toxicology 37 th Annual Meeting, March 1998, see 1998 Toxicol. Sci. 42 (1-S):232, abstr. no. 1145), but the invention need not be so limited.
  • the MISP is AK.
  • adenylate kinase is a ubiquitous enzyme involved in maintaining the homeostasis of cellular adenine and guanine nucleotide pools (Schulz, 1987 Cold Spring Harbor Symp. Quant. Biol. 52: 429-39; Tomasselli et al, Eur. J. Biochem. 93: 263-67,1979).
  • AK1, AK2, AK3, and AK4 are ubiquitous enzyme involved in maintaining the homeostasis of cellular adenine and guanine nucleotide pools.
  • AK1, AK2 and AK3 are nuclear encoded proteins, but localize to different subcellular compartments: AK1 is present mainly in the cytosol, whereas mature AK2 and AK3 are imported into mitochondria, with AK2 distributing to the mitochondrial intermembrane space while AK3 is found in the mitochondrial matrix. AK2 is therefore a MISP, although in some tissues a subpopulation of the total intracellular AK2 pool may also be found in the cytosol (Nobutomo et al, 1998). At least two isoforms of vertebrate AK2 have been identified, cloned and sequenced ⁇ e.g., Nakazawa et al., 1990 Prog. Clin. Biol.
  • AK4 is believed to localize to the mitochondrial matrix based on its primary structure (Yineda et al, 1998).
  • MISPs include full length proteins and polypeptides, fragments, and variants thereof, and further include MISP fusion polypeptides as provided herein.
  • the portion of a MISP fusion polypeptide that is a MISP domain as provided herein may be a full length MISP or any analog, fragment or portion thereof, including a truncated MISP or a MISP variant.
  • the fusion polypeptides of the present invention comprise at least one MISP protein domain and at least one affinity domain as provided herein; in preferred embodiments the MISP protein domain is fused in-frame to the affinity domain.
  • a truncated molecule for example a truncated MISP polypeptide or nucleic acid, may be any molecule that comprises less than a full length version of the molecule.
  • the present invention provides truncated MISP polypeptides, and in certain embodiments the invention provides nucleic acids encoding such truncated polypeptides.
  • Truncated nucleic acid molecules have less than the full length nucleotide sequence of a known or described nucleic acid molecule, where such a known or described nucleic acid molecule may be a naturally occurring, a synthetic or a recombinant nucleic acid molecule, so long as one skilled in the art would regard it as a full length molecule.
  • truncated nucleic acid molecules that correspond to a gene sequence contain less than the full length gene where the gene comprises coding and non- coding sequences, promoters, enhancers and other regulatory sequences, flanking sequences and the like, and other functional and non-functional sequences that are recognized as part of the gene.
  • truncated nucleic acid molecules that conespond to a mRNA sequence contain less than the full length mRNA transcript, which may include various translated and non-translated regions as well as other functional and non-functional sequences.
  • truncated molecules are polypeptides that comprise less than the full length amino acid sequence of a particular protein.
  • deletion has its common meaning as understood by those familiar with the art, and may refer to molecules that lack one or more portions of a sequence from either terminus or from a non-terminal region, relative to a corresponding full length molecule, for example, as in the case of truncated molecules provided herein.
  • Truncated molecules that are linear biological polymers such as nucleic acid molecules or polypeptides may have one or more of a deletion from either terminus of the molecule or a deletion from a non-terminal region of the molecule, where such deletions may be deletions of 1-1500 contiguous nucleotide or amino acid residues, preferably 1-500 contiguous nucleotide or amino acid residues and more preferably 1-300 contiguous nucleotide or amino acid residues.
  • MISP deletion mutants as provided herein permits identification of MISP structural domains that are responsible for particular functional properties, including, for example, the polypeptide regions that may mediate translocation of a particular MISP.
  • MISP truncation deletion mutants may permit molecular fine regulation of mitochondrial function in vivo.
  • detectably altered ⁇ e.g., increased or decreased) MISP translocation in response to one or more translocation-inducing agents in cells transfected with MISP fusion polypeptides, or deletion or other mutants thereof permits correlation of the presence of a particular MISP structural domain with a particular mitochondrial or cellular function or event ⁇ e.g., altered mitochondrial permeability, induction of apoptosis, stimulation of necrosis, etc.).
  • the affinity domain of the MISP fusion polypeptide permits reliable localization of the fusion polypeptide, including, for example, verification that the expressed fusion polypeptide is delivered to the mitochondrial intermembrane space and determination of whether the fusion polypeptide has translocated and may be detected as an extramitochondrial fusion polypeptide.
  • Such extramitochondrial fusion polypeptides may include any mitochondrial intermembrane space protein (MISP) fusion polypeptide as provided herein that is not present in the mitochondrial intermembrane space, for example, a MISP fusion polypeptide that is present in the cytosol or a MISP fusion polypeptide that is present in a subcellular fraction prepared according to any of a variety of cell fractionation methods well known in the art and which is substantially free of mitochondria.
  • MISP mitochondrial intermembrane space protein
  • the affinity domain may also be useful for isolation and/or detection of the fusion polypeptide according to methodologies with which those having ordinary skill in the art will be familiar.
  • the affinity domain of the fusion polypeptide is a small peptide that comprises fewer than 20 amino acids, more preferably fewer than 15 amino acids and still more preferably 12 or fewer amino acids.
  • the present invention provides a MISP fusion polypeptide comprising an affinity domain that is a small ⁇ e.g., ⁇ 20 amino acids) peptide, such that the influence of the affinity domain on MISP fusion polypeptide translocation may be minimized where the translocation mechanism may be sensitive to MISP molecular mass.
  • the affinity domain of the MISP fusion polypeptide provides a specific and reliable detectable translocation marker.
  • the present invention provides a method of detecting MISP translocation that may be qualitative ⁇ e.g., determination of whether MISP fusion polypeptide localizes mitochondrially or extramitochondrially under particular conditions), and in certain embodiments the invention provides a method of quantitatively detecting MISP translocation ⁇ e.g., quantifying mitochondrial and/or extramitochondrial MISP fusion polypeptide levels under particular conditions).
  • MISP fusion polypeptides described herein may be detected, for example, by fluorescence, phosphorescence, radiography, bioluminescence, chemiluminescence or any other method for detecting such polypeptides with which those having ordinary skill in the art will be familiar.
  • MISP fusion polypeptides include fusion proteins that may in certain embodiments be detected, isolated and/or purified by protein-protein affinity ⁇ e.g., receptor- ligand), hydrophobicity, metal affinity or charge affinity methods.
  • detection of the affinity domain is employed, for example, through the use of a detectable ligand that specifically binds to the affinity domain under conditions that can be readily identified by a person having ordinary skill in the art without undue experimentation.
  • Detectable ligands may include antibodies that are specific for the affinity domain, and may also include other ligands able to specifically interact with an affinity domain, as described in greater detail below.
  • the subject invention fusion proteins may be detected by specific protease cleavage of a fusion protein having a sequence that comprises a protease recognition sequence, such that the MISP domain polypeptide may be separable from the affinity domain polypeptide sequence.
  • each MISP polypeptide sequence is fused in-frame to an affinity domain polypeptide sequence.
  • isolated means that the material is removed from its original environment ⁇ e.g., the natural environment if it is naturally occurring).
  • nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • nucleic acids could be part of a vector and/or such nucleic acids or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • Polypeptide sequences present in MISP fusion polypeptides may thus facilitate affinity detection and isolation of MISP fusion polypeptides and may include, for example, affinity domains that are poly-His or the defined antigenic peptide epitopes described in U.S. Patent No. 5,011,912 and in Hopp et al., (1988 Bio/Technology 5:1204), or the XPRESSTM epitope tag (Invitrogen, Carlsbad, CA), or the myc epitope tag ⁇ e.g., Roche Molecular Biochemicals, Indianapolis, IN).
  • the affinity domain sequence may also be a hexa-histidine tag as supplied, for example, by a pBAD/His (Invitrogen) or a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host.
  • the affinity domain sequence may be a hemaglutinin (HA) tag.
  • the HA tag conesponds to an antibody defined epitope derived from the influenza hemaglutinin protein (Wilson et al., 1984, Cell 37:161) and which is useful when mammalian host cells, for example COS-7 cells, are used.
  • MISP fusion polypeptides may further comprise immunoglobulin constant region polypeptides added to MISP domain sequences to facilitate detection, isolation and/or localization of MISPs.
  • the immunoglobulin constant region polypeptide preferably is fused to the C-terminus of a MISP domain polypeptide.
  • General preparation of fusion proteins comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al. (1991 Proc. Nat. Acad. Sci. USA, 88:10535) and Byrn et al. (1990 Nature, 344:611).
  • MISP:Fc fusion polypeptides may be allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between Fc polypeptides, yielding dimeric MISP fusion proteins.
  • MISP fusion polypeptides having specific binding affinities for pre-selected antigens by virtue of affinity domain polypeptides comprising immunoglobulin V-region domains encoded by DNA sequences linked in-frame to sequences encoding a MISP are also within the scope of the invention, including variants and fragments thereof, as provided herein.
  • Fusion proteins may in certain embodiments comprise a MISP domain polypeptide fused to one or more other affinity domain polypeptides having desirable affinity properties ⁇ e.g., receptor-ligand).
  • additional affinity domain polypeptides having affinity properties include, without limitation, enzymes such as glutathione-S-transferase (GST) and Staphylococcus aureus protein A polypeptide.
  • GST glutathione-S-transferase
  • Staphylococcus aureus protein A polypeptide Staphylococcus aureus protein A polypeptide.
  • affinity domain polypeptides for construction of MISP fusion polypeptides may include streptavidin fusion proteins, as disclosed, for example, in WO 89/03422; U.S. 5,489,528; U.S. 5,672,691; WO 93/24631; U.S. 5,168,049; U.S. 5,272,254 and elsewhere, and avidin fusion proteins (see, e.g., EP 511,747).
  • MISP domain polypeptide sequences may be fused to affinity domain polypeptide sequences that may be full length fusion polypeptides and that may alternatively be variants or fragments thereof.
  • the detectable ligand may be any molecule, receptor, counterreceptor, antibody or the like with which the affinity tag may interact through a specific binding interaction as provided herein.
  • the detectable ligand is an antibody.
  • Antibodies that are specific for a MISP fusion polypeptide affinity domain are readily generated as monoclonal antibodies or as polyclonal antisera, or may be produced as genetically engineered immunoglobulins (Ig) that are designed to have desirable properties using methods well known in the art.
  • antibodies may include recombinant IgGs, chimeric fusion proteins having immunoglobulin derived sequences or "humanized" antibodies that may all be used for detection of a human mesothelin polypeptide according to the invention.
  • Many such antibodies have been disclosed and are available from specific sources or may be prepared according to well known methodologies.
  • antibodies includes polyclonal antibodies, monoclonal antibodies, fragments thereof such as F(ab') 2 , and Fab fragments, as well as any naturally occurring or recombinantly produced detectable ligands, which as provided herein are molecules that specifically bind to the affinity domain of a MISP fusion polypeptide, for example, an anti- HA monoclonal antibody that binds to an AK2-HA fusion polypeptide.
  • Antibodies are defined to be "immuno specific" or specifically binding if they bind a cognate antigen ⁇ e.g., an affinity domain of a MISP fusion polypeptide) with a K a of greater than or equal to about 10 4 M" 1 , preferably of greater than or equal to about 10 5 M' 1 , more preferably of greater than or equal to about 10 6 M" 1 and still more preferably of greater than or equal to about 10 7 M" 1 .
  • Affinities of detectable ligands such as antibodies can be readily determined using conventional techniques, for example those described by Scatchard et al., Ann. N. Y. Acad. Sci. 51:660 (1949).
  • Determination of other proteins as detectable ligands that bind to an affinity domain of a MISP fusion polypeptide can be performed using any of a number of known methods for identifying and obtaining proteins that specifically interact with other proteins or polypeptides, for example, a yeast two-hybrid screening system such as that described in U.S. Patent No. 5,283,173 and U.S. Patent No. 5,468,614, or the equivalent.
  • the present invention also includes the use of a MISP fusion polypeptide, and peptides based on the amino acid sequence of a MISP fusion polypeptide, to prepare binding partners and antibodies that specifically bind to such a fusion polypeptide.
  • antibodies may generally be prepared by any of a variety of techniques known to those of ordinary skill in the art ⁇ see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
  • antigen-binding fragments of antibodies may be preferred.
  • Such fragments include Fab fragments, which may be prepared using standard techniques ⁇ e.g., by digestion with papain to yield Fab and Fc fragments).
  • the Fab and Fc fragments may be separated by affinity chromatography ⁇ e.g., on immobilized protein A columns), using standard techniques. See, e.g., Weir, D.M., Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston.
  • Single chain antibodies for use in the present invention may also be generated and selected by a method such as phage display ⁇ see, e.g., U.S. Patent No. 5,223,409; Schlebusch et al., 1997 Hybridoma 16:47; and references cited therein).
  • the assay may be performed in a Western blot format, wherein a protein preparation from the biological sample is submitted to gel electrophoresis, transferred to a suitable membrane and allowed to react with the antibody. The presence of the antibody on the membrane may then be detected using a suitable detection reagent, as described below.
  • a “biological sample” for use according to the invention may comprise any tissue or cell preparation in which a cell comprising a mitochondrion capable of maintaining a membrane potential when supplied with one or more oxidizable substrates such as glucose, malate or galactose is present; or in the case of isolated mitochondria or permeabilized cells, glucose, galactose, pyruvate, succinate, glutamate, malate, acetoacetate or ⁇ -hydroxybutyrate; and wherein the mitochondrion comprises at least one MISP fusion polypeptide as provided herein.
  • Mitochondrial membrane potential may be determined according to methods with which those skilled in the art will be readily familiar, including but not limited to detection and/or measurement of detectable compounds such as fluorescent indicators, optical probes and/or sensitive pH and ion-selective electrodes ⁇ See, e.g., Ernster et al., 1981 J. Cell Biol. 91:221s and references cited therein; see also Haugland, 1996 Handbook of Fluorescent Probes and Research Chemicals- Sixth Ed., Molecular Probes, Eugene, OR, pp. 266-274 and 589-594.).
  • detectable compounds such as fluorescent indicators, optical probes and/or sensitive pH and ion-selective electrodes ⁇ See, e.g., Ernster et al., 1981 J. Cell Biol. 91:221s and references cited therein; see also Haugland, 1996 Handbook of Fluorescent Probes and Research Chemicals- Sixth Ed., Molecular Probes, Eugene, OR, pp.
  • TMRM TMRM
  • a biological sample may, for example, be derived from a normal ⁇ i.e., healthy) individual or from an individual having a disease associated with altered mitochondrial function.
  • Biological samples may be provided by obtaining a blood sample, biopsy specimen, tissue explant, organ culture or any other tissue or cell preparation from a subject or a biological source.
  • the subject or biological source may be a human or another biological organism, including a genetically engineered organism, such as a non-human animal, a plant, a unicellular organism or a multicellular organism or mitochondria prepared therefrom.
  • the subject or biological source may also be a primary cell culture or culture adapted cell line including but not limited to genetically engineered cell lines that may contain chromosomally integrated or episomal recombinant nucleic acid sequences, immortalized or immortalizable cell lines, somatic cell hybrid or cytoplasmic hybrid "cybrid" cell lines, differentiated or differentiatable cell lines, transformed cell lines and the like.
  • a biological sample may, for example, be derived from a recombinant cell line or from a transgenic animal.
  • the biological sample comprises a cell that is a host cell or mitochondria prepared therefrom. Host cells are genetically engineered (transduced, transformed or transfected) with one or more vectors and/or expression constructs as described herein.
  • Engineered host cells may be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying particular genes such as genes encoding MISP fusion proteins and/or genes encoding Bcl-2 family members. Suitable culture conditions for particular host cells will be readily apparent to the ordinarily skilled artisan.
  • a host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell.
  • appropriate host cells include, but need not be limited to, fungal cells, such as yeast; insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells, such as MDCK, Hep-2, CHO or COS; human cells such as Jurkat or 293 cells; plant cells, or any suitable cell already adapted to in vitro propagation or so established de novo.
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as MDCK, Hep-2, CHO or COS
  • human cells such as Jurkat or 293 cells
  • plant cells or any suitable cell already adapted to in vitro propagation or so established de novo.
  • the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • various mammalian cell culture systems can be employed that express a recombinant MISP fusion polypeptide as provided herein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:115 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • the cell line is a neural cell line and in another such embodiment the cell line is a neuroblastoma cell line, for example, the human SH-SY5Y cell line ⁇ e.g., Biedler et al, 1978 Cancer Res. 38:3751).
  • host cells may be cybrids ⁇ e.g., cytoplasmic hybrid cells comprising a common nuclear component but having mitochondria derived from different individuals).
  • cybrids Methods for preparing and using cybrids are described in U.S. Patent No. 5,888,438, published PCT applications WO 95/26973 and WO 98/17826, King and Attardi ⁇ Science 246:500-503, 1989), Chomyn et al. ⁇ Mol Cell. Biol. 77:2236-2244, 1991), Miller et al. (J. Neurochem. (57:1897-1907, 1996), Swerdlow et al.
  • Mammalian expression vectors suitable for use in preparation of the nucleic acid expression construct of the present invention typically will comprise an origin of replication, a suitable promoter and enhancer, and also any additional necessary sequences, for instance, ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences and/or 5' flanking non-transcribed sequences.
  • DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • Introduction of the construct into the host cell can be effected by a variety of methods with which those skilled in the art will be familiar, including but not limited to, for example, calcium phosphate transfection, liposome-mediated transfection, transfection with naked DNA, biolistic particle-mediated transfection, DEAE- Dextran mediated transfection, vector-mediated gene delivery or electroporation ⁇ e.g., Davis et al., 1986 Basic Methods in Molecular Biology; Ausubel et al., 1993 Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston, MA; Sambrook et al., 1989 Molecular Cloning, Second Ed., Cold Spring Harbor Laboratory, Plainview, NY).
  • the invention is directed in pertinent part to a method comprising contacting a biological sample with an agent that is, in certain embodiments, an apoptogen.
  • an apoptogen A variety of apoptogens are known to those familiar with the art and may include by way of illustration and not limitation apoptogens that, when added to cells under appropriate conditions with which those skilled in the art will be familiar, require specific receptors such as the tumor necrosis factor, FasL, glutamate, NMDA (N-methyl-D-aspartic acid), IL-3, corticosterone, mineralcorticoid or glucocorticoid receptor(s).
  • Apoptogens may further include other agents, and thus include atractyloside; bongkrekic acid; thapsigargin; carbachol (carbamoylcholine chloride); herbimycin A (Mancini et al., 1997 J. Cell. Biol.
  • any of a variety of well established assays for determining the induction of apoptosis may be employed to verify readily and without undue experimentation that a biological sample and an apoptogen have been contacted under conditions ⁇ e.g., concentration, media formulation, temperature, pH, cell density, etc.) and for a time sufficient to induce apoptosis, based on the disclosure herein.
  • cells may be examined for morphological, permeability or other changes that are indicative of an apoptotic state.
  • Such changes include, but are not limited to, altered morphological appearance (such as plasma membrane blebbing, cell shape change, loss of substrate adhesion properties or other morphological changes that can be readily detected by those skilled in the art using light microscopy); fragmentation and disintegration of chromosomes (which may be apparent by microscopy and or through the use of DNA specific or chromatin specific dyes that are known in the art, including fluorescent dyes); and/or altered plasma membrane permeability properties, as may be readily detected through the use of vital dyes ⁇ e.g., propidium iodide, trypan blue) or by the detection of lactate dehydrogenase leakage into the extracellular milieu.
  • altered morphological appearance such as plasma membrane blebbing, cell shape change, loss of substrate adhesion properties or other morphological changes that can be readily detected by those skilled in the art using light microscopy
  • fragmentation and disintegration of chromosomes which may be apparent by microscopy and or through the use of DNA specific or chromatin specific dye
  • translocation of cell membrane phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane may be evaluated by measuring outer leaflet binding by the PS-specific protein annexin (Martin et al., J. Exp. Med. 182:1545, 1995; Fadok et al., J. Immunol. 148:2201, 1992).
  • PS cell membrane phosphatidylserine
  • induction of specific protease activity in a family of apopto sis-activated proteases known as the caspases may be measured, for example by determination of caspase-mediated cleavage of specifically recognized protein substrates.
  • substrates may include, for example, poly-(ADP-ribose) polymerase (PARP) or other naturally occurring or synthetic peptides and proteins cleaved by caspases that are known in the art ⁇ see, e.g., Ellerby et al., 1997 J. Neurosci. 77:6165).
  • the synthetic peptide Z-Tyr-Val- Ala-Asp-AFC (SEQ ID NO:l), wherein "Z” indicates a benzoyl carbonyl moiety and AFC indicates 7-amino-4-trifluoromethylcoumarin (Kluck et al., 1997 Science 275:1132; Nicholson et al., 1995 Nature 376:31), is one such substrate.
  • Other substrates include nuclear proteins such as Ul-70 kDa and DNA-PKcs (Rosen and Casciola-Rosen, 1997 J. Cell. Biochem. 64:50; Cohen, 1991 Biochem. J. 326:1).
  • the present invention thus provides certain advantages with regard to regulation of mitochondrial function, and in particular regulation of the mitochondrial permeability "pore" and MPT, as described herein.
  • the invention also pertains to compositions and methods that relate to the mitochondrial adenine nucleotide translocator (ANT).
  • ANT mitochondrial adenine nucleotide translocator
  • This membrane potential drives ANT-mediated stoichiometric exchange of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) across the inner mitochondrial membrane, and provides the energy contributed to the phosphate bond created when ADP is phosphorylated to yield ATP by ETC Complex V, a process that is "coupled” stoichiometrically with transport of a proton into the matrix.
  • Mitochondrial membrane potential, ⁇ m is also the driving force for the influx of cytosolic Ca 2+ into the mitochondrion.
  • the inner membrane Under normal metabolic conditions, the inner membrane is impermeable to proton movement from the intermembrane space into the matrix, leaving ETC Complex V as the sole means whereby protons can return to the matrix.
  • ETC Complex V the integrity of the inner mitochondrial membrane is compromised, as occurs during MPT that may accompany a disease associated with altered mitochondrial function, protons are able to bypass the conduit of Complex V without generating ATP, thereby "uncoupling" respiration because electron transfer and associated proton pumping yields no ATP.
  • mitochondrial permeability transition involves the opening of a mitochondrial membrane "pore", a process by which, inter alia, the ETC and ATP synthesis are uncoupled, ⁇ m collapses and mitochondrial membranes lose the ability to selectively regulate permeability to solutes both small ⁇ e.g., ionic Ca 2+ , Na + , K + , H + ) and large ⁇ e.g., proteins).
  • ANT adenine nucleotide translocator
  • these interactions include binding to ANT by atractyloside, carboxyatractyloside, palmitoyl-CoA, bongkrekic acid, thyroxin, eosin Y and erythrosin B.
  • ANT by atractyloside
  • carboxyatractyloside palmitoyl-CoA
  • bongkrekic acid bongkrekic acid
  • thyroxin bongkrekic acid
  • thyroxin bongkrekic acid
  • thyroxin bongkrekic acid
  • thyroxin bongkrekic acid
  • thyroxin bongkrekic acid
  • thyroxin bongkrekic acid
  • thyroxin bongkrekic acid
  • thyroxin bongkrekic acid
  • thyroxin bongkrekic acid
  • thyroxin bongkrekic acid
  • thyroxin
  • one or more of such agents may be contacted with a biological sample as provided herein.
  • this pore is a physically discrete conduit that is formed in mitochondrial membranes, for example by assembly or aggregation of particular mitochondrial and/or cytosolic proteins and possibly other molecular species, or whether the opening of the "pore" may simply represent a general increase in the porosity of the mitochondrial membrane.
  • MPT may also be induced by compounds that bind one or more mitochondrial molecular components.
  • Such compounds include, but are not limited to, atractyloside and bongkrekic acid, which are known to bind to ANT.
  • Methods of determining appropriate amounts of such compounds to induce MPT are known in the art ⁇ see, e.g., Beutner et al., 1998 Biochim. Biophys. Ada 1368:1; Obatomi and Bach, 1996 Toxicol. Lett. 89:155; Green and Reed, 1998 Science 281:1309; Kroemer et al., 1998 Annu. Rev. Physiol. 60:619; and references cited therein).
  • ANT mitochondrial molecular components
  • adenine nucleotide translocator ANT
  • ANT inhibitors such as atractyloside or bongkrekic acid induce MPT under certain conditions.
  • structural and functional assays that provide, for example, ANT ligands and other agents that interact with ANT, which will be useful for therapeutic management of mitochondrial pore activity. See also U.S.
  • the mitochondrial permeability transition "pore" may not be a discrete assembly or multisubunit complex, and the term thus refers instead to any mitochondrial molecular component (including, e.g., a mitochondrial membrane per se) that regulates the inner membrane selective permeability where such regulated function is impaired during MPT.
  • mitochondria are comprised of "mitochondrial molecular components", which may be any protein, polypeptide, peptide, amino acid, or derivative thereof; any lipid, fatty acid or the like, or derivative thereof; any carbohydrate, saccharide or the like or derivative thereof, any nucleic acid, nucleotide, nucleoside, purine, pyrimidine or related molecule, or derivative thereof, or the like; or any other biological molecule that is a constituent of a mitochondrion.
  • Mitochondrial molecular components includes but is not limited to “mitochondrial pore components".
  • a “mitochondrial pore component” is any mitochondrial molecular component that regulates the selective permeability characteristic of mitochondrial membranes as described above, including those responsible for establishing ⁇ m and those that are functionally altered during MPT.
  • any of a variety of well known criteria may be employed for determining whether a fusion polypeptide is an extramitochondrial fusion polypeptide as provided herein.
  • cell fractionation techniques for the enrichment and detection of mitochondria and/or biochemical markers characteristic of these organelles may be used to determine that a particular subcellular fraction containing a detectable MISP fusion polypeptide as provided herein is substantially free of mitochondria (see, e.g., Ernster et al., 1981 J. Cell Biol. 97:227s).
  • Such methods may be particularly preferred in embodiments of the invention that may be usefully performed on a large scale, for example high throughput screening assays for identifying an agent that alters MISP translocation as provided herein.
  • intracellular localization of a MISP fusion polypeptide may be conducted to detect an extramitochondrial localizaiton of a MISP fusion polypeptide, such as by immunoelectron microscopy, immunofluorescence microscopy ⁇ e.g., including laser- scanning confocal microscopy) or the like.
  • the present invention provides a method of identifying an agent that alters ⁇ e.g., increases or decreases in a statistically significant manner) mitochondrial intermembrane space protein translocation, by detecting such translocation, as described above, and comparing the level of extramitochondrial fusion polypeptide detected in the absence of a candidate agent to the level of extramitochondrial fusion polypeptide detected in the presence of the candidate agent.
  • a method of identifying an agent that alters translocation comprises a high throughput screening assay, for example, where conditions are determined that permit contacting the detectable ligand with extramitochondrial but not with mitochondrial MISP fusion polypeptide.
  • selective solubilization conditions may permeabilize the plasma membranes of cells without altering mitochondrial permeability, such that absolute ⁇ e.g., absence versus presence) or relative ⁇ e.g., quantitative) comparison may be made of the level of extramitochondrial ⁇ e.g., cytosolic) MISP fusion polypeptide.
  • absolute ⁇ e.g., absence versus presence or relative ⁇ e.g., quantitative comparison may be made of the level of extramitochondrial ⁇ e.g., cytosolic) MISP fusion polypeptide.
  • a candidate agent may alter MISP translocation directly ⁇ e.g., by physical contact with one or more MISPs) or indirectly ⁇ e.g., by interaction with one or more additional molecular components, such as mitochondrial molecular components present in a host cell, where such additional components alter MISP translocation in response to contact with the agent).
  • the candidate agent may be a peptide, polypeptide, protein or small molecules.
  • candidate agents are provided as "libraries" or collections of compounds, compositions or molecules.
  • Such molecules typically include compounds known in the art as "small molecules” and having molecular weights less than lO 3 daltons, preferably less than 10 4 daltons and still more preferably less than 10 3 daltons.
  • members of a library of test compounds can be administered to a plurality of biological samples as provided herein, and then assayed for their ability to alter MISP translocation in a cell-based assay.
  • Candidate agents further may be provided as members of a combinatorial library, which preferably includes synthetic agents prepared according to a plurality of predetermined chemical reactions performed in a plurality of reaction vessels.
  • various starting compounds may be prepared employing one or more of solid-phase synthesis, recorded random mix methodologies and recorded reaction split techniques that permit a given constituent to traceably undergo a plurality of permutations and/or combinations of reaction conditions.
  • the resulting products comprise a library that can be screened followed by iterative selection and synthesis procedures, such as a synthetic combinatorial library of peptides (see e.g., PCT/US91/08694 and PCT/US91/04666) or other compositions that may include small molecules as provided herein (see e.g., PCT/US94/08542, EP 0774464, U.S. 5,798,035, U.S. 5,789,172, U.S. 5,751,629).
  • a diverse assortment of such libraries may be prepared according to established procedures, and tested using a MISP translocation assay according to the present disclosure.
  • Agents that alter MISP translocation, and that preferably also inhibit or delay the onset of apoptosis, may be used for a variety of purposes.
  • such agents may be used to alter ⁇ e.g., enhance or inhibit) initiation of an apoptotic cascade by a mitochondrion.
  • the mitochondrion may be isolated or may be present within a cell. Briefly, a mitochondrion is contacted with an agent as described above under conditions and for a time sufficient to translocate one or more MISPs.
  • any of a variety of standard techniques may be used to detect MISP translocation by the mitochondrion (including determination of extramitochondrial MISPs) and/or to detect apoptosis.
  • any experimentally measurable consequence for cells containing mitochondria undergoing MISP translocation may be used, including, for example, detection of the loss of MISPs as provided herein to the cytoplasm, activation of one or more caspases as a downstream event in the apoptotic signaling cascade (see above), cell death and any other phenotypic, biochemical, biophysical, metabolic, respiratory or other useful parameter the alteration of which may depend on MISP translocation.
  • Agents identified according to the methods of the present invention that are suitable for treatment of a disease associated with altered mitochondrial function may potentiate, impair or alter the frequency and/or occurrence of MISP translocation and/or translocation-related regulatory mechanisms.
  • agents that inhibit the appearance of one or more of the above indicators of MISP translocation are particularly preferred. Such agents may also be used to alter survival of a cell. Briefly, a cell is contacted with an agent under conditions and for a time sufficient to modulate cell survival. Cell survival may then be assayed using standard techniques.
  • an agent may be administered to a patient for treatment or prevention of diseases associated with altered mitochondrial function.
  • Preferred agents for such uses inhibit MISP translocation.
  • Diseases associated with altered mitochondrial function include, but are not limited to, AD, diabetes mellitus; Parkinson's Disease; Huntington's disease; dystonia; Leber's hereditary optic neuropathy; schizophrenia; mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS); cancer; psoriasis; hyperproliferative disorders; mitochondrial diabetes and deafness (MIDD) and myoclonic epilepsy ragged red fiber syndrome.
  • Such diseases may be diagnosed using standard clinical criteria, which are well known in the art.
  • the agents that alter MISP translocation are preferably part of a pharmaceutical composition when used in the methods of the present invention.
  • the pharmaceutical composition will include at least one of a pharmaceutically acceptable carrier, diluent or excipient, in addition to one or more MISP translocation-altering agents and, optionally, other components.
  • “Pharmaceutically acceptable carriers” for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remingtons Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985).
  • sterile saline and phosphate-buffered saline at physiological pH may be used.
  • Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
  • sodium benzoate, sorbic acid and esters of jo-hydroxybenzoic acid may be added as preservatives.
  • antioxidants and suspending agents may be used.
  • “Pharmaceutically acceptable salt” refers to salts of the compounds of the present invention derived from the combination of such compounds and an organic or inorganic acid (acid addition salts) or an organic or inorganic base (base addition salts).
  • the compounds of the present invention may be used in either the free base or salt forms, with both forms being considered as being within the scope of the present invention.
  • compositions that contain one or more MISP translocation-altering agents may be in any form which allows for the composition to be administered to a patient.
  • the composition may be in the form of a solid, liquid or gas (aerosol).
  • routes of administration include, without limitation, oral, topical, parenteral ⁇ e.g., sublingually or buccally), sublingual, rectal, vaginal, and intranasal.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrastemal, intrathecal, intracavemous, intrameatal, intraurethral injection or infusion techniques.
  • the pharmaceutical composition is formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient.
  • Compositions that will be administered to a patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of one or more compounds of the invention in aerosol form may hold a plurality of dosage units
  • an excipient and/or binder may be present.
  • examples are sucrose, kaolin, glycerin, starch dextrins, sodium alginate, carboxyme hylcellulose and ethyl cellulose.
  • Coloring and/or flavoring agents may be present.
  • a coating shell may be employed.
  • the composition may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension.
  • the liquid may be for oral administration or for delivery by injection, as two examples.
  • preferred compositions contain, in addition to one or more MISP translocation-altering agents, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • a liquid pharmaceutical composition as used herein, whether in the form of a solution, suspension or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • a liquid composition intended for either parenteral or oral administration should contain an amount of MISP translocation-altering agent such that a suitable dosage will be obtained. Typically, this amount is at least 0.01 wt% of an MISP translocation- altering agent in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Preferred oral compositions contain between about 4% and about 50% of MISP translocation-altering agent(s). Preferred compositions and preparations are prepared so that a parenteral dosage unit contains between 0.01 to 1% by weight of active compound.
  • the pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.
  • the base for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.
  • Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.
  • Topical formulations may contain a concentration of the MISP translocation -altering agent of from about 0.1 to about 10%) w/v (weight per unit volume).
  • the composition may be intended for rectal administration, in the form, e.g., of a suppository which will melt in the rectum and release the dmg.
  • the composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient.
  • bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
  • the MISP translocation-altering agent(s) may be administered through use of insert(s), bead(s), timed-release formulation(s), patch(es) or fast-release formulation(s).
  • the optimal dosage of the MISP translocation-altering agent(s) may depend on the weight and physical condition of the patient; on the severity and longevity of the physical condition being treated; on the particular form of the active ingredient, the manner of administration and the composition employed. It is to be understood that use of a MISP translocation- altering agent in chemotherapy can involve such an agent being bound to another compound, for example, a monoclonal or polyclonal antibody, a protein or a liposome, which assist the delivery of said compound.
  • the RNA was purified using TriZOL (Gibco/Life Technologies, Rockville, MD) essentially according to the manufacturer's instructions, followed by treatment with RNase-free DNase I (Roche Molecular Biochemicals, Indianapolis, IN) using 1 ul of DNase I (10 U/ul) in a buffer containing 40 mM Tris-HCl, pH 7.0, 6 mM magnesium chloride and 2 mM calcium chloride for 30 minutes at 37°C. This treatment was followed by two phenol/chloroform extractions, one chloroform extraction and an ethanol precipitation in the presence of sodium acetate.
  • RNA pellet was collected by centrifugation, washed with 70% ethanol, air dried, and resuspended in RNase-free sterile water.
  • the RNA was reverse transcribed to generate cDNA using RNase H-deficient Reverse Transcriptase (SUPERSCRIPTTM; Life Technologies).
  • AK2 cDNAs were amplified by polymerase chain reactions (PCR) in a thermal cycler using the following primers, AMPLITAQTM DNA Polymerase (Perkin-Elmer, Norwalk, CT), and reagents and buffers supplied in a GENEAMPTM PCR Reagent Kit (Perkin-Elmer), according to the manufacturer's instructions.
  • PCR polymerase chain reactions
  • PCR products were cloned into the PCR 2.1 TA cloning vector (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions and the inserts were sequenced using ABI 373 Sequencer (P-E Applied Biosystems Division, Foster City, CA) to confirm their identities.
  • AK2 inserts were recovered from the PCR 2.1 TA vector by digesting the vector with the restriction endonucleases Xhol and Notl (both enzymes from Roche Molecular Biochemicals). The digestion was carried out according to the manufacturer's recommendations using manufacturer-supplied reaction buffers. Restricted DNAs were purified by horizontal agarose gel electrophoresis and band extraction using the GENE CLEAN® kit (Bio 101, Vista, CA).
  • AK2 inserts were ligated into the mammalian expression vector pCDNA3 (Invitrogen, Carlsbad, CA).
  • the vector was first digested with the restriction endonucleases Kpnl and Xhol (Roche Molecular Biochemicals) and ligated with an AK2 insert (prepared as described above) and a DNA fragment encoding an HA tag.
  • the DNA fragment encoding the HA tag was obtained by annealing a pair of overlapping oligos, which formed the restriction sites for the restriction endonucleases Notl and Xhol at its 5' and 3' termini, respectively.
  • the overlapping oligos were:
  • AK2A-HA/pCDNA3 The resulting pCD ⁇ A3 vector containing AK2-HA construct is termed AK2A-HA/pCDNA3.
  • EXAMPLE 2 EXPRESSION OF AK2A-HA FUSION PROTEINS
  • 293T cells (American Type Culture Collection, Manassas, VA) transformed with SV40 T antigen, and COS-1 cells (ATCC; accession numbers 45504 and CRL-1650, respectively) were transfected with the AK2-HA/pCDNA3 expression construct described in Example 1 using lipofectamine (BRL/Life Technologies, Rockville, MD) essentially according to the manufacturer's instructions. The transfected cells were selected for G418 (Sigma, St. Louis, MO) resistance and expanded in culture. The AK2-HA transfected 293T and COS-1 cells were lysed and the total protein concentration of each lysate was determined using the BCA Protein Assay kit (Pierce Chemical Co., Rockford, IL).
  • Figure 1 show that the AK2-HA fusion protein is expressed in 293T and COS-1 cells.
  • the fusion protein was also detected using standard immunofluoresence microscopy (Harlow and Lane, Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor Laboratory, NY) with a murine anti-HA as the primary antibody and a goat anti-mouse FITC-conjugate (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) as the secondary antibody (FITC, fluorescein isothiocyanate, is a fluorescent tag).
  • FITC fluorescein isothiocyanate
  • MC3 a pool of 3 mixed control cybrid cell lines produced from SH-SY5Y cells that have been repopulated with mitochondria from platelets of 3 healthy donors (Sheehan et al., 1997, J. Neurosci. 17: 4612) was co-transfected with the AK2-HA/pCDNA3 expression construct described in Example 1 and with the pBP-bcl-2 expression construct which directs the expression of the Bcl-2 gene and which contains a puromycin resistance genetic selection marker (Kane et al., 1993, Science 262: 1274). G418 and puromycin (Sigma) were used to select doubly transfected cells expressing both AK2-HA/pCDNA3 and pBP-bcl-2 constructs. Three stable co-transfected cell lines were obtained.
  • AK2A-HA expression of AK2A-HA in these cell lines was detected by western blot analysis ⁇ see Fig. 2A) and immunofluorescence analyses, as described in Example 2. Similarly, the expression of the Bcl-2 gene was detected using rabbit polyclonal anti-human Bcl-2 antibody (Pharmingen, San Diego, CA) and goat anti-rabbit Ig conjugated with HRP (Amersham) as the primary and secondary antibodies, respectively ⁇ see Fig. 2B). All three cell lines overexpressed Bcl-2 protein. One cell line (Fig. 2, lane 9) also overexpressed the AK2A-HA fusion protein.
  • SH-SY5Y neuroblastoma cells were transfected with the AK2-HA/ pCDNA3 expression construct as described in Example 1 using lipofectamine (BRL-Life Technologies). The transfected cells were selected for G418 resistance and further screened for overexpression of the AK2A-HA fusion protein using western blot analysis and immunofluorescence analysis, as described in Example 2.
  • ATP and Mg 2+ are believed to be generally protective to mitochondria for multiple reasons, only some of which are understood. For instance, ATP can co-precipitate with Ca , effectively taking Ca out of solution. Although preferably greater than about 120 ⁇ M, and more preferably greater
  • the multiparameter chamber is an instmment that simultaneously measures several aspects of mitochondrial activity in a sample.
  • the multiparameter chamber comprises a first electrode that is responsive to changing levels of TPP + , which reflects the mitochondrial membrane potential (the "TPP + electrode”); a
  • the 94- 9+ second electrode that responds to changes in Ca concentrations (the "Ca electrode”); a third electrode that is responsive to changes in levels of oxygen in the media, which reflects mitochondria-mediated cellular respiration (the “O 2 electrode”); and a light pipe that detects absorption at 660 nm (A 660 ), which reflects mitochondrial swelling.
  • non-MPT conditions 9+ herein as "non-MPT conditions”. Specifically, in response to the highest load of Ca (1.2 mM), the mitochondria were able to slowly reestablish some membrane potential (as indicated by readings from the TPP + electrode with 2 ⁇ M TPP + in the medium; Fig. 4, upper
  • the suspensions were centrifuged at 12,000g, and the pellets and supernatants were assayed for cytochrome c and HA-tagged AK2 release by Western blot analysis.
  • the HA-tagged AK2 was detected as described in Example 2, and cytochrome c was detected using an antibody specific therefor (Promega Corp., Madison, WI).
  • Alamethicin (Sigma Chemical Co., St. Louis, MO; 40 ⁇ g/mg protein) was used as a positive control.
  • This agent permeabilizes phospholipid bilayers, thereby inducing the efflux of proteins by either direct release, or via osmotic swelling following formation of a non-specific pore.
  • Measurement of the signaling kinase AKT was used to confirm similar loading of the gel.
  • the results of the Western analyses confirmed that, in the absence of ATP and
  • MPT pore comprises one or more isoforms of the adenine nucleotide translocator (ANT) protein (for a review, see Green and Reed, Science 281 :1309-1312, 1998).
  • ANT adenine nucleotide translocator
  • BkA bongkrekic acid
  • CAtr carboxyatractyloside
  • Bcl-2 is an "anti-apoptotic" member of a family of related proteins that includes "pro-apoptotic” proteins such as Bax (for reviews of the Bcl-2/Bax family of proteins, see Adams and Cory, Science 281 :1322-1326, 1998, and Kramer, Nature Medicine 3:614-620, 1997).
  • Bax has been implicated in stimulating cytochrome c release from mitochondria both with and without MPT-like events (Narita et al., Proc. Natl. Acad. Sci. U.S.A. 95:146881-14686, 1998, and J ⁇ rgensmeier et al, Proc. Natl. Acad. Sci.
  • the Bcl-2-overexpressing cells were also evaluated for their response to the ANT ligand carboxyactractyloside (CAtr) under the "non-MPT" conditions described above.
  • CAtr carboxyactractyloside
  • Treatment with 1 mM CAtr induced a change in the calcium tracing for the control cells ("puros").
  • puros the control cells
  • SY5Y cells stably expressing HA tagged adenylate kinase were treated with various apoptogens, harvested, and analyzed for cytochrome c and adenylate kinase release from the mitochondria.
  • Cells were treated with apoptogens in growth media while still adherent to plastic culture dishes.
  • Apoptogens included: staurosporine (200 nM, Sigma, St. Louis, MO), etoposide (10 ⁇ M, Sigma), thapsigargin (15 ⁇ M, Calbiochem, San Diego, CA), and actinomycin D (0.5 ⁇ g/ml, Calbiochem).
  • the cells were harvested following trypsinization, and an aliquot of harvested cells was pelleted by centrifugation and resuspended at a concentration of 2.7 x 10 7 cells/ml in a KCl-sucrose media containing respiratory substrates and digitonin (150 mM sucrose, 50 mM KC1, 20 mM Hepes, 2 mM K 2 HPO 4 , pH 7.0 containing 5mM glutamate, 5 mM malate, 0.03% digitonin, 4 mM MgCl 2 , 1 mM EGTA, 3.0 mM ATP).
  • a KCl-sucrose media containing respiratory substrates and digitonin (150 mM sucrose, 50 mM KC1, 20 mM Hepes, 2 mM K 2 HPO 4 , pH 7.0 containing 5mM glutamate, 5 mM malate, 0.03% digitonin, 4 mM MgCl 2
  • the cell suspension was incubated for 20 minutes at room temperature while being stirred in a disposable spectrophotometer cuvette to keep the cells in suspension. After the incubation period, cytosol was separated from the pellet (which contained mitochondria, other organelles and plasma membrane) by centrifugation at 20,800 x g in a refrigerated microfuge for 10 minutes at 4°C.
  • the supernatant was transferred to a new tube and Complete Protease Inhibitors (Roche Molecular Biochemicals, Inc., Indianapolis, IN) were added according to the supplier's recommendations; the pellets were solubilized in PLC lysis buffer (50 mM Hepes, 150 mM NaCl, 10% glycerol, 1% Triton X- 100, 1.5 mM MgCl 2 , 1 mM EGTA, 100 mM NaF) with complete protease inhibitors in a volume equal to the original volume of the sample.
  • PLC lysis buffer 50 mM Hepes, 150 mM NaCl, 10% glycerol, 1% Triton X- 100, 1.5 mM MgCl 2 , 1 mM EGTA, 100 mM NaF
  • Proteins in the cytosol and pellet samples were separated by SDS-PAGE (Novex 4-20% Tris Glycine gels, Invitrogen, Inc., San Diego, CA), transferred to nitrocellulose membranes using a Novex transfer module, and analyzed by western blotting essentially as described above in Example 5.
  • Membranes were initially probed with an anti- cytochrome c antibody (1:2000, Pharmingen, Inc., San Diego, CA) and then sequentially stripped and probed with an anti-HA antibody (1 ⁇ g/ml, Santa Cruz Biotech, Inc., Santa Cruz, CA) to detect the HA-adenlyate kinase fusion protein, and an anti protein kinase B (PKB) antibody (Phospho-Akt antibody diluted 1 :2000, New England Biolabs, Inc., Beverly, MA) to detect PKB/Akt, a soluble cytosolic marker used to confirm that individual gel lanes had been comparably loaded.
  • PKB protein kinase B
  • Figure 8 shows the western blot analysis of cytosolic and pellet fractions from cells that had been treated with 10 ⁇ M etoposide for the indicated amounts of time
  • Figure 9 shows western blot analysis of cytosolic and pellet fractions from cells that had been treated with 15 ⁇ M thapsigargin for the indicated times.
  • the pellet containing all cellular organelles (Fiskum et al., 1980) was transferred to the multiparameter chamber and assayed in digitonin-free incubation medium with respiratory substrates ⁇ e.g., glutamate/malate) as described in the preceding examples.
  • respiratory substrates e.g., glutamate/malate
  • cyclosporin A (CsA, Sigma, St. Louis, MO) was present at 1 ⁇ M as indicated (Fig. 12; Fig. 13, lane 4).
  • Other additions were made at indicated times (Figs. 11-12) of Ca 2+ (300 ⁇ M), of the electron transport chain inhibitor antimycin (Fig. 11, AntA), or of the permeabilizing agent alamethacin (40 ⁇ g/ml) to determine maximal cytochrome c release.
  • Figure 11 shows calcium and mitochondrial membrane potential ( ⁇ ) traces for digitonin- permeabilized (but not cytosol depleted) cells ("Dig”), or for digitonin-permeabilized and cytosol depleted cells (“CD"). Traces were from readings taken as described in Example 5. with additions of calcium or antimycin A at indicated times. Ca 2+ uptake in the absence of cytosol appeared to be solely driven by mitochondrial respiration, whereas permeabilized cells that retained cytosol appeared to utilize ATPase and respiratory activities to provide energy for Ca 2+ uptake (Fig. 11).
  • Figure 12 shows changes in light absorbance by cytosol depleted (Fig.
  • Cytochrome c detected in the supernatants from cytosol depleted cells is shown in Figure 13, lane 1 (control cells following exposure to 1 mM EGTA with no added Ca 2+ ); lane 3 (300 ⁇ M Ca 2+ per Fig. 12, upper panel); lane 4 (300 ⁇ M Ca 2+ plus 1 ⁇ M CsA per Fig. 12, upper panel); lane 5 (0.06% digitonin permeabilization control cwt-multiparameter chamber); lane 6 (0.09% digitonin permeabilization control />o.yt-multiparameter chamber); lane 7 (0.03% digitonin permeabilization control /r ⁇ st-multiparameter chamber); and lane 8 (alamethacin per Fig. 12, upper panel).
  • lane 2 shows cytochrome c detected in the supernatant from digitonin- permeabilized (but not cytosol depleted) cells following exposure to 300 ⁇ M Ca 2+ .
  • Fig. 13, lane 9 shows a cytochrome c protein standard used as a positive control for reactivity of the anti-cytochrome c antibody.
  • detecting MISP translocation for identifying an agent that alters MISP translocation, and for identifying a compound that alters the activity of an agent that alters MISP translocation, each of said methods comprising in pertinent part the use of a sample comprising a mitochondrion contained within a cell that is permeabilized and that is depleted of cytosol. Determination of when a cell is depleted of cytosol may be accomplished by any of a variety of methods well known in the art, for example, those described in Fiskum et al.
  • cytosolic depletion including quantitative methods for monitoring the degree of cytosolic depletion by determining any of a number of known cytosolic markers, for example, the enzyme lactate dehydrogenase (LDH), or by monitoring the effects of the depletion method on cellular architecture.
  • a cell depleted of cytosol is essentially completely depleted of cytosol, which refers to depletion of cytosol that results in there being no remaining detectable cytosolic marker associated with the cell, according to criteria such as those described Fiskum et al. (1980).
  • a cell that is depleted of cytosol may be substantially depleted of cytosol, which may include a cell from which greater than 40 percent, preferably greater than 60 percent, and more preferably greater than 75 percent of at least one detectable cytosolic marker ⁇ e.g., LDH) is no longer associated with the cell using criteria known to the art, relative to control cells from which cytosol has not been depleted.
  • cytosolic marker e.g., LDH
  • compositions and methods for detecting MISP translocation and for detecting agents that alter ⁇ e.g., increase or decrease in a statistically significant manner) MISP translocation, and for detecting compounds that alter the activity of such agents, which methods may relate to reintroducing to a sample comprising a mitochondrion ⁇ e.g., a cytosol depleted cell as provided herein) one or more cytosolic molecular components.
  • cytosolic components may include, for example, ATP or other biolochemical molecules such as metabolites, catabolites, intermediates, cofactors, substrates, catalysts and the like.
  • Such cytosolic components may also include, for example, one or more of a protein, peptide, glycopeptide or glycoprotein, nucleic acid or polynucleotide (including, for example, DNA or RNA), lipid including a glycolipid, proteolipid or phospholipid, or a carbohydrate, or any combination of such species, that may be present in cytosol.
  • a protein peptide, glycopeptide or glycoprotein
  • nucleic acid or polynucleotide including, for example, DNA or RNA
  • lipid including a glycolipid, proteolipid or phospholipid, or a carbohydrate, or any combination of such species, that may be present in cytosol.
  • Isolation of cytosolic molecular components may be achieved according to any of a number of well known biochemical and chemical separation strategies known to the art, including but not limited to radiolabeling or otherwise detectably tagging cytosolic components in a biological sample, or to cell fractionation, density sedimentation, differential extraction, salt precipitation, ultrafiltration, gel filtration, ion-exchange chromatography, partition chromatography, hydrophobic chromatography, electrophoresis, affinity techniques or any other suitable separation method.
  • Antibodies to partially purified components may be developed according to methods known in the art and may be used to detect and/or to isolate such components.
  • Affinity techniques may be particularly useful in the context of the present invention, and may include any method that exploits a specific binding interaction between a cytosolic component and an agent identified according to the invention that interacts with the cytosolic component. For example, because agents that influence MPT and/or MISP translocation can be immobilized on solid phase matrices, an affinity binding technique for isolation of the cytosolic component(s) may be particularly useful.
  • affinity labeling methods for biological molecules in which a PT-active (or a MISP-active) agent may be modified with a reactive moiety, are well known and can be readily adapted to the interaction between the agent and a cytosolic component, for purposes of introducing into the cytosolic component a detectable and/or recoverable labeling moiety.
  • a PT-active (or a MISP-active) agent may be modified with a reactive moiety
  • Characterization of cytosolic component molecular species may be accomplished using physicochemical properties of the cytosolic component such as spectrometric absorbance, molecular size and/or charge, solubility, peptide mapping, sequence analysis and the like.
  • Additional separation steps for biomolecules may be optionally employed to further separate and identify molecular species that co-purify with such cytosolic components that influence MPT and/or MISP translocation and/or other mitochdondrial activities such as those detected using the multiparameter chamber described above.
  • MPT and/or MISP translocation and/or other mitochdondrial activities such as those detected using the multiparameter chamber described above.
  • MISP translocation and/or other mitochdondrial activities such as those detected using the multiparameter chamber described above.
  • Examples of such methods include RP-HPLC, ion exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, native and/or denaturing one- and two-dimensional electrophoresis, ultrafiltration, capillary electrophoresis, substrate affinity chromatography, immunoaffinity chromatography, partition chromatography or any other useful separation method.
  • cytosolic protein may be obtained for partial structural characterization by microsequencing.
  • sequence data so generated, any of a variety of well known suitable strategies for further characterizing the cytosolic components may be employed.
  • nucleic acid probes may be synthesized for screening one or more appropriately chosen cDNA libraries to detect, isolate and characterize a cDNA encoding such component(s).
  • Other examples may include use of the partial sequence data in additional screening contexts that are well known in the art for obtaining additional amino acid and/or nucleotide sequence information. See, e.g., Molecular Cloning: A Laboratory Manual, Third Edition, edited by Sambrook, Fritsch & Maniatis, Cold Spring Harbor Laboratory, 1989.
  • Such approaches may further include nucleic acid library screening based on expression of library sequences as polypeptides, such as binding of such polypeptides to PT-active or MISP-active agents identified according to the present invention; or phage display screening approaches or dihybrid screening systems based on protein-protein interactions with known mitochondrial proteins, and the like, any of which may be adapted to screening for mitochondrially active cytosolic components provided by the present invention, using routine methodologies with which those having ordinary skill in the art will be familiar. ⁇ See, e.g., Bartel et al., In Cellular Interactions in Development: A Practical Approach, Ed. D.A. Harley, 1993 Oxford University Press, Oxford, United Kingdom, pp.
  • extracts of cultured cells may be sources of novel mitochondrially active cytosolic proteins or other cytosolic factors.
  • Preferred sources may include blood, brain, fibroblasts, myoblasts, liver cells or other cell types.

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Abstract

L'invention concerne des compositions et des techniques d'identification d'agents modifiant la translocation des protéines de l'espace intermembranaire mitochondrial (MISP). Les techniques de criblage permettent généralement de détecter des agents qui modifient le niveau détectable de MISP extra-mitochondriales, à la suite de l'exposition d'une cellule à un agent connu pour induire une translocation des protéines de l'espace intermembranaire mitochondrial ou susceptible d'induire une telle translocation. Ces agents peuvent s'utiliser notamment dans le traitement de plusieurs états associés à une altération de la fonction mitochondriale.
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