WO2013192298A1 - Apoptosis biomarkers - Google Patents

Abstract

The present invention provides novel biomarkers for apoptosis. The apoptosis biomarkers comprise a polypeptide with a caspase cleaved terminus and also a phosphotylaCed amino acid residue located close to the terminus (e.g., within about 15 residues). The invention also provides methods of using such biomarkers to monitor apoptotic activities and methods for identifying apoptosis biomarkers in various cellular systems.

Description

APOPTOSIS BIOMAR ERS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The subject patent application claims the benefit of priority to US.

Provisional Patent Application No. 61/663,076 (filed June 22, 2012). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes.

STATEMENT OF GOVERNMENT SUPPORT

[0Θ02] This invention was made in part with the U.S. government support by the National Institutes of Health Grant No. CA087660. The U.S. Government therefore has certain rights in the invention.

BACKGROUND OF THE IN VENTION

[0003] Apoptosis, or programmed cell death, is orchestrated by a family of cysteine proteases called caspases, which cleave their protein substrates after aspartic acid residues. It is the process whereby the body can rid itself of unwanted, old, or damaged cells. Apoptosis is the physiological counterpart of cell proliferation, it is essential for both biological processes such as normal tissue turnover, embryonic development, and maturation of the immune system, including pathological processes. such as hormone deprivation, thermal stress and metabolic stress.

|OO04] When apoptosis is unregulated, disease results. Unregulated apoptosis is involved in diseases such as cancer, heart disease, neurodegenerative disorders, autoimmune disorders, and viral and bacterial infections. For example, defective apoptosis represents a major causative factor in the development and progression of cancer. The ability of tumor ceils to evade engagement of apoptosis can play a significant role in their resistance to conventional therapeutic regimens. Our understanding of the complexities of apoptosis and the mechanisms evolved by tumor cells to resist engagement of cell death has focused research effort on the development of strategies designed to selectively induce apoptosis in cancer cells.

I [0005] There is a great need in the art for better biomarkers of apoptosis and more effective means for monitoring tumor development and treatment. The instant invention addresses this and other needs. S UMMARY OF THE IN VENT30N

[0006] in one aspect, the invention provides recombinant or isolated polypeptides which are apoptosis biomarkers. These polypeptides comprise a caspase cleaved terminus and a phosphorylated residue within about 15 amino acids of the term inus. Typically, the caspase cleaved terminus in these polypeptides is generated by a caspase after an aspartate residue. In some preferred embodiments, the phosphorylated residue is within about 6, 5, 4 or 3 amino acids of the caspase cleaved terminus, in some embodiments, the caspase cleaved terminus is C-terminus of the polypeptides. Some of these polypeptides comprise a sequence selected from the group consisting of SEQ ID NOs:22-46 and 58-65. In some other apoptosis biomarkers, the caspase cleaved terminus is N-terminus of the polypeptides. Some of these polypeptides comprise a sequence that is the same as or substantially identical to a sequence selected, from the group consisting of SEQ ID NOs:47-57 and 66-76.

[0007] In another aspect, the invention provides recombinant or isolated apoptosis biomarker polypeptide. These polypeptides comprising a caspase cleaved terminus, a phosphorylated residue that is within about 15 amino acids of the caspase cleaved terminus, and a second terminus thai is generated, by cleavage of a second protease. For example, the polypeptides can have a second terminus that is generated by trypsin cleavage. In some of these apoptosis biomarker polypeptides, the caspase cleaved terminus is the C-terminus and the terminus generated by cleavage of a second protease is the N-terminus. Some these polypeptides comprise a sequence that is the same as or substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 77- 101. In some other apoptosis biomarker polypeptides, the caspase cleaved terminus is the N-terminus and the terminus generated by cleavage of a second protease is the C-terminus. Some of these polypeptides comprise a sequence that is the same as or substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 102- 1 12,

0008] In a related aspect, the invention provides methods for monitoring apoptotic activity of a cell. These methods involve detecting and quantifying an apoptosis biomarker in the cell, the apoptosis biomarker being a polypeptide comprising a caspase cleaved terminus and a phosphorylated residue located within about 15 amino acids of the terminus. In some methods, the apoptosis biomarker to be detected is a polypeptide comprising a sequence that is the same as or substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 22-1 12. in some methods, the cei! to be examined, is present in a biological sample obtained from a subject. Some methods of the invention are directed to detecting an apoptosis biomarker in a ceil from a subject suffering afflicted with a tumor.

[0009] in another aspect, the invention provides methods for identifying novel apoptosis biomarkers in a cell, These methods entail (a) inducing apoptosis of the cell, and (b) detecting in the ceil one or more polypeptides comprising a caspase cleaved terminus and a phosphorylated residue within about 15 amino acids of the terminus, if the detected polypeptides are absent in a non-apoptotic control cell, the polypeptides are identified as apoptosis biomarkers of the cell. In some embodiments, the novel apoptosis biomarkers are identified by proteomic analysis of caspase cleavage and phosphorylation of proteins in the cell In some preferred embodiments, the proteomic analysis is performed via the qP-PROTQMAP method described herein,

j 001 J A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.

DESCRIPTION OF THE DRAWINGS

| 111 Figures i A-l H show global crosstalk between phosphor lation and proteolytic pathways in apoptosis. (A) Phosphorylation events are enriched near sites of caspase cleavage in: apoptotic celis. 210 proteins observed in our data, that contain known sites of caspase cleavage were aligned around their scissiie aspartate residues (PI ) and the number of phosphorylation sites detected^■!./- 200 residues are shown. The region of enrichment of phosphorylation surrounding scissiie aspartate residues is shaded, (B) Caspase-cieaved proteins are more likely to be phosphorylated than uncleaved proteins. (C) Kinase activity profiles in Jurkat T-cells as measured by

KtNativ analysis at 2 hr post-STS treatment relative to kinase activities measured in control cells. (D) The most highly over-represented motifs for apoptosis-speeific phosphorylation events as determined b the motif-x algorithm . (E) Anti-p(S T]-Q western blot showing increased phosphorylation of [S/Tj-Q motifs following induction of apoptosis. (F) Anti-p[ S/T}-Q blot demonstrating that DNA-PK, but not ATM or ATfl kinases, is responsible for the apoptosis-related increase in p[S/T]-Q events. (G) Quantitative peptograph showing proteolysis of DNA-PK at 2 hr post-STS treatment The C-terminal persistent fragment contains the ΡΪ-3-kinase-like catalytic domain, (H) Confirrnation by western blotting that, upon induction of apoptosis, DNA-PK is cleaved and a C-terminal fragment containing the kinase domain translocates from the nucleus (Nuc) to the cytoplasm (Cyto). The cleavage kinetics for DNA-PK match closely the kinetics of DNA-PK activation as measured by KiNativ (bar graph) or p[S/T]-Q immunoreactivity (E).

{0012] Figures 2A-2F show that caspase cleavage exposes new sites for phosphorylation. (A and B) Quantitative peptographs showing SF3B2 at 2 hr (A) and 4 hr (B) post-STS treatment. A C-terminal apoptosjs-specific phosphorylation event at Ser861 occurs at the P2 position relative to the caspase cleavage site, at Asp862 (AQVEKBDFS*D; SEQ ID NO: 151). An additional apoptosis-specific

phosphory lation event is observed at Ser289 on the parental form of SF3B2

(S*QQEEEEMETDAR; SEQ ID NO: 152) at 2 hrs, which is 10 residues from another site of caspase cleavage (Asp299). Cleaved product from the latter site is detected at 4 hrs (S * QQEEEEMETD ; SEQ ID NO: 153).. (C) MS-based quantitation showing that the cieaved/unphosphorylated (Ser8 1 ) SF3B2 peptide is generated prior to the

cleaved/phosphorylated (pSer861 ) peptide during apoptosis. Quantified peptides:

uncleaved/unphosphory!ated - EQQAQVE EDFSDMVAEHAAK (SEQ ID NO:4), uncleaved phosphorylated^■· EQQAQVEKEDFS*DMVAEHAAK (SEQ ID NO: 5) (endogenous form not detected), cieaved/unphosphorylated - EQQAQVEKEDFSD (SEQ ID NO:6), cleaved/phosphorylated - EQQ A Q VE^'KEDF S * D (SEQ ID NO: 101). (D) In vitro peptide substrate assays demonstrating that phosphorylation of SF3B2 at Ser881 prevents cleavage by caspases. Peptide substrates:

EQQAQVEKEDFSDMVAEHAAK (SEQ ID NO:4) and

EQQAQVEKEDFS*DMVAEHAA (SEQ ID NO:5). (E) Quantitative peptograph of HCLS1 showing an apoptosis-specific phosphorylation event at Serl 12 occurring at the P4 position of a caspase cleavage site at Aspl l.5. (F) In vitro peptide substrate assays demonstrating that phosphorylation of HCLS 1 at Serl l2 prevents proteolysis by caspase-3 and hinders proteolysis by caspa.se-8. Peptide substrates; SA VGHEYVAEVEKHSSQTDAAK (SEQ ID NO:7) and

S A V GHE Y V AE VEK HS S * QTD A AK (SEQ ID N0:8). See also Figure 4.

[0013] Figures 3A-3H show that phosphorylation at the P3 position of caspase cleavage sites promotes caspase-8-mediated proteolysis. (A and B) Quantitative peptographs showing an apoptosis-specific phosphorylation event at Thrl 00 on the parental form of KHSRP (A, band 6, SEQ ID NO: 1 53) at 2 hr, and on a half-tryptic, aspartate (Asp 103 germinating peptide of a stable fragment of this protein at 4 hr (B, band 21, SEQ 3D NO: 154). Note that this half-tryptic peptide is shown in gray because it lacks an isotopically labeled amino acid. (C) MS-based quantitation showing a rapid increase in the uncleaved/phosphorylated (pThrlOO) KHSRP peptide (yellow line) from 0-2 hr post-STS treatment, which is ~1 hr prior to the appearance of the cleaved forms of this peptide. Note that the uncleaved/unphosphorylated KHSRP peptide was found at 10 times higher levels than the other peptides and was therefore not shown in the figure for the sake of clarity (see Figure 4). Quantified peptides: uncleaved/unphosphorylated -IGGDAATTVNNSTPDFGFGGQK (SEQ ID NO:9), uncleaved/phosphorylated - I GG DA ATT VNN ST* PDFG FG GQK (SEQ ID MO; 10), cleaved/unphosphorylated - IGGDAATTVNNSTPD (SEQ ID NO: 1 1), cleaved/phosphorylated - 1^'GGD A AT W N ST* PD (SEQ ID NO:97). (D) In vitro peptide substrate assays demonstrating that phosphorylation at Thrl 00 of KHSRP enhances cleavage by caspase-8. Peptide substrates: IGGDAATTVNNSTPDFGFGGQK (SEQ ID NO: 9) and IGGDA ATT VNN ST*PDFGFGGQK. (SEQ ID NO: 10). (E) Structure of caspase-8 (PDB: 1QTN) with the tetrapeptide ST*PD (SEQ ID NO:20) modeled into the active site. See Example 8 and Figure 5 for additional details. (F) In vitro peptide substrate assays demonstrating that phosphorylation at Ser882 of RB I promotes cleavage by caspase-8 and, to a lesser extent, by caspase-3. Peptide substrates: TLQTDSIDSFETQR (SEQ ID NO: i 2) and TLQTDS*IDSFETQR (SEQ ID NO: 13). (G) Quantitative peptograph showing caspase-3 ai 2 hr post-STS treatment, revealing an apoptosis- specific phosphorylation event at Ser26, which is the P3 position relative to the known caspase cleavage site at Asp28. (H) In vitro peptide substrate assays demonstrating that phosphorylation at Ser26 promotes cleavage of caspase-3 by caspase-8 and, to a lesser extent by caspase-3. Peptide substrates: IIHGSE SMDSGI S LDNS YK (SEQ ID NO: 15) and IIHGSES*MDSG1SLDNSYK (SEQ ID NO: 16). See also Figure 5. [0014] Figures 4A-4C show in vitro substrate assays indicating linearity of product formation over the tested range of substrate concentrations. (A) SP3B2 peptide substrates: EQQAQVEKBDFSDMVAEHAAK (SEQ ID NO:4) and

EQQAQ VE EDF S * DMV AEH A AK (SEQ ID NO:5). Note that the phosphory!ated peptide substrate is not turned over at any concentration. (B) HCLS1 peptide substrates: SAVGHEYVAEVEKHSSQTDAAK (SEQ ID NO:7) and

S A VGHE Y V AE VEKHS S * QTD A A K (SEQ ID HQ: 8). Note that the phosphorylated peptide is not turned over at any concentration, by caspase-3, although caspase-8 displays moderate activity with this substrate (see Figure 2). (C) Phosphorylation events occurring within six amino acids of scissile aspartate residues in apoptotic proteomes are overrepresented in previously unreported phosphorylation sites.

[0015] Figures 5A-5G show that phosphorylation at the P3 position relative to the scissile aspartate enhances substrate hydrolysis by caspase-8. Phosphorylated and unphosphoryiated tetrapeptide substrates representing the caspase-3 sequence containing pSer26 (ES*MD (SEQ ID NO:21 ), A) or the KHSRP sequence containing pThrlOO (ST*PD (SEQ ID NO:20), B) were modeled into the active sites of caspase-8 or caspase-3 (PDB : IQTN and 1 PAU, respectively, see Supplemental Experimental Procedures for details). Hydrogen bonding interactions with the P3 residues are shown as dashed yellow lines. The lower panels in (A) show schematic representations of the interactions with the phosphorylated substrates. Hydrogen bonding interactions (< 4A) are shown as dashed lines. Notably, Argl 77 in caspase-8 interacts with the

phosphorylated, but not unphosphoryiated substrates, and caspase-3 does not contain a homologous cationic residue. Note that the left panels of part B are identical to the ones shown in Figure 3E, and are reproduced here for clarity. (C-F) Representative in vitro substrate assays with a KHSRP peptide containing the ThrlOO residue showing linear response of product formation by caspase-3 and -8 over the tested range of substrate concentrations. Caspase-8 was unable to turn over the unphosphoryiated peptide at any concentration (C and E). Caspase-8 proteolytic activity was completely blocked by preincubation with the caspase inhibitor z-VAD-FMK (D and F). Peptide substrates: IGGDAATTVNNSTPDFGFGGQK (SEQ ID NO:9) and

IGGDAATTVNNST*PDFGFGGQK (SEQ ID NO: 10). Similar assays were conducted for RB I and caspase-3 substrate peptides with both enzymes, but are not shown due to space constraints. (G) Quantitation of endogenous KHSRP peptides shows that the absolute amounts of uncleaved/phosphorylated peptide and cieaved/unphosphory!ated peptide were similar at their respective peak accumulation valises (2 and 4 hr time points, respectively). Peptide sequences are described in Figure 3.

DETAILED DESCRIPTION

I. Overview

10016] The present invention is predicated in part on the present inventors' discovery of functional crosstalk between phosphorylation and caspase proteolytic pathways that lead to enhanced rates of protein cleavage and the unveiling of new sites for phosphorylation. As detailed in the Examples below, the inventors developed a quantitative proteomic platform that enables simultaneous analysis of proteolytic and phosphorylation processes in cells and direct integration of phosphorylation sites into the topographical maps of cleaved proteins during apoptosis in cells. Employing this proteomic platform, the inventors observed that phosphorylation events are enriched on cleaved proteins in apoptotie cells and occur near sites of caspase proteolysis. The inventors also identified examples where caspase cleavage exposes new

phosphorylation sites that are found exclusively in apoptotie cells. As a specific example, it was found that phosphorylation at the +3 position of caspase recognition sites can directly promote substrate proteolysis by caspase-8.

[0017] In accordance with these discoveries, the present invention provides novel biomarkers for apoptosis, as well as methods for identifying apoptosis biomarkers n various cellular systems. The following sections provide more detailed guidance for practicing the invention. II. Definitions

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed,), Academic Press (i^s! ed., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmoi Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et ai. (Eds.), John Wiley & Sons (3^r ed., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (l^sl ed,, 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Teios (1994); Dictionary of Organic

Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4^th ed., 2000). Further clarifications of some of these terms as they apply specifically to this invention are provided herein.

[0019] The singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise,

[0020] The term "analog" or "'derivative" is used herein to refer to a molecule that structurally resembles a reference molecule (e.g., an apoptosis biomarker) but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, an analog would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Synthesis and screening of analogs to ^'identify variants of known compounds having improved traits (such as higher binding affinity for a target molecule.) is an approach that is well known in pharmaceutical chemistry, [0021 ] Caspases, or cysteine-aspartic proteases or cysteine-dependent aspartate- directed proteases are a family of cysteine proteases that play essential roles in apoptosis (programmed cell death), necrosis, and inflammation. Caspases are essential in cells for apoptosis, or programmed cell death, in development and most other stages of adult life, and have been termed "executioner" proteins for their roles in the cell. Some caspases are also required in the immune system for the maturation of lymphocytes. Failure of apoptosis is one of the main contributions to tumor

development and autoimmune diseases.

[0022] At least twelve caspases have been identified in humans. There are two types of apoptotic caspases: initiator (apical) caspases and effector (executioner) caspases. Initiator caspases (e.g., CASP2, CASP8, CASP9, and CASP I 0) cleave inactive pro-fonns of effector caspases, thereby activating them. Effector caspases (e.g., CASP3, CASP6, and CASP7) in turn cleave other protein substrates within the cell, to trigger the apoptotic process. The initiation of this cascade reaction is regulated by caspase inhibitors, Some of the final targets of caspases include: nuclear lamins, ICAD DFF45 (inhibitor of caspase activated DNase or D A fragmentation factor 45), PARP (poly-ADP ribose polymerase), and PAK2 (P 21 -activated kinase 2).

(0023] A caspase cleaved terminus refers to either the N-terminus or the C- terminus of a peptide or polypeptide that is generated by caspase cleavage or that mimics the sequence of a terminus generated by caspase cleavage. Thus, caspase cleaved termini as used herein encompass N-terminus or C-terminus of a synthetic or isolated polypeptide that has the same sequence as or substantially identical sequence to that of a polynucleotide terminus generated by caspase cleavage.

[0024] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essentia! or not. (0025] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional character! stic(s) of that embodiment of the invention.

[0026] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

(0027] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences, For polypeptide sequences, "conservatively modified variants" refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge.

Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginme, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamme, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine. Lsoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0028] As used herein, a_. "derivative" of a reference molecule (e.g., an apoptosis bioraarker polypepiide) is a molecule that is chemically modified relative to the reference molecule while substantially retaining the biological activity. The modification can be, e.g., oligomerization or polymerization, modifications of amino acid residues or peptide backbone, cross-linking, cyciizafion. conjugation, fusion to additional heterologous amino acid sequences, or other modifications that substantially alter the stability, solubility, or other properties of the peptide.

[0029] The term ''engineered cell" or "recombinant host ceil" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced, it should be understood that such terms are intended to refer not only to the particular subject cell b t to the progeny of such a cell Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0030) Unless otherwise specified, a "fragment" of an apoptosis biomarker polypeptide refers to any peptide or polypeptide having an amino acid residue sequence shorter than that of an apoptosis biomarker polypeptide or protein described herein. Relative to the reference apoptosis biomarker polypeptide or protein sequence (e.g., SEQ ID NOs: 22-76), the fragment typically contains the caspase cleaved N-terminus or C-terminus terminus including the nearby phosphorylated residue. Other than the terminal residues of the reference apoptosis biomarker polypeptide, the fragment can additionally contain 5, 1 0, 25, 50, 100, 200, 300 or more consecutive residues corresponding to the residues beyond the phosphorylated residue in the reference biomarker polypeptide.

[0031] The term "isolated" with respect to a protein or polypeptide means the protein or polypeptide is removed from its natural surrounding. However, some of the components found with it may continue to be with an "isolated" protein. Thus, an "isolated polypeptide" is not as it appears in nature but may be substantially less than 100% pure protein.

[0032] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e„ 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about SO nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0033] Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c, 1970; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol, 48:443, 1970; by the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad, Sci, USA 85 :2444, 1988; by computerized implementations of these algorithms (GAP, BESTFIT, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wl); or by manual alignment and visual inspection (see, e.g., Brent et ai., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003)). Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et aL Nuc. Acids Res.

25 :3389-3402, 1977; and Altschul et aL, J. Mol. Biol. 215:403-410, 1990, respectively.

[0034 j Other than percentage of sequence identity noted above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication thai two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence,

[0035] Unless otherwise specified, the terms "polypeptide" and "peptide" are used interchangeably herein (e,g., "apoptosis biomarker polypeptide" and "apoptosis biomarker peptide") to refer to a polymer of amino acid residues. They encompass both short oligopeptides (e.g., peptides with less than about 25 residues) and longer

U po!ypeptide molecules (e.g., polymers of more than about 25 or 30 amino acid residues). Typically, apoptosis biomarker peptides (oligopeptides) or polypeptides (proteins) of the invention can comprise from about 5 amino acid residues to about 1500 or more amino acid residues in length. In some embodiments, the peptides or polypeptides comprise from about 10 amino acid residues to about 200 amino acid residues in length. In some other embodiments, the peptides or polypeptides comprise from about 8 amino acid residues to about 50 amino acid residues in length. The apoptosis biomarker peptides or polypeptides of the invention can include naturally occurring amino acid polymers and non-naturally occurring amino acid polymer, as well as amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants or derivatives thereof.

[0036] The term "operably linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulator sequence to a transcribed sequence. For example, a promoter or enhancer sequence Is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regiilatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance,

[0037] As used herein, the term "orthoiogs" or "homologs" refers to polypeptides that share substantia! sequence identity and have the same or similar function from different species or organisms. For example, a specific apoptosis biomarker protein (e.g., SF3B2) from human, rabbit, rat, mouse and many other animal species are orthologs due to the similarities in their sequences and functions.

[0038] The term "subject" refers to any animal classified as a mammal, e.g., human and non-human mammals. Examples of non-human animals include dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, and etc. Unless otherwise noted, the terms "patient" or "subject" are used herein interchangeably. Preferably, the subject is human. [0 39J The term "treating" or "alleviating" includes the administration of compounds or agents to a subject to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease (e.g., a tumor), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Subjects in need of treatment incl de those already suffering from the disease or disorder as well as those being at risk of developing the disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the

manifestation of clinical or subclinical symptoms thereof) o therapeutic suppression or alleviation of symptoms after the manifestation of the disease,

[0040 J As used herein, the term "variant" refers to a molecule that contains a sequence that is substantially identical to the sequence of a reference molecule (e.g., an apoptosis biomarker). In some embodiments, the variant can share at least 50%, at least 70%, at least 80%, at least 90, at least 95% or more sequence identity with the reference molecule. In some other embodiments, the variant differs from the reference molecule by having one or more conservative amino acid substitutions, in some other embodiments, a variant of a reference molecule has altered amino acid sequences (e.g., with one or more conservative amino acid substitutions) but substantially retains the biological activity of the reference molecule. III. Novel biomarkers for apoptosis

[0041] The invention provides novel apoptosis biomarkers which are identified via proteomic analysis of phosphorylation events in caspase cleaved proteins in cells undergoing apoptosis. The inventors developed a proieomic tool, qP-PROTOMAP, which enabled investigation of interactions between phosphorylation and proteolytic pathways in apoptosis at a global level. qP-PROTOMAP quantifies phosphorylation events in proteomes and incorporates these modifications into the topographical maps of proteins such that their relationship to proteolytic processing can be directly inferred. As detailed in the Examples below, the inventors uncovered several ways that phosphorylation and proteolytic pathways intersect in apoptotic ceils. This crosstalk is evident on a global level by the enrichment of phosphorylation events on proteolyzed proteins at locations that are in close proximity to caspase cleavage sites. As demonstrated herein, the qP-PROTOMAP method represents a versatile proteomic platform for addressing such questions through its ability to generate global, quantitative, and integrated profiles of phosphorylation and proteolytic pathways in any biological system.

(0042] From a functional perspective, it was found that caspase cleavage can unveil new sites for phosphorylation on proteins and that, apoptosis-specific

phosphorylation events close to caspase recognition sites (e.g., P3 position) can directly promote the cleavage of proteins. In addition, it was revealed that caspase cleavage can also activate kinases, like DNA-PK, that contribute to the creation of a network of phosphorylation events that are specific to apoptotic cells. As exemplifications, it was found that phosphorylation events that promote proteolysis occur, e.g., at the P3 position relative to caspase cleavage sites, where they dramatically enhanced substrate hydrolysis by caspases (e.g., caspase-8). This finding is unexpected, and important because phosphorylation events within caspase consensus motifs (P4-P1 ' residues) have, in the past, been exclusively found to hinder caspase cleavage.

[0043] Not intended to be bound in theory, ii is possible that the kinases responsible for these phosphorylation events cannot gain access to their substrates due to steric hindrance. Caspase cleavage at a proximal location along the protein backbone could then relieve this steric blockade to expose sites for phosphorylation.

Alternatively, there may be kinases that selectively phosphor late proteins near their N- or C-termini. It is also possible that cleavage promotes the redistribution of kinases (e.g., DNA-PK) to distinct subcellular compartments where they phosphorylate new- set of substrates.

[ΘΘ44] As disclosed herein, the apoptosis biomarkers of the invention are isolated or recombinant polypeptides which comprise a caspase cleaved terminus and also at least one phosphorylated residue that is close to the caspase cleaved terminus. 'The phosphorylated residue in the various specific polypeptide sequences disclosed herein is denoted with an asterisk symbol thereafter. The caspase cleaved terminus can be either the N-terminus or the C -terminus of the polypeptide. In some embodiments, the polypeptide comprises the N-terminal fragment of a caspase cieaved protein, which accordingly has a caspase cieaved C-terminus. In othe embodiments, the polypeptide comprises the C-terminal fragment of the cleaved protein, which therefore has a caspase cleaved N-terminus. In still some other embodiments, the polypeptide comprises one caspase cleaved terminus and another terminus resulting from digestion with another protease (including another caspase). The latter protease can be any endopeplidases and exopeptidases present in various cells such as serine proteinases, cysteine (thiol) proteinases, aspartic proteinases, or metalloproteinases. Specific examples include, e.g., trypsin, chymotrypsin, pepsin, papain, elastase, thrombin, plasmin, Hageraan factor, cathepsm G, aminopeptidases, and carboxypeptidase A.

[0045] As noted above, the apoptosis biomarkers of the invention correspond to polypeptides generated from caspase cleavage after an aspartate residue in a protein. The pliosphorviated residue is typically located within about 25 amino acid residues of the caspase cleavage site, preferably within about ] 5 amino acid residues of the cleavage site, and more preferably within 10 amino acid residues of the cleavage site. In various embodiments, the phosphorylated residue is located within 9, 8, 7, 6, 5 or 4 residues of the cleavage site, in some embodiments, the phosphorylated residue in the apoptosis biomarkers of the invention is located within 3 or 2 residues from, or immediately next to, the aspartate residue at the cleavage site.

[0046] The apoptosis biomarkers of the invention can comprise a caspase cleaved C -terminus, a caspase cleaved N-terminus or both. For apoptosis biomarkers with a caspase cleaved C-terminus, the polypeptide will usually have an aspartate residue (PI ) at its C-terminus. The phosphorylated residue in these biomarkers can be the residue located at any position from F2 to P2Q or P25, Preferably, the phosphor lated residue is located at a position from P2 to PI 0 or PI 5. More preferably, the phosphorylated residue is present at P2, P3, P4, P5, P6, P7, P8, P9 or P10 position. For apoptosis biomarker polypeptides or peptides with a caspase cleaved N-terminus, the first N- terminal residue of the polypeptide is the residue that immediately follows the aspartate residue at the cleavage site in the uncleaved protein (i.e., PI ' position). In these biomarkers, the phosphorylated residue can be the residue located at any position from ΡΓ to P20' or P25\ Preferably, the phosphorylated residue is located at a position from PI ' to PI 0' or P15 '. More preferably, the phosphorylated residue is present at PI \ P2\ P3 ', P4', P5\ Ρό', P7\ P8\ P9' or P! O' position.

[0047] The apoptosis biomarkers of the in vention can comprise the intact N- terminal fragment or C -terminal fragment of a caspase cleaved protein that harbors the phosphorylated residue, e.g., the fragment bearing the phosphorylated residue of caspase cleaved proteins shown in SEQ ID NOs:22-76. In some embodiments, the apoptosis biomarkers of the invention comprise the caspase cleaved terminus and the nearby phosphorylated residue, as well as an amino acid sequence that is not the same as, but substantially identical to, the sequence of the caspase cleaved fragment of a wiidtype or naturally existing protein (e.g., SEQ ID NOs:22~76), including orthoiog or variant sequences (e.g., conservatively modified variants). In some other embodiments, the apoptosis biomarkers can comprise the caspase cleaved terminus and the nearby phosphory lated residue but otherwise just a portion of the intact N-terminal fragment or C-terminal fragment of a caspase cleaved protein (or a substantially identical sequence). Thus, the apoptosis biomarkers of the invention can contain at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 100, 200, 300, 500, 750, 1000 or more amino acid residues in length. Some of the polypeptide markers comprise from about 10 amino acid residues to about 500 amino acid residues. Some of the markers comprise from about 25 amino acid residues to about 250 amino acid residues. Some other biomarkers of the invention comprise from about 50 amino acid residues to about 150 amino acid residues.

[0048] Some of the speci fic apoptosis biomarkers of the invention are l isted in Tables 1 and 2 (SEQ ID NOs: 22-76). These include the intact C-terminal or N- terminal fragment of a caspase cleaved protein bearing a phosphorylated residue close to the cleavage site (e.g., within 10 or 15 residues from Asp residue at the cleave site). Apoptosis biomarkers exemplified herein also include some polypeptides generated from both caspase cleavage and digestion with another protease (e.g., trypsin). For example, polypeptides with a sequence shown in SEQ ID NOs: 77-1 12 (Table 1) are half-tryptic peptides (one tryptic terminus and one aspartate terminus, indicative of direct caspase cleavage) identified by the present inventors via qP-PROTOMAP analysis (see Examples below). In some, other embodiments, the apoptosis biomarker of the invention has at least the first 5, 10, 15, 20, 50, 100, 250 or more residues at its caspase cleaved terminus that include the phosphorylated residue and are substantially identical (e.g., 75%, 85%, 90%, 95% or 99% identical) to the corresponding residues of any of the specific apoptosis biomarkers exemplified herein (e.g., SEQ ID NOs:22- 1 1:2). Some apoptosis biomarkers of the invention, while having an overall sequence that is substantially identical to that of a specific polypeptide exemplified herein (e.g., SEQ ID NQs:22-- l 12), have one or both of their terminal residues that are identical to that of the exemplified polypeptide. In some preferred embodiments, the apoptosis biomarkers of the invention have at least the first 5, 10, 15, 20, 50, 100, 250 or more residues at its caspase cleaved terminus that are 100% identical to the corresponding residues of any of the specific apoptosis biomarkers exemplified herein. Caspase cleaved proteins with phosphostte close to cleavage site

IP100455210. CHD4 P4' 5.3 GYET*D^[-iQDYCEVCQQGGEHLCDTCPR (108)

! IPI00550821.2 P4⁷ 54 GRAT*PSENLWSSAR?j09)

1PI00000856.7 PLEKHC ! P4' 55 AALS*DLEFFLEGGK (110)

HTATSFS PT 56 AGGEPDS* LGQQPTDTP YE WDLDKK (1 J 1) Ip 00l64949j ΤΗΤΪΓ GGQQEDDS * GEGEDDAE VQQECLH ( ! 12)

IV. Monitoring apoptotic activities with apoptosis biomarkers

[0049] The apopiosis biomarkers described here can be readily used for detecting and monitoring apoptotic activities in ceils. The methods of monitoring or detecting

apopiosis in a cell or group of cells typically entail obtaining from a subject a biological sample comprising an individual ceil or a group of cells. The cells can be from any

sample obtained from the subject e.g., blood sample, tissue sample, biopsy, or tissue

culture. For example, the biological sample to be examined can be a biological fluid

such as extracellular or intracellular fluid or a cell or tissue extract or homogenate. The biological sample can also be an isolated ceil (e.g., in culture) or a collection of cells

such as in a tissue sample or histology sample. The sample can be suspended in a

liquid medium or fixed onto a solid support such as a microscope slide for detection of an apoptosis biomarker as described herein.

[0050] To detect abnormal apoptotic activities, the biological sample is analyzed

for the presence of one or more specific apoptosis biomarkers described herein.

Detected level of a biomarker is then compared to the level of the same marker in a

control cell (i.e., control level). The control ceil can be the same type of cell obtained

from a normal healthy subject or a different type of cell with normal apoptotic. activities from the same subject. Alternatively, for monitoring any change of apoptotic activities

(e.g., during tumor treatment), the detected level of the biomarker is compared to le vel of the biomarker in the same type of ceil sample obtained at a different tim e point from

the subject (control level). The different time point can be, e.g., various points during

the treatment process o prior to treatment. A substantial departure of the delected level of the biomarker in the subject relative to the control level or a level detected at another time point would be indicative of an abnormal or changing apoptotic activity, it would be indicative of the presence or progression/improvement of a disease or disorder in the subject from whom the biological sample is obtained. For example, an increase in the level of one or more apoptosis biomarkers in a subject undergoing treatment of a tumor could be correlated with effectiveness of the treatment.

[0051] In some embodiments, monitoring efficacy of therapeutic treatments (e.g., tumor treatment) can be performed via measuring level of one or more apoptosis biomarkers disclosed herein in combination with any known monitoring means or diagnostic tests. For example, in addition to detecting and measuring level of the apoptosis biomarkers, the subject undergoing treatment of a solid tumor can also be examined with any imaging technique, e.g., FDG positron emission tomography (FDG- PET), magnetic resonance imaging (MRJ), and optical imaging, comprising bioluminescence imaging (BLI) and fluorescence imaging (FLI), Other tests that can be employed in conjunction with methods of the present invention include diagnostic tests with other known tumor markers (e.g., CEA for colorectal tumor) or blood tests that examine circulating tumor cells (CTCs) in the subject afflicted with metastatic cancer (e.g., metastatic breast, colorectal tumor, and prostate tumor).

[0052] Detection and quantification in a cell sample of one or more apoptosis biomarkers disclosed herein can be accomplished with the methods described herein (e.g., LC-MS/MS) or other techniques routinely practiced in_. the art. See, e.g.. Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, inc. (ringbou ed„ 2003)). For example, the apoptosis marker can be detected and quantified via the use of a phospho-specific antibody. Phospho-specific antibodies for a target apoptosis biomarker of the invention can be developed via standard immunology protocols. For example, rabbits can be immunized with synthetic phosphopeptides representing the amino acid sequence surrounding the phosphorylation site of the target biomarker polypeptide. The immune serum can be then applied to a peptide affinity column to generate a highly specific immunoreagent. The phospho-specific antibodies can be used for detecting and quantifying the target apoptosis biomarker in several immunoassays and analysis tools. These include Western blot ELISA, cell-based ELISA, intracellular flow cytometry, mass spectrometry, and proteome profiling. Some of these methods equire the cells to be lysed or processed to isolate proteins therefrom prior to the detection step, e.g., Western blot or ELISA. Other methods of the present invention for detecting apoptosis in cells do not require the step of acquiring a protein sample or !ysate, but rather detect apoptosis in the cells or tissues themselves. Such methods include immuriohistochemistry, imniunocytpchemistry, and flow cytometry. l^'0053^'j By way of exemplification, the apoptosis biomarker of the invention can be analyzed quantitatively via ELISA. ELISA has become a powerful method for measuring protein phosphorylation, ELiSAs are more quantitative than Western blotting and show great utility in studies that modulate kinase activity and function. The format for this microplate-based assay typically utilizes a capture antibody specific for the desired protein, independent of the phosphorylation state. The target protein, either purified or as a component in a complex heterogeneous sample such as a cell lysate, is then bound to the antibody-coated plate. A detection antibody specific for the phosphorylation site to be analyzed is then added. These assays are typically designed using colorimetric or fluoromeiric detection. The intensity of the resulting signal is directly proportional to the concentration of phosphorylated protein present in the original sample. The phospho-specific ELISA technique confers several advantages over more traditional immunobiotting in the measurement of protein phosphorylation. First, results are easily quantifiable by utilizing a calibrated standard. Second, high specificity is possible due to the use of two antibodies specific for the target protein employed together in the sandwich format. Finally, the higher sensitivity often accomplished using ELiSAs allows for smaller sample volumes and the detection of low abundance proteins,

[0054] As exemplified herein, mass spectrometry may also be used in the practice of the present invention. Large-scale phospho-protem analysis in complex protein mixtures involves identification of phospho-proteins and phosphopeptides and sequencing of the phosphorylated residues. Mass spectrometry (MS) techniques are useful tools for these tasks. Although MS can be used with excellent sensitivity and resolution to identify a single protein, there are several inherent difficulties for the analysis of phospho-proteins. First, signals from phosphopeptides are generally weaker, as they are negatively charged and poorly ionized by electrospray MS, which is performed in the positive mode. Second, it can be difficult to observe the signals from low-abundance phospho-proteins of interest in the high-background of abundant non- phosphoryiated proteins. To overcome these drawbacks, several enrichment strategies for phospho-protein analysis by MS have been developed including immobilized metal affinity chromatography (1MAC), phosphospecific antibody enrichment, chemical- modification-based methods such as beta-elimination of phospho-serine and -threonine, and replacement of the phosphate group with biotinylated moieties. [0055] The novel apoptosis biomarker disclosed herein can be readily employed in various diagnostic applications. For example, subjects undergoing treatment of diseases or conditions associated with or mediated by abnormal apoptotic activities (e.g., tumor) can be monitored with one or more apoptosis biomarkers of the invention. A great number of diseases and conditions are amenable to monitoring with methods and compositions of the present invention. Examples of tumors that can be monitored with methods and compositions of the present invention include but are not limited to skin, breast, brain, cervical carcinomas, and testicular carcinomas. They encompass both solid tumors and metastatic tumors. Cancers that can be monitored by the compositions and methods of the invention include cardiac cancer (e.g., sarcoma, myxoma, rhabdomyoma, fibroma, lipoma and teratoma); lung cancer (e.g..

bronchogenic carcinoma, alveolar carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatosis hamartoma, mesothelioma); various gastrointestinal cancer (e.g., cancers of esophagus, stomach, pancreas, colon, small bowel, and large bowel);

genitourinary tract cancer (e.g., kidney, bladder and urethra, prostate, testis; liver cancer (e.g., hepatoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma); bone cancer (e.g., osteogenic sarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Swing's sarcoma, malignant lymphoma, multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma, benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant ceii tumors); cancers of the nervous system (e.g., of the skull, meninges, brain, and spinal cord); gynecological cancers (e.g., uterus, cervix, ovaries, vulva, vagina);

hematologic cancer (e.g., cancers relating to blood, Hodgkin's disease, non-Hodgkin's lymphoma): skin cancer (e.g., malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysp!astic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis); and cancers of the adrenal glands (e.g., neuroblastoma).

[0056] Disease states other than cancer may also be monitored by the methods and compositions of the invention. These include restenosis, autoimmune disease, arthritis, graft rejection, inflammatory bowel disease, proliferation induced after medical procedures such as surgery, angioplasty, and the like. For examples, subjects suffering from thrombotic thrombocytopenic purpura (TTP) or hemolytic-uremic syndrome (HUS) are also amenable to monitoring with the apoptosis biomarkers of the present invention.

V · Identify i n _, novel apoptos ΐ s markers

[0057] Utilizing the quantitative phospho-PROTQMAP (or qP-P OTO AP) platform disclosed herein, the invention provides methods for identifying novel biomarkers of apoptosis in an)' cellular systems. PROTOMAP (Protein Topography and Migration Analysis Platform) characterizes proteolytic events in cells by detecting shifts in protein migration through a combination of 5DS-PAGB and mass spectrometry (MS)-based proteomics. The proteomic measurement of dynamic post-translational modifications, like phosphorylation, requires quantification of individual peptides. As described herein (e.g., Example 1), qP-PROTOMAP combines PROTOMAP with stable isotopic labeling methods for detecting protein phosphorylation. Detailed protocols for performing qP-PROTQMAP analysis of a target apoptosis cell are described herein, e.g., Example 8.

[0058] To identify novel apoptosis biomarkers in a target cell, the cell undergoing (or induced to undergo) apoptosis and a control non-apoptotic cell are both subject to qP-PROTOMAP analysis. As detailed in the Examples below, the control ceil and the apoptoiic target cell can be grown in media containing isotopically light and heavy amino acids, respectively. Equal quantities of each cell proteome can then be combined and separated by SDS-PAGE. The gel lanes can then be sliced into evenly spaced bands digested in-gel with trypsin to extract peptides, Phosphopeptides are then enriched via immobilized metal-affinity chromatography (IMAC) and subjected to reverse-phase liquid chromatography and MS analysis, Unphosphorylated peptides present in flow-through from the IMAC step are also analyzed. The combined S1LAC ratios of unphosphorylated and phosphorylated peptides are integrated into quantitative peptographs to provide a complete picture of protein phosphorylation and proteolysis. As described herein, (e.g., Examples 1 -3), by analyzing and comparing peptographs obtained with the control cell and the target, apoptotic cell, polypeptides with apoptosis- related phosphorylation events can be identified, importantly, qP-PROTOMAP integrates phosphorylation sites into the topographical maps of cleaved proteins, which allows determination of the precise protein isoforms that possess individual phosphorylation events. The method can therefore identify phosphorylation events that

11 may occur exclusively on full-length proteins or, alternatively, on fragments of these proteins generated during apoptosis.

[0059] As exemplified herein for Jurkat T-ceils, the qP-PROTOMAP analysis described herein ca be employed to identify phosphory fated proteins in any other ceils undergoing apoptosis, As all cell types can undergo apoptosis under normal physiological conditions, the methods of the invention are suitable for identifying apoptosis markers in any cellular system. These include cells from the skin, breast, brain, cervical tissue, cardiovascular tissue, lung, gastrointestinal tract (e.g., esophagus, stomach, pancreas, colon, small bowel, and large bowel), genitourinary tract (e.g., kidney, bladder and urethra, prostate, testis); liver, the nervous system (e.g., of the skull, meninges, brain, and spina! cord), gynecological system {e.g., uterus, cervix, ovaries, vulva, vagina), and the hematologic system (i.e., blood ceils), and the endocrine system (e.g., adrenal glands). EXAMPLES

(0060) The following examples are offered to illustrate, but not to limit the present invention.

Example 1 . Analysis of phosphorylation, and proteolysis by qP-PROTOMAP

[0061 ] The proteomic measurement of dynamic post-transiationai modifications, like phosphorylation, requires quantification of individuai peptides, and we therefore sought to combine PROTOMAP with stable isotopic labeling methods for this purpose.

We also needed to incorporate a phosphopeptide enrichment step without sacrificing the protein size and topography information provided by the SDS-PAGE step of the original PROTOMAP method. The workflow for the resulting quantitative phospho-

PROTOMAP (or qP-PROTOMAP) platform was therefore as follows; Control and apoptotic cells were grown in media containing isotopically light and heavy amino acids, respectively. Equal quantities of each cell proteorne were then combined and separated by SDS-PAGE. Next, as in the original PROTOMAP method, gel lanes were sliced into 22 evenly spaced bands that were digested in-gel with trypsin to extract peptides. Phosphopeptides were then enriched via Immobilized metal-affinity

chromatography (IMAC) and subjected to reverse-phase liquid chromatography and

MS analysis on an LTQ-Velos Orbitrap. Flow-through from the IMAC step (containing unphosphorylated peptides) was also analyzed, and the combined SILAC ratios of unphosphorylated and phosphorylated peptides were integrated into quantitative peptographs to provide a complete picture of protein phosphorylation and proteolysis, [0062] Peptographs for qP-P OTOMAP experiments display detected peptides from left-to-right based on their position in the primary sequence of their proteins, and from top-to-bottom depending on the gel band in which they were detected (the vertical dimension thus represents molecular weight). Phosphopeptides are marked by a circle. For the purposes of quant itation, each peptide is assigned a color on a continuum from red to blue reflecting the light/heavy ratio: peptides exhibiting no-change (1 : 1 ratio) are displayed in purple; control- and apoptosis-specific peptides are shown in red and blue, respectively. To facilitate visual interpretation of these quantified peptide data, a box plot is provided in the middle panel {to the right of the peptograph) that displays the distribution of ratios found, in each band. Spectral-count information, is displayed in a third pane! to enable estimation of the relative abundance of each protein isoform. Most importantly, because qP-PRGTOMAP integrates phosphorylation sites into the topographical maps of cleaved proteins, the approach can determine the precise protein isoforms that possess individual phosphorylation events. Thus, we are able to identify phosphorylation events that may occur exclusively on full-length proteins or, alternati vely, on fragments of these proteins generated durin apoptosis.

[0063] For our assessment of crosstalk between proteolysis and phosphorylation, we induced the intrinsic apoptotic pathway in Jurkat T-celis with staurosporine ("STS"). While it might initially seem counterintuitive to use a broad-spectrum kinase inhibitor like STS to study phosphorylation events in apoptosis, we hypothesized that this drug, through the inhibition of many kinases, might simplify the phosphoproteome to facilitate the characterization of phosphorylation events that were important for programmed cell death. Indeed, STS induces a highly efficient apoptotic cascade in Jurkat T-celis_.that is essentially complete by -6 hours. We therefore expected that any kinase pathways relevant for this rapid cell death process would necessarily be insensitive- to this drug. We therefore analyzed STS-treated Jurkat cells at an 'early' (2 hr) and 'late' (4 hr) stage of apoptosis by qP-PROTGMAP. in total 4,521 proteins were detected across both time points, and 5,034 sites of phosphorylation were quantified on serine, threonine, or tyrosine residues from 1 ,624 of the proteins (36% of all proteins detected). Peptographs were generated for each protein at both time points, enabling rapid visual interpretation of their (1) cleavage status, (2) cleavage magnitude, and (3) phosphorylation status on individual protein isoforms in a time -dependent manner.

Example 2. Analysis of cleaved proteins in apoptotic ceils by qP-PROTOMAP

[0064] We initially evaluated the performance of qP-PROTOMAP as a global method for characterizing cleaved proteins, which were expected to: (1) possess multiple peptides in the parental protein band that substantially deviate from the 1 : 1 SILAC ratio observed for unc leaved proteins, and/or (2) display persistent protein fragments selectively in apoptotic cells. We developed abundance thresholds for quantifying the cleavage state of proteins, and, out of 2,867 proteins that met these thresholds, 744 of them (26%) showed strong evidence of cleavage (see Example 8 for details), A majority of the cleaved proteins identified in our original PROTOMAP study were also observed to be cleaved by qP-PROTOMAP (77%), and we detected 349 proteins that had not been previously described in the literature as caspase substrates (some of which are shown in Table 1). Considerably more proteins were found to be cleaved than has been previously reported, both in absolute number and as a percent of the proteome. This can be ascribed to the higher accuracy and sensitivity that is achievable using SILAC quantitation, enabling high-confidence assessments of lower-abundance proteins. The majority of cleaved proteins (67%) displayed one or more persistent fragments.

Exam pi e 3. Analysis of the phosphoproteome in apoptotic ceils by qP-PROTOMAP

[0065] We next assessed the performance of qP-PROTOMAP as & global method for characteriz n phosphorylation events in apoptotic cells. Phosphorylation events that showed > 2-fold SILAC ratios in control or apoptotic cells were defined as

"control-specific" or "apoptosis-specific", respectively. We should note the potential for these phosphopeptide SI LAC ratios to be■influenced by the cleavage of proteins. For instance, reductions in protein abundance during apoptosis could indirectly cause a loss of phosphopeptide signals. Conversely, the stochastic nature of peptide detection in individual data-dependent S runs could result in the identification of a static phosphorylation event exclusively on one isoform of a protein. We attempted to address at least some of the complexities by performing numerous replicates for our phosphoproteomic experiments (see Example 8 for details), which yielded rapidly diminishing returns for unique phosphorylation-siie identification with each replicate.

[0066] A global analysis of SILAC ratios for the 5,060 phosphorylation events identified in our combined qP-PROTO AP datasets led to several important discoveries. First, a majority of phosphorylation events (> 85%) either showed no change or were elevated in control cells. This is not a surprising result given that we induced apoptosis with the broad-spectrum kinase inhibitor STS. Even with this mode of inducing apoptosis. however, we still identified 53 1 phosphorylation sites that were apoptosis-specific. Striking examples included the pSer347 and pSer882 events in the retinoblastoma protein (RB 1 ), a well-studied tumor suppressor that is known to be cleaved at two distinct caspase sites following the induction of apoptosis. We identified 18 additional sites of phosphorylation on RB I , virtually all of which were control- specific, Most of these control-specific phosphorylation events occurred at well- characterized sites identified in numerous (> 10) independent studies as determ ined by searches of the PhosphoSite database; interestingly, however, the apoptosis-specific pSer347 and pSer882 events had either not been previously reported (pSer347) or detected in only one other phosphoproteomic analysis (pSer882), These initial data suggested that apoptosis might activate a special phosphorylation network that is distinct in content from other cellular processes. We more systematically assessed this possibility by comparing the "novelty" of static, control-specific, or apoptosis-specific phosphorylation events, as estimated by the absence of annotation of these events in the PhosphoSite database. Strikingly, close to half of the apoptosis-specific

phosphorylation sites were previously unreported in the literature, while less than 15% of the static/control-specific phosphorylation sites fell into this category. Apoptosis- specific phosphorylation events were also underrepresented among phosphorylations that were frequently detected in the literature (> 5 citations). We conclude from these data that apoptosis leads to the activation of a specific set of kinases (and/or inactivation of phosphatases) to create a rare pool of phosphorylation events that are not observed in healthy cells.

[0067J A closer examination of the apoptosis-specific phosphorylation sites on RB I uncovered another provocative feature - both of these events are proximal to known sites of caspase cleavage that generate persistent fragments detectable by qp- P OTOMAP and western blotting. The pSer347 event, for instance, occurs just two residues upstream (the P3 position) from the scissiie aspartate, The pSer882 event is located four residues upstream of a known scissiie aspartate (at the P5 position) and was identified by qP-PROTOMAP on a haif-tryptic peptide ending at this residue, indicating thai the phosphorylation event resides on a caspase-cleaved fragment of RB I (in this case, the cleavage event is not expected to produce a shift in gel migration of RBI , since cleavage occurs near the C-terminus of the protein). These observations led us to wonder if such cleavage-site-proximai phosphorylation events were unique to RB I, or whether they might represent a more general phenomenon that occurs during apoptosis.

Example . Crosstalk between proteolysis and phosphorylation during apoptosis [0068] To evaluate how phosphorylation events that occur during apoptosis might globally intersect with caspase-rnediated proteolysis, we compiled all of the known sites of caspase cleavage, including 75 sites that were newly identified in the current study (some of which are shown in Table 1), to give 679 total sites on 566 distinct proteins. 413 of these proteins were detected in our analysis of Jurkat T-cells, and we aligned their sequences such that they were all anchored around the scissiie PI aspartate residue. We then searched for phosphorylation events in our data sets that were located 200 residues up- or down-stream of the PI residues, resulting in the discovery of -675 such phosphorylation events on 21 0 proteins. These phosphorylation sites were strikingly clustered around the region immediately surrounding the scissiie aspartate, in particular from the P6 to P6' residues (shaded region in Figure ί A; also see Table 2). This clustering was evident not only for apoptosis-specific phosphorylation events, but also for static and control-specific phosphorylation events. We furthermore found that known caspase substrates were more likely to be phosphorylated in Jurkat T-cells than were uncieaved proteins (Figure I B).

[0069] We next asked whether the phosphorylation events that occurred in apoptotic cells were catalyzed by a specific set of kinases. To discover kinases that might be activated during apoptosis, we employed a functional proteomic platform termed KiNativ (Patriceili et al„ Chemistry & biology 18, 699-710, 20 Π ; see Example 8 for details.). The majorit of kinases showed reduced KiNativ signals in apoptotic cells (Figure 1 C), likely reflecting inhibition by STS. However, a handful of kinases showed stronger KiNativ signals in STS-treated cells, the most dramatic of which was DNA-depcndent protein kinase (DNA-PK) (Figure 1C). DNA-PK is known to preferentially phosphorylate serines and threonines that are located before gSutamine residues on proteins ([S/T]-Q motif). Consistent with the activation of DNA-PK during apoptosis, a motif-x analysis revealed that S-Q phosphorylations were the most overrepresented motifs among the apoptosis-speeific phosphorylation events in our datasets (Figure I D). No such enrichment of S-Q motifs was observed for static or co trol -specific phosphorylation events. These proteoraic data were confirmed by- western blotting using an antibody that recognizes p[S/T]-Q motifs, which showed a time-dependent increase in p[S/T]-Q-immunoreactive proteins in apoptotic cells compared to control cells that peaked at 2 hr post-STS treatment (Figure 3 E).

[0070] The [S/T]-Q substrate motif is utilized by other kinases, most notably ATM and ATR, which, along with DNA-PK, are important regulators genome stability and the DNA-damage response. No change in ATM or ATR activity was seen in our KiNativ data, but this finding does not rule out a contribution of these kinases to phosphorylation events in apoptosis. We more directly tested for this possibility by treating J urkat T-eells with selective inhibitors of DNA-PK (NU-7441 and NU-7026), ATM (KU5633), or ATM/ATR (CGK733) for 1 hour prior to induction of apoptosis. Western blotting revealed a near-complete block of p[S/T]-Q events upon treatment with DNA-PK inhibitors, while inhibitors of ATM and/or ATR were without effect (Figure IF). We also generated two Jurkat T-ceil lines with stable shRNA-mediated knockdowns of DNA-PK and found that these cells showed substantially blunted [S/T]- Q phosphorylation following induction of apoptosis (Figure I F). Finally, we performed a qP-PROTOMAP study of apoptotic Jurkat cells pre-treated with NU-7443 , which resulted in a two-fold or greater reduction in the majority of p[S/T]-Q events (-60%), with other non-p[ST]-Q events being minimally affected. Interestingly, of the p[S/T]~Q events reduced by NU-7441 treatment, -80% were apoptosis-speeific, which, when combined with our immunoblotting results (Figure 3 F), indicate that DNA-PK is responsible for a large fraction of the plS/Tj-Q events observed in apoptotic cells.

[0071 } We next investigated how DNA-PK might be activated during apoptosis. We found that, early in the apoptotic cascade, DNA-PK relocated from the nucleus to the cytoplasm, where, interestingly, the enzyme was cleaved to generate a stable -150 kDa C-terminal fragment that contains the kinase domain (Figures 1G, H). The appearance of a cleaved form of DNA-PK in the cytoplasm directly correlated with the increased p[S/T]-Q immunoreactive proteins (Figure IE) and the enhanced KiNativ signals for this kinase observed 2 hr after induction of apoptosis (Figure 1 H, top panel). Pre-treatment of cells with the caspase inhibitor Z-VAD-FM blocked STS-indnced cleavage of DNA-PK and p[S/T]-Q events. Previous studies have also reported the easpase-mediaied cleavage of DNA-PK in apoptotic cells (Casciola-Rosen et al., J. Exp. Med. 182: 1 625- 1634, 1995). but have mostly interpreted this proteolytic event to inactivate DNA-PK. The assays used in such studies, however, typically measured DNA-dependent DNA-PK activity with a peptide substrate. Our data support an alternative model wherein caspase cleavage releases DNA-PK from genomic DNA to generate an active, truncated form of the enzyme that traverses into the cytoplasm to catalyze a large number o f apoptos is -spec i He phosphorylation events.

Example 6. Caspase cleavage can expose phosphorylation sites

[0072] Previous studies that have examined the functional effects of

phosphorylation on caspase cleavage with individual protein substrates in vitro have mostly uncovered instances where phosphorylation blocks caspase cleavage (Duncan et al., Biochimica et Biophysica Acta 1804: 505-510, 2010; Kurokawa and Kornbiuth, Cell 138, 838-854, 2009; Tozser et al., Biochem. J. 372: 137- 143, 2003). These studies led to a model where phosphorylation serves to 'protect' proteins from proteolytic processing. However, the apoptosis-specific phosphorylation events identified in our study did not appear to conform to this scenario. As shown in Table 1 , man of the apoptosis-specific phosphorylation events were located on half-tryptic peptides ending in C-terminai aspartates, the hallmark of caspase cleavage. One such example is SF3B2, which contains an apoptosis-specific phosphorylation event at Ser86 I that is located at the P2 position adjacent to a site of caspase cleavage (Figures 2A, B). To ascertain whether this phosphorylation event occurs before or after easpase-mediated proteolysis, we used a targeted MS approach with isotopically-labeled peptides to measure the four possible forms of the SF3B2 peptide: (1)

uncleaved/unphosphorylated. (2) cleaved/unphosphorylated, (3)

uncleaved/phosphory!ated. and (4) cleaved/phosphorylated. These experiments provided two key lines of evidence supporting that phosphorylation of Ser863 occurs after easpase-mediated proteolysis. First, the cleaved/unphosphorylated peptide appeared at an earlier time point than the cleaved/phosphorylated peptide (Figure 2C), Second, the uncleaved/phosp oryiated peptide was not detected at any time point, suggesting that the full-length (parental) form of SF3B2 is not phosphoryiated at Ser86 i . These predictions were also supported by in vitro substrate assays, where we found that the unphosphorylated, bu not phosphoryiated peptide served as a substrate for caspases (Figure 2D and Figure 4) , Finally, a search of the PhosphoSite database revealed that phosphorylation of Ser861 has not been detected in any previous phosphoproteomic studies, even though more than 20 other phosphorylation sites have been identified in SF3B2.

10073] A broader search of our qP-PROTOMAP data set identified several additional apoptosis-specific phosphorylation events that meet the expected criteria for exclusively occurring after caspase cleavage: (1 ) being present on half-tryptic, aspartate -terminating peptides, and (2) not being detected in previous

phosphoproteomic studies of non-apoptotic cells (Figure 4). One candidate was found at the P4 position (S*QTD; SEQ ID NO: l) on an N-terminal fragment of HCLS i (Figure 2E), Similar to what was observed for pSer861 in SF3B2, phosphorylation of Serl 12 completely blocked caspase- 3 cleavage of the HCLS I peptide (Figure 2F and Figure 4). Caspase-8 cleavage was also significantly reduced by phosphorylation of Serl 12, although residual hydrolytic activity was detected (Figure 2F). These data, taken together, are consistent with phosphorylation of Serl 12 occurring after caspase- mediated cleavage of HCLS 1 .

Example 7. Phosphor lation can promote caspase cleavage

[0074] We next wondered whether phosphoryiation might also, in certain instances, directly promote (rather than block) caspase-mediated proteolysis. We accordingly searched our qP-PRGTOM AP data sets for apoptosis-specific

phosphorylation events that were located on the parental forms of cleaved proteins in close proximity to sites of caspase-mediated proteolysis. A compelling example was found in the protein HSRP (Figure 3A), where we observed an apoptosis-specific phosphorylation event at Thrl OO at 2 hr post-STS treatment on the parental 74 kDa form of the protein (band 6), and at 4 hr on a half tryptic, aspartate (Asp 103}- terminating peptide in an N-terminal -15 kDa fragment (band 21/22) (Figure 3A and 5B, respectively). pThrl OO was thus located at the P3 position relative to the Asp 103 caspase cleavage site. We also detected the unphosphorylated version of the half- tryptic, Asp 103 -terminal peptide in the KHSRP fragment,

[0075] To determine the relative kinetics ofThrlOO phosphorylation versus caspase-mediated proteolysis at Asp 103 in KHSRP, we performed a targeted MS analysis using isotopically labeled peptides following the protocol outlined above for the SF3B2 protein. In band 6, where the parental form of KHSRP migrates, we detected the uncleaved/phosphorylated form of the peptide, which was strongly increased over the first 2 hrs following STS treatment and then decreased thereafter (Figure 3C), In contrast, the cleaved forms of the peptide in bands 21/22 did not appear until 2.5 hr and continued to accumulate throughout the remainder of the time course (Figure 3C). These data indicate that phosphorylation at Thr.100 precedes proteolysis by a substantial time window during the apoptotic cascade. We should note that the vast majority of the cleaved peptide was found in the unphosphorylated form, with only trace levels of the c!eaved/phosphoryiated peptide being detected throughout the time course.

[0Θ76] Nonetheless, we were intrigued by the complementary time courses for phosphorylation versus proteolysis, as well as the similar stoichiometrics of the uncleaved/phosphorylated and cieaved/unphosphorySated peptides, both of which peaked at ~10% of the total quantity of uneleaved/unphosphorylated peptide (Figure 5). These data correlate well with the low overall magnitude of cleavage for KHSRP (see peptographs in Figures 3A and B) and suggest further that phosphorylation and proteolysis may have a quantitative relationship wherein phosphorylation at Thri OO promotes caspase proteolysis at Asp l 03, Irs this model, the lack of accumulation of the cleaved phosphorylated peptide could be explained by rapid dephosphoryiation of pThrlOO following caspase cleavage.

|0077] We tested whether phosphorylation at Thri OO directly affects caspase cleavage at Aspl03 using in vitro peptide substrate assays, Caspase-3 hydroiyzed the phosphor fated and unphosphorylated KHSRP peptides at equivalent rates (Figure 3D and Figure 5); caspase-8, however, exhibited a dramatic increase in hydroiytic activity (> 20-fold) for the phosphorylated form of the peptide (Figure 3D and Figure 5), The increased hydroiytic activity of caspase-8 could be. completely blocked by

preincubation with the inhibitor z-VAD-ftnk (Figure 5). These findings intrigued us because it is known that caspase-8, but not caspase-3, displays a strong preference for glutamic acid, which is an approximate isostere of phosphorylated serine/threonine residues, at the P3 position. These data suggested that caspase-8 may have evolved a special capacity to accommodate and even prefer phosphorylated residues at the P3 position. To further explore this concept, we modeled the interaction of phosphorylated and unphosphoryiated KHSRP peptides in the active sites of caspase-3 and caspase-8 (Figure 3E and Figure 5). These models predict a clear interaction between the pThri OO of the KHSRP substrate and an arginine residue (Arg i77) in caspase-8 that is not found in caspase-3 (Figure 3E and Figure 5), Argl 77 has also been found to interact with the P3 glutamic acid residue of inhibitors in caspase-8 co-crystal structures, inspired by this discovery, we searched our qP-PROTOMAP data for additional examples of apoptosis-specific Ser/Thr phosphorylation events occurring at the P3 position of known caspase cleavage sites (Table 2). We have already briefly discussed another such example - the apoptosis-specific pSer347 in RB I , which is located at the P3 position adjacent to the Asp350 cleavage site. Utilizing synthetic RB I peptides, we again found that the phosphorylated peptide served as a much better caspase-8 substrate compared to_. the unphosphoryiated variant (Figure 3F). In this case, caspase-3 also showed improved activity for the phosphorylated peptide, but exhibited a less dramatic increase than caspase-8 (Figure 3F). Finally, we noticed that caspase-3 itself possesses an. apoptosis-specific phosphorylation event, pSet26 (detected at 2 hr posi-STS treatment}, that is located at the P3 position relative to the known caspase- cleavage site Asp2 between the prodomain and the large catalytic subunit (Figure 3G). Cleavage at this site is thought to occur primarily by autocatalytic processing, however there is some evidence that caspase-8 also proteolyses this site. As we found for KHSRP and RB I , caspase-8 displayed markedly greater hydrolytic activity for the phosphorylated versus unphosphoryiated caspase-3 peptide (Figure 3H}.

|0078j These results, taken together, indicate that phosphorylation can promote the caspase cleavage of proteins during apoptosis primarily through a mechanism involving the P3 position of caspase proteolytic sites, which, upon phosphorylation, dramatically increases substrate hydrolysis by caspase-8.

(113)

PALM2- *QEELD j SGTJDEI, 25 tP!00032064 ΑΚΑΡ2 698 (11 ) P6

S*TDSVDj SKRLTV 31

IPi0079 l35 SPTBN1 1454 (115) P6

S* DDEM j E SSEG 58

IPi00004363 STK39 387 (116) P6

S*LDGADj STGVVA 59

ΙΡί00438229 TRIM28 883 (117) P6

S*QDVHDi S NTLT 28

ΙΡΙ00465428 VPS13C 1400 (118) P6

HS *SQTD 1 AAKGFG 60

ΙΡΙ00026156 HCLS1 111 (119) P5

ES*DLVD i GRH3PP 34

J J 0003..¹ ...„ PRPSAP2 219 (120) P5

GS * DEAD 1 GSKHLP 33

ΙΡΙ00302829 RB1 882 (121) P5

H3S*QTD 1 AAKGFG 36

ΙΡΙ00026156 HCLS1 112 (122} P4

PAS*LPD] SSLATS 35

ΙΡΙ00376199 IRF2BP2 492 (123.) P4

DVS*EVD i ARHIIE 38

ΙΡΙ00029822 SMARCA4 699 (124) P4

GSES* DjSGISLD 61

ΙΡΙ00292140 CASP3 26 (125) P3

NNST*PD i FGFGGQ 42

ΙΡΙ00855957 KHSRP 100 illll P3

LKES*FD j GSVRAR 40

ΙΡΙ00604620 NCL 591 (127) P3

TEDT*FD|KKMEVA 62

ΙΡΙ00026940 NUP50 246 (128) P3

Q DS * I D ί S ETQR 63

IPS0Q302829 RBI 347 (129) P3

KGDS*I>D 1 SVEALI 44

ΙΡΙ00745092 SPTAN1 1484 mil P3

DDET* D [ MAKLEE 64

ΙΡΙ00178440 EEF1B2 153 ( 31) P2

EA ES*D! SEVQQ? 65

IPI00395014 RSRC1 237 (132) P2

KEDFS* 0 S VAEHA 46

IPI00221106 SF3B2 861 (133) P2

DDDD *D| DLDELD 45

IPI00329528 VPR8P 1421 (134) P2

KAASAD [ S*TTEGT 66

IPI00304171 H2AFY 173 (135) _Ρ1·

TEDKGD ! S*LDSVE 67

IPI00745092 SPTAN1 1484 ^■136» P1'

SVKE Dj S*SSASA 68 ίΡΙ00219913 US 1 228 illll ΡΎ

LKESFD } GS*VRAR 69

IPI00604620 NCL 595 (138; P2'

GEDALD j FT*QESE 70

SPI00397904 NUP93 159 (139·; P2'

SVKSTEM SS* SASA 71

ΙΡΙ00219913 USP14 229 (140) P2'

PEDLTD ! GSY*DDV 72 iPIOOI 86394 AR C10 89 (141; P3'

IP100747447 EIF3B 1 164 __, PE. F D ! DVS* EEE P3' 52

Table 2, Apoptosis-specific phosphorylation sites found within six residues (P6-P6') of caspase cleavage sites. The 1ΡΪ number refers to International Protein Index which is an integrated proteonte database providing data relating to many eukaryotic proteins, including their sequence information. See Kersey et al., Proteomics 4: 1985-1988, 2004; and also http://www.ebi, c.uk/IPl/.

Example 8. Experimental Procedures

f0079| Ceil Culture and Induction of Apoptosis: Jurkat cells were grown at 37 °C under 5% C0₂ in RPMi 1640 media supplemented with 10% fetal calf serum (FCS) and 2 mM ghitamine. For metabolic labeling (SILAC), cells were maintained in RPMI media containing 2 mM ghitamine and light or heavy arginine and lysine (Sigma) were supplemented at a concentration of 100 Lig/ml. Cells were passaged six times in heavy media before testing for full incorporation of the heavy amino acids. Prior to induction of apoptosis, Jurkat cells were seeded to a. density of 1 x 10⁶ cells/ml in RPMI 1640 media containing 10% FCS and 2 mM giutamine. DMSO or staurosporine (1 μΜ final concentration, Sigma) was added and the cells were incubated for 1, 2, or 4 hr at 37°C under 5% CO₂. For kinase inhibition experiments, 1 10 cells/mi were pretreated with inhibitor for 1 hr prior to addition of staurosporine. Kinase inhibitors [CGK 733 (10 μΜ final), KU55933 ( 10 μΜ final), NU-7441 ( 1 μΜ final), and NU-7026 (10 μΜ final)] were purchased from Tocris, with the exception of CGK 733, which was purchased from Calbiochem. For caspase inhibition experiments, I x 10⁶ cells were pre-treated with 60 μΜ z-VAD-FM (Roche) or DMSO for 1 hr prior to STS addition. 0 8Q] Preparation of Ceil Lysates: Soluble and particulate fractions were prepared as described in Jessani ei al . Proc. Nail. Acad. Sci. 99:10335-10340, 2002. Briefly, cells were washed 3 times in cold PBS and resuspended in 200 μΐ of PBS containing protease inhibitors (complete EDTA-free protease inhibitor cocktail, Roche), phosphatase inhibitors (PHOSTOP, Roche), and z-VAD-FMK (Roche). Ceils were then sonicated to lyse and centrifuged at 100,000 x g for 45 min. The supernatant was collected as the soluble fraction. For experiments containing cytosolic and nuclear fractions, samples were prepared according to manufacturers instructions (NE-PER Nuclear and Cytosolic Extraction Kit Pierce).

[00811 Sample Preparation, SDS-PAGE, and IMAC Enrichment: For

phosphopepiide enrichment, 200 μg of each control (l ight) and apoptotic (heavy) soluble fraction were combined and separated via a 10% SDS-PAGE gel for 850 volt hr. The gel was washed in water and manually excised into twenty-two 0.5 cm bands. Bands corresponding to the migration of molecular-weight markers were noted and this information was used to estimate the molecular weights of proteins migrating in each band. Bands were subjected to in-gel trypsin digestion as previously described (Rosenfeld et ah, Anal. Biochem. 203 : 173-179, 1992) with minor modifications.

Briefly, bands were washed in 100 mM ammonium bicarbonate and proteins were reduced in 10 mM tris(2-carboxyethyl)phosphine (TCEP) at 37 °C for 0,5 hr and then alkylated with 55 mM iodoacetamide in the dark for 0.5 hr. The bands were then dehydrated, by washing in 50:50 acentonitrile: 100 mM ammonium bicarbonate, followed by 100% acetonitriie. Gel bands were then dried and resuspended in 40 μΐ of trypsin at 10 ng/μΐ. U pon re-swelling of the gel bands, 25 mM ammonium bicarbonate was added to a final volume of 200 μί and the gel bands were placed at 37 °C overnight. Supernatants containing peptides were removed, and the gel bands were further extracted with 5% formic acid and acetonitriie and dried down via speed vac. For phosphopepiide enrichment, the dried peptides were resuspended in 350 μΐ of IMAC binding buffer (250 mM acetic acid, 30% acetonitri ie). 30 μΐ of equilibrated IMAC slurry (PHOS-Select, Sigma) was added to each band. Samples were then placed on a rotator at room temperature for 3 .5 hr. The peptide/slurry mixture was then washed twice with 800 μΐ binding buffer followed by one wash with 300 μΐ water. Peptide ei tion was accomplished with 300 μΐ of 400 mM NH₄OH and then dried in a speed vac and stored at -80 °C prior to use. The 4-hr dataset was derived from five separate biological replicates and consisted of three IMAC flow-through (non- phosphopeptide) samples and six IMAC eluate (enriched for phosphopeptides) samples, The 2-hr dataset was derived from two separate biological replicates and consisted of one IMAC flow-through sample and two IMAC eluate samples.

[0082] Mass Spectrometric Analysis: Phosphopeptides and unenriched peptides were analyzed separately via LC-MS/MS in the same way; peptides from each band were resuspended in 10 μΐ buffer A (95% I LO. 5% .acetonitriie, 0.1 % formic acid) and loaded via autosampier onto a 100 μιη (inner diameter) fused silica capillary coiumrs with a 5 μΐΏ tip that was packed with 1 0 cm of CI.8 resin (aqua 5μ.Γη, Phenomcnex). LC-MS MS analysis was performed on an LTQ-Velos Orbitrap mass spectrometer (ThermoFisher) coupled to an Agilent 1200 series HPLC, Peptides were eiuted from the column using a 2-hr gradient of 5-100% buffer B (5% H₂0, 95% acetonitriie, 0.1% formic acid). The flow rate through the column was 0.25 μΐ/min and the spray voltage was 1.7 kV, The mass spectrometer was operated in data-dependant scanning mode, with one full MS scan (400-1,800 m/z) occurring in the Orbitrap (60,000 resolution) followed by ten MS2 scans of the nth most abundant ions with dynamic exclusion enabled (20 s duration).

[0083] Data Processing. Analysis, and Deposition: Raw mass spectrometry data were stored as RAW files generated by XCalibur version 2.1 ,0.1 139 running on a Thermo Scientific LTQ-Velos Orbitrap mass spectrometer. RAW files were converted to MS2 format (McDonald et al,₅ 2004) using RAW-Xiract version 1 ,8 and these MS/MS data were searched using ProLuCiD (Xu et al. Mol. Ceil. Proteomics 5:S 174, 2006). ProLuCiD searches were performed using a reverse-concatenated non- redundant variant of the human IP1 database version 3.33. Cysteine residues were required to be carboxyamidomethy^'lated (+57.02146 Da) and up to three differential phosphorylation marks (+79.9663 Da) were permitted on serine, threonine, or tyrosine residues in each peptide. Peptides were required to have at least one tryptic termi us. ProLuCiD data from each gel band were quality-filtered and sorted with DTASelect version 2.0.25 which performs linear discriminant analyses within each charge- and modification-state to achieve a peptide false-positive rate below 1% (Tabb et aL, J Proteome Res. 1 :21~26, 2002).. Actual peptide false-positive rates were below 1 % at this stage (0.9% and 0,7% in the 2- and 4~hr datasets, respectively). RAW data were also converted to mzXML format using ReAdW version 4.3.1 and SILAC quantitation was performed using an in-house software package called Cimage (described in

Weerapana et al., Nature 468:790-795, 2010). Cimage was run using a 10 ppm mass- window and requiring an R² correlation value for light/heavy co-etution of 0.8 or greater. Peptides that were found exclusivel in healthy or apoptotie cells (only light or heavy peak observed) were considered candidate singlet peptides and were subject to additional filters to remove singlet peaks with insufficient abundance or excessive background noise. At this stage the peptide false positive rates were 0.6% and 0.3% in the 2- and 4-hr datasets, respectively, indicating thai incorporation of MSI -based SILAC information improves accuracy of peptide identifications. Peptides were then assembled into quantitative peptographs using in-house software. This process involved additional unbiased peptograph-level noise-flHering steps such as requirement that at least two distinct peptides be observed in a given band across a!! replicates. No reverse (decoy) peptides remained in either dataset following application of these filters. All peptographs detected in both datasets can be viewed at:

http://www.scripps.edu/chemphys/oravatt DixSimon2012 using username : "guest" and password: "peptide" (without the quotes). SILAC ratios and chromatographs from every peptide can also be found at this website. All raw data generated in this study was deposited in the Proteome Commons repository (Smith et ai., Methods Mol, Biol. 696: 123- 145, 201 1 ), and can be accessed at https;//proteomec mmons.qrg using the password "cdjajqjx" and the following hashes:

BFKUGbPTPJBJQIchJCIUgSsF7nQVaMfsJf2be kuBiKBts4J6SworZyln/U92D9RlZJ HHHohg024CtAyl+ptQpieu/HkAAAAAAAA09A=== and

Bz6bOoFiA2Ho7acYv8jXfB6u2rvxcpUh8qdKxaVYVkkiC3tT sGOvsfkSD0MxBH42

G54G4g5j6/gQsXzp/Kvq9fi3 UAAAAAAACa I Q== for the 2- and 4-hr datasets, respectively. Assessments of phosphosite novelty were derived from the

"phosphorylation site dataset" from the PhosphoSitePlus database (Hornbeck et al,, Nucleic Acids Res. 40:0261 -70, 2012). The CASBAH database, which catalogues the known substrates of apoptotie proteolysis (Luthi and Martin, Cell Death &

Differentiation 14:641 -650, 2007) was downloaded and used fo assessments of caspase-substrate novelty.

|ΘΘ84] identification of Cleaved Proteins: Cleaved proteins were identified on the basis of the distribution of peptide-ratios in each band. Only proteins and fragments of sufficient abundance were considered: two spectral counts from at least two distinct peptide sequences were required in a given band and eight spectral counts from at least four distinct peptides were required for each protein, The distribution of peptide ratios in each band were organized into quartiies and if the ratios in the upper three quartiles were more than 3-fold elevated in the control-cells then the band was flagged as control-specific, indicative of a parental degradation event. If the ratios in the lower three quartiies were more than 3 -fold elevated in the apoptotic cells then the band was flagged as apoptosis-specific, indicative of a c leaved fragment , Proteins that did not display apoptosis-specific fragments were further categorized as partially cleaved

(where some bands were control-specific and others were not) versus completely cleaved (where alt bands showed control -specific ratios). Entries were flagged as candidate cleaved proteins if: ( ! ) they displayed apoptosis-specific fragments at either iimepoint, or (2) they showed complete parental cleavage at the 4-hr tiniepoint. Of the 2,867 proteins that met. the abundance thresholds, 837 proteins were flagged as potential substrates of apoptotic proteolysis using this algorithm. This list was then manually pruned to remove ambiguous or mis-categorized protein entries resulting in a final high -confidence list of 744 protesns that are cleaved or degraded during apoptosis.

[0085] Classification of Phosphorylation Sites and Motif-x Analysis: SILAC ratios for ail peptides containing a given phosphorylated residue were extracted from the 2- and 4-hr datasets. Those sites with peptides displaying SILAC ratios that were all at least 2-fold enriched in apoptotic cells were deemed apoptosis-specific. The remaining phosphosites had SILAC ratios that were either unchanged ("static", either displaying less than 2-fold change in either direction or displaying both control -specific and apoptosis specific ratios indicative of a static phosphorylation event on a cleaved protein) or control-specific (at least 2-fold enriched in control-cells). For the DNA-P inhibitor experiments, phosphorylation sites were categorized using the same algorithm described above, except that sites displaying two-fold or greater reduction upon treatment with NU-7441 were designated 'Suppressed by NU-7441 ' and ail other sites were classified as 'insensitive'. For motif analysis, sequences surrounding each phosphosite (+/- 9 residues, referred to as "sequons") were extracted from our 2- and 4- hr datasets and analyzed with the motif-x algorithm (Schwartz and Gygi. Nat,

Biotechnol. 23: 1391- 1398, 2005) using either serine or threonine as the central residue. Apoptosis-specific sequons were submitted (Figure I D) in one batch and control- specific and static sequons were submitted separately, The human proteome (ipi.HUMAN.fasia) was used as a background database and the significance and number-of-occurrences and significance thresholds were set at 20 and 0.000001 , respectively,

[0086] Western Blotting: Either the soluble or the cytoplasmic and nuclear fractions of both control and staurosporine-treated Jurkat cells were analyzed via western blotting using standard methods. Blots were probed with antibodies for caspase-3, PA P1, DNA-PK, pS/T-Q, aetin, lamin, and RB I (Cell Signaling

Technology #s 9662, 9542, 4602, 285 L 4970, 2032, and 9313, respectively). The GRAP2 antibody was from R&D Systems (AF4640).

[0087J Lentiviral Knockdown: DNA-PK shRNA pLKQ.l lenti viral contructs were purchased from Open Biosystems. Short hairpin-plasmid DNA, along with envelope protein (psPAX2) and coat protein (CMV-VSVG) vectors were co-transfected into HEK293T cells. The vims-containing media containing was collected and filtered . Po!ybrene was added to the filtered media to a final concentration of 10 pg/m!, Varying amounts of virus-containing media was then used to infect Jurkat cells. Two days post-infection, Jurkat cells were resuspended in selection media containing 1 μ§/ηι! puromycin. 7 days post-selection, cells were collected and nuclear fractions were prepared. DNA-PK knockdown efficiency was measured by western blot.

Sequences for the clones that gave the best knockdowns were

CCAGTGAAAGTCTGAATCATT (SEQ ID NO:2) and

GCAGCCTTATTACAAAGACAT (SEQ ID NO:3).

10088 j Peptide Quantification by LC- S: 1 10* cel!s/ml of Jurkat cells were treated with 1 μΜ staurosporine and collected every- 30 min for 4 nr. The cells were then washed three times with PBS and resuspended in PBS containing protease inhibitors, phosphatase inhibitors, and z-VAD-FMK. The soluble fraction was isolated via high-speed centrifugation (see preparation of cell lysates) and then 200 μ was subjected to SDS-PAGE. Gel bands corresponding to relevant molecular weights were excised and digested in-gel with trypsin (as described above). Isotopically-labeied peptides (incorporating heavy lysine and arginine residues where present) were then added to tryptic digests prior to analysis via LC-MS/M S. When quantification of phosphopeptides was needed, an 1MAC enrichment step was performed prior to LC- MS/MS analysis. LC-MS MS analyses utilized targeted fragmentation by targeting the mass-to-charge ratios of relevant peptides for MS2 fragmentation. Peptides masses were then extracted, and. a diagnostic MS2 ion was selected for quantitation via

"pseudo-MRM". This quantification method is referred to as pseudo-MRM because, unlike true MEM (multiple reaction monitoring, typically performed on a iripte- quadrupole mass spectrometer) all of the fragment ion masses are measured in the trap, rather than isolating a single daughter ion for quantification. Quantitation is then performed at the software level, after-the-fact, by measuring peaks consisting of a "transition" from parent ion to one of several diagnostic daughter ions (a similar approach is described in detail in Schorl et al. Anal. Chem. 80: ^'! 1 82-1 1 1 . 2008).

[0089] iNativ Profiling of Active Kinases: Jurkat cells were plated at 1 x 10⁶ cells/ml and treated with Ι Μ STS for 1 , 2, or 4 hrs. Cells were then washed, pelleted, lysed in ceil lysis buffer (25 mM Tris pH 7.6, 150 mM NaCl, 1% CHAPS, 1 % Tergitoi NP-40 type, 1 v/v phosphatase inhibitor cocktail Π [EMD/Calbiochem, #524625]), and sonicated. Lysates were filtered and probe reactions were performed at room temperature with a final probe concentration of 5 μΜ. Samples were labeled with both Biotin-Hex-Acyl-ATP and Biotin-Hex-Acyi-ADP probes, as previously described

(PatricelH et al., Chemistry & Biology 18:699-710, 201 1). All reactions were performed in duplicate. Labeled lysates were denatured and reduced, alkylated, and was digested with for 1 hr at 37°C. Desthiobiotinylated peptides were captured using high-capacity streptavidin resin (Thermo Scientific), and spectrometry and data ana! vsis was performed as previously described,

10090] Caspase Activity Assays with Synthetic Peptide Substrates: Synthetic peptides were purchased from Thermo (HeavyPeptide AQUA standards) and diluted in assay buffer containing 50 mM HEPES, 100 mM NaCl, 0.1 % CHAPS, 1 mM EDTA. 10% glycerol, and 10 mM DTP. Recombinant human caspase-8 or caspase-3 (carrier free, R&D Systems) was diluted to 100 nM into assay buffer containing substrate peptide as well as an internal standard peptide that does not serve as a caspase substrate. Samples were incubated for varying lengths of time at 37 °C. Assays were then quenched by acidification with formic acid (0.5% final) and then subjected to ZipTip purification (Mil!ipore) before mass spectrometry analysis on an LTQ-Velos Orbitrap mass spectrometer using a gradient that consisted of a 30 mm loading phase followed by a 30 mitt gradient from 5% to 00% B (95% acetonitriie, 5% water, 0.1 formic acid), ionization efficiencies of the product peptides relative to the interna! standard were calculated using synthetic standards. These values were then used to calculate absolute rates of product formation. All assays were performed under non-saturating substrate concentrations for a period of time that resulted in less than 20% turnover of the substrate (typically 1 μΜ substrate concentration for 60- 120 min). Peptide substrates used for the assays are listed here, with the phosphorylated residue indicated with an asterisk. SF3B2: substrate - EQQAQVE EDFS*DMVAEHAA (SEQ ID NO:5), product - EQQAQVEKEDFS*D (SEQ ID NO: 101). HCLS l : substrate - SAVGHEYVAEVEKHSS*QTDAAK (SEQ ID NQ:8), product - SAVGHEYVAEVEKHSS*QTD (SEQ ID NO:91.). RB 1 : substrate - TLQTDS * IDS FETQR (SEQ ID NO: 13). product ~ TLQTDS*ID (SEQ ID NO: 14). KHSRP: substrate - IGGDAATTVNNST* PDFGFGGQ (SEQ ID NO: 10), product - IGGDAATTVNNST*PD (SEQ ID NO:97). Caspase-3: substrate - IIHGSES*MDSGISLDNSYK (SEQ ID NG: i6), product - IIHGSES*MD (SEQ ID NO: 17).

{0091 j Structural Modeling: Models of caspase-substrate peptide interaction were generated using the flexible peptide docking program released in the Rosetta 3.0 software suite (Leaver-Fay et al, Methods Enzymo!. 487, 545-574, 201 1). Crystal structures of caspase-8 (PDB: IQTN) and caspase-3 (PDB: IPAU) bound with

tetrapeptide analogues were used as the starting template for modeling. First, the unphosphorylated substrate peptide sequences (STPD (SEQ ID NO: .18) for KHSRP or ESMD (SEQ ID NO: 19) for caspase-3) was grafted onto the peptide backbone in the crystal structure at the catalytic cysteine. Second, the rigid-bod orientation between the catalytic cysteine in the caspase and the PI aspartlc acid in the peptide substrate was fixed to mimic the acyl-enzyme intermediate, as observed in the crystal structures. The rest of enzyme-peptide interaction across the binding interface was locally refined using Monte^Carlo energy minimization (MCM) by varying torsion angles in all protein sidechalns as well in the peptide backbone and sidechains. Out of the 500 structural models generated, the one ranked by the best total energy was selected as the final model. Based on the models with unphosphorylated peptides, the phosphorylated peptides (ST*PD (SEQ ID NO:20) or ES*MD (SEQ ID NO:21)) were generated by replacing the P3 serine or threonine residue with a phosphorylated variant that has an additional sidechain torsion angle allowing the position of the phosphate group to be energetically optimized. The same MCM -based flexible peptide docking method was applied to refine the local interaction across the caspase-peptide interface and after 50 structures were generated, the best-ranked mode! by total energy was subjected to a further round of gradient-based energy minimization to optimize the electrostatic interaction between the phosphoryiated residue and its neighboring residues.

* * *

[0092] The invention thus has been disclosed broadly and illustrated in reference o representative embodiments described above. It is understood that various

raodifications can be made to the present invention without departing from the spirit and scope thereof. It is further noted that all publications, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for ail purposes as if each is individually so denoted. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

Additional sequences disclosed (SEQ ID NOs:22~76)

DHX9 (SEQ ID NQ:22)

MGDVKNFLYAWCOK KMTPSYElRAVGNKN QKFMCEVQVEOYNyTGMGNSTN DAQSN

AARDFVNYLVRI EiKSEEVPAFGVAS'PPPLTDTPD

ZYX (SEQ ID NO:23)

MA APRPSF A IS VS VSAP AF YAPQ KKFGPV VAPKPKV PFRPG DSEPPPAPG AQR AQMGRV

GEIPPPPPEDFPLPPPPLAGDGDDAEGALGGA PPPPPPIEESFPPAPLEEF.IFPSPPI'P

PEEEGGPEAPiPPPPQPRE VSS*IDLEII

STK3 (SEQ ID O:24)

MEQPPAP SKLK LSEOSLTKQPFiiVFDVLH LGEGSYGSVF^' AIH ESGQVVAi QVPV

ESDLQfill EISl OQCDSPYVV YYGSYF N DL IVMEYCGAGSVSDlfRLRNKTL!E

DElATiL STLKGLEYLI-n-MR IHRDI^GNILLNTEGHA LADFGVAGQLTDTMAKR T

VIGTPF^VMAPEVIQEIGYNCVADiWSLGrrSiE AEG PPYADiHPMRAiFMlPTNPPP'T

FR PEI.AVSDDFTDFVKKCLVKNPEQRATATQLI^FIPFIKNAKPVSiLRBLri¾AME!KAK

RHEEQQRELEEEEENS*I)EDELD

PAL 2-AKAP2 (SEQ ID NO.25)

MAEAEUI ERLQAIAE R RQTEIEG RQQLDEQILLLQHSKSKVLRE WLLQGIPAGTA

EEEEARRRQSEEDEFRV QEBDNIQRTEQELQTLESBESQLSAKEQIA-EKUETEKSKK

DFQKGFSSTDGDAVNYISSQLPDLPILCSRTAEPSPGQDGTSRAAGVGWEN^'VLL EGESA

SNA.7^'ETSGPDM ^'FXKPPQLSE0DIWL SEGDNYSA^' LLEPAASSLSPDH NMEIEVSVAE CKS GRRSTPHPMDHPSAFYSPPH GLLTDHHESLDNDVAREFRYLOEVLEANCCDSAV

DG NGI¾SPEPGAVVLVGGI^,SPPVHEATQPEPTF;RTASRQAPPFIIEI.S SSPDP AEAE RTNGHSPSQPRDALG»SLQVPVSPSSTTSSRCSSRDGEFTLTTL KEAKFELRAFHEDK PS LPEDDEHFXEQYCI¾KVRPSEE I LEKERRELIRSQAV K PGL¾A WNPPQE T lEEQLDEEHLESHKKYKERKERRAQQEQLLLQKQLQQQQQQPPSQLC APASSHERASMl DKAKEDIVTEQiDFSAAR QFQLMENSRQAVA OQSTPRLFST PFYRPlXJSV SDKPLT NPRPPSVGGPPEDSGASAA GQKSPGALETPSAAGSQGNTASQGKEGPYSEPS ROPLS LW AEDGEFTS ARA VLTW DDDHGILDQFSRS-V VSLT* QEELD

MDC1 (SEQ ID NO:26)

MEDTQAffiWDVEEEEETEQSSESLRCSVEPVG LHIFSGAHGPE DFFLiSLGKNVVGR P

DCSVALPFPSISKQHAVilEILAW^'D APILRDCGSLNG^'rQiLRPPKVLSPGVSHRIJtDQE^

ILFADLLCQYHRLDVSLPF 'SRGPLTVEETPRVOGETQPQRLLLAEDS*EiiEVD

SAPS3 (or PP6R3) (SEQ ID NO:27)

i¾¾FDLHSSSHIDTLUEREDVTLK^

HEEPPQDMDEKffiYKYP iSCELLTSDVSQMNDRLGEDESI.LM LYSFLLNDSPL.NPt,f,

ASFFS VLSiLlSRKPEQiVDfLKKKHDFVOLitKHlGTSAiMDLLLRLLTCIEPPQPRQ

DVi..N\VL^^EEKjiQRl..VEiV'FlPSQEEDRHS ASQSLCL-r\¾LSRDQMLQiQMSTEPDPLL.A

TLEKQaiEQLl.SN(FHitE NESAfVSAIQItLTLLETRRPTFEGHlElCPPGMSHSACS

VNKSVLEAIRGRLGSFHELl.LEPPK SV^'MK JAVGVLDPPVGNTRLNVlRLfSSLLQTN^'I^'S

SWGDLMELNSIGVlLN FF YTWNNFLHTQVEIClALlLASPFENTENATn QDSTGD

NLLLKHLFQ jLiERltEAWEMNEK QAEGGRRHGYMGHLTRlANClVHSTDKGPNSAL

VQQLIKDIPDEVRERWETFCTSSIXiE7T'KR,NTVDLV^'I^"VCHiHSSS*DDElD

VPS!iC (SEQ5DNO:28)

MV1XSVVAOLLNRFLGDYVENLN SQLKLGIWGGNVALDNLQIKE ALSELDVPFKVKAG

Q5⁾KLTlXIPWK LYGEAVYATLEGLYLLVVPGASIKY AVKEEKSLQDV QKEl.SRiEE

ALQiiAAEKGTHSGEF!YGLE FVYKDIKPGRKRK HKKHFK i - GLDRS DKPKEARKD

TFVEKLATQVlKNVQVKITDlHniYEDDVTDPiCRPLSFGVTLGELSLLTANEiiWTPCiL ;

EADKnYKLlRLDSUAYVVNVNCSMSYQRSREQiLDQLKNElL -SGN'IPPNYQYIFOPiS

ASAKLY NPYAESELK PKi CNJElQNlAiELT PQYLS iDLEESVDYMVRNAPYRKY

KPYLPLHTNGPJiWWKYA rJSVii;VHiRRyTQ. WSWSNi imQU, SYKfAYKNKLTQSK

VSEElQ EIQr ,.E Tl.DVFNIILARQQAQVEVlRSGQKLR XSAD^'rGEKRGGWFSGL GK

KES KKDEESLlPETTDiJIJVi PEE fJKLFTAlGYSESTHNLTLPKQYVAHI TLKLVSTS

VT!RENK iPEiLKiQliGiG^'IQVSQRPGAQALKYEAKLEHWy!TGl.RQQDF\^rPSLVASI GDTTSSLLKniFCTNPEX>SPADQTLIVQSQPVEVIYDAKTVNAVVEFFQSN GLDl.EQiT

SATLM LEEBiERTATGLTI-IiETR VLDLRIN KPSYLWPOTOFHHEKSDL LDFOT

FQtNSKDQGLQKTTNSSl-T^IMD AYD FDVEI NVQLLFARAEETWKKCRFQHPST HI

LQPMDfHVBIAKA VEKDlRMARFKVSGGLi^>LMHVRLSDQKM iH"LYL SlPLPQ SSA

QSPERQVSSiPrfSGGT GLLGTSLLLDTVESESDDEYFDAEDGEPQTC S KGSELKKA

AEVPNEELINLLL FEi EVTLEFT QQ EEDTiLVF VTQLGTEATMRTFDLTVVSYL

KiSLDYHEIEGSKRKPLHUSSSDKPGLDLLKVEYIKADKNGPSFQTAFG TEQTVKVAF

SSLNLi.LQTQALVA S ΓΝ YLTTIIPSDDQSISVA EVQ IS TEKQQKN STLPK A i VSS RD SD

IIDFRLFA L N A FCVIVCNEK N IAEJ3K 1QG1.DSS LSLQSR QS l.FA RLEN 1 iVTD VD PK

TVH KAVSIMGNEVFRFNLDLYPDATEGDLYTDMSKVDGVLSLNVGCIQIVYLHKFLMSL

L FLNiNFQTAKESLSAATAQAAERAATSVKDLAQRSFRVSI IDl-KAPVIVIPQSSiSTN

AVVVDLGLiRVH QFSI^^DEDYL PPVmRMDVQLTKLTLYRTVlQPGlYHPDIQLLFlP n^l^FLV RNEAASWYHKWVVElKGHLDS NVSLNQEDLNLLFRlL EKl TEGTEDIJJK

VKPRVQETGE1 EPLEISIS*QDVHD

PML (SEQ ID O::29)

MEPAPARSPRPQQDPARPQEPTMPPPETPSEGRQPSPSPSPVERAFASEEEFQFLRCQQC QAEAKCPKLLPCLHTLCSGCLEASGMQCPICQAIWPLGADTPALDNVPFESLQRRLSVYR

Qr\'DAQAVCTRC ESABFWCFECEQLLCA CFEAFlQWFLKIlFARPLAELRNQSVREFLDG

TRKT NIFCSNPNHRTFrL SlYCRGCSKPtC SCAIXDSSHSELKCDISAElQQRQEEL

DAMTGALQEQDSAFGAVIIAGMHAAVGQLGRARAETBELIRERVRQVVA.HVRAQERELLEA

VDARYQRDYEEMASRLGRLDAVLQRIRTGSALVQRMKCYASDQEVLDt MGFLRQALCRLR

QEEPQSLQAAVRTDGFDEFKVRU5DLSSCITQG DAAVS KASPEAASTPRDPIDVDLPE

EA ERV AQ VQA LGi . A EAQPMA V V QS VPG AMPVPVYA FSI GPS YGl- DVS TTT AQKRKCS

QTQCPR V{KMESEEG EARLARSSPEQPRPSTSKAVS*PPHLD

ANP32B: (SEQ ID O'30)

DM RRWLF RNRTPAAVRELVtD.VCKS>3DGKiEGLTAEFVNLEFLSUNVGLISVSNI.

P LPKLK. LEL3EN iFGGLDMLAEKLPNETHLNl.SG LKD$STLEPLK LEGL SLDE

FNCEVTNLNDYRESVFX PQLTYLDGYDREDQEAPDS»DAEVX»

SPTBNS (SEQ ID NO 31}

MTT VATDYDNIEIQQQYSDVNNR DVDDWDNE SSARLFERSRIKALADEREAVQK TF TKW\¾SHLARVSC rrDLy DU¾DGRMJ..n LLEYLSGERLPKPT G^'RM iHCL£NVDKAL

QFLKEQRWIXNMGSHDrVDGNHRLTlXJUWTULRFQIQDlSVETEDNKE SAKDAlX

LWCQM TAGYPNVNfliNFTOWRDG TOALa!KFIRPDLroFDKLKKSNAliYNLQNAFNt

AEQHLGi;r IiVDPEDJSVDHP.DEKSBTYVVTYYHYFS M Al.AVEGK iG VLDN!AIET KKMlE YESi-ASpi EWiEQTHlLNNR FANSLVGVQQQLQAFNTYRTVEKPPKFTEKG

N EYLLFTIQSKMRANNQKVYMPREGKLiSDrNKAWHRLEKAEHBRELAERNELTRQEKL EQLAKJ FDRKAAMiETWLSE QRLVSQ

Y^'AVARELEAENYHDKRri ARjKDNViRLWEYLl LLRARRQRLE NLGLQKIFQE LYl

MDW lDE K.VLVLS¾DYG HL GVEDLLQKHΐ^Λ iADίGIQAERV GV^ASAQKFATDGEG

YKPCDPQVIRDRVAii EFCYQELCQLAAER¾ARLEESRRIA¥KFFtVl¾lAEEEGWlRE £ !

LSSDD YGKDLTS V RLLSKHRAFEDEMSGRS GHFEQ A IKEGEDM1AEEHFG S E JRERil

YlREQWANLEQLSAIR KRLEEASU-HQFQADADDIDAWMLDll- iVSSSDVGHDEYSTQ

SLVKKH DVAEI¾ANYRPTLDTLHEQASA!,PQEHAESPDVRGRLSG1EERYKEVAELTRL

RKQALQDTlALYKMFSEADACELWlDEmjWLNNMQIPEKLEDLEVIQHRFBSLi^B N

QASRVAVVNQIARQL HSGHPSEKEKAQQDKLNTR SQFRELVDR DALLSALSIQNY

HLECNET SWIRE TKVIESTQDLGNDLAGVMALQR LTG ERDLVAIEAKLSDLQKEAE

KI_JESEBT¾>QAQAILSRLAE!SDV¾'EEM 1 EK REASLGEAS LQQFLRDLDDFQSWLSR QTAIASEDMP TLTEAE LLTQHENIKNEIDNYEFJJYQ M D QEMVTQGQ PAQYMFL

RQl QALDIOWNELHKMWE RQNLLSQSHAYQQFLRDTKQAEAFI.NNQEYVI.AiiTEMP'rT

LEGAEAAiKKQEDFMTT DANEE mAVVETGRRl-VSDG INSDRiQE VDSIDDRHRKN

RETASELLMR1.KD RDLQ FLQDC ?F SI.WINEKM1.^'1^'AQD!V1SYDEAR LHS LKHQAFM

AELASN EWLDKlEXEGMQLiSEKPETEAW E UTGLHK EVLESTTQT AQRLFDAN

KAELFTQSCADLD LHGLESQIQSDDYGKDLTSWILLK OOMLENQMEVR XEIEELQ

SQAQALSQEGK.8*TDEVD

NO:32)

MAAAAVSESWPELELAERERRRELLLTGPGLEERVRAAGGQLPPRLFTLPLLHYL VSGC

GSLRAPGPGLAQGLPQLHSLVLRRNALGPGLSPELGPLPALRVLDLSGNALEALPPGQGL

GPAEPPGLPQi.QSLNLSG RLRELPADEARCAPRiQSLNL^'i^'G CLDSFPAELFRPGALPL

LSELAAAD^'NCLRELSPDJAHLASL TI.DlvSNNQLSElPAEl.ADCP JL. ErNF^'RGN ERDK.

RLEK VSGCQTRSILEYLRVGGRGGG G GRAEGSEKEESRIUCRRERKQRREGGDGEEQD

VGDAGRELLRVEHVSENFVPE-TVRVSPEVRDVRPYiVGAWRGMDEQPG AL RFLTSQT KLHBDLCE RTAATLATOELRAV QPLLYCARPPOOLKiVpLGRKEAKAKELVRQl-QLEA

EEQR QK RQSVSGLiiRYLHLLDG ENYPCLVDADGDVISFPP!TNSEKTKV TrSDLF LEVTSATSLQICKDVMDAL1L AE K YTLE KEEGSLS*DTEAD

RBI (SEQ ID NO.33)

MPPKTPR TAATAAAAAAEPPAPPPPPPPEEDPEQDSGPEDLPLVRLEFEETEEPDFTAL

CQKLKlPDViVRERAWL^'r\VEK^'VSSVDGVLGGYIQ EI,WG ^,IFLAAVDLDEMSFTFTEl.

Q NIEISVH FFNLL JElDTST VDNAMSRLLKKYDVUFALfS LERTCEUYLTQPSSS iSTESNSALVLKVSWnTLLAKGEVLQ EDDLVISFQLMLCVLDYF!KLSPPMLLKEPY

TAVIPINOSPRTPRRGQNRSAR!AKQLE DTRHEVLCKEHEC IDEVKNVYPKNFIPFM

NSLOLVTSNGlJPBVENLS RYEBYlJ NKDI A! ,Ff-DHp Tl.QTDSIDSFETQRTPRKS

NLDEEVNVM'MTPVRW NTIQQI^MILNSASDQPSE LISYFT^NCTVNPKRSIL RVK

D!GYiFiiE FAI¾VGQGCVF GSQRY LGVRLVYRVMESMLKSEEERLSSQNFS LLND>i iFH SLl ACAi..EVVMATYSRSTSQNLDSGTD LS FPWILNVLNLKAFDFyK. VIES FIKAEG

NLTREMIKHLERCEHRI BSi-AWLSDSPLroLIKQSKDREGPTDHLESACPLNLPLQN H

TAADMYLSPVRSP GS71'RV^"NS ^¾NAETQATSAFQTQ PL STSLSLFY KVYRLAYL

RLNTLCERLLSEilPELEH!TWTLFQl-rrLQfiEYEUvlRDRHLDQlMMCSMYGICKV NtDL

F UVrAYKDLPHAVQETFKRVLlKEEEYDSIiVFYNSVFMQRLKTNttQYASTRPPTLS

PiPHlPRSPYKFPSSPIA!PGGNmSPTJKSPYKISEGLPTPU M PRSRlLVStGESFG

TSEKFQ INQMVC SDRVLKRSAEGSNPP PF. KLRFDIEGS*DEAD

PRPS AP2 (SEQ ID NO:34)

MFCV PPEI-E KMNnT GGLVLFSANSNSSC ELSK lAERUjVEMGKVQVYQEPNRETR

VQTQESVRGKJDVFUQl SKI>WlTlMEU.lMVYACKTSCA SUGVtPYFPYSKQCKMR RGSIVSRLLASM CKAGLTHUT¾4DLHQ EiQGFF IPVDNLRASPFLLQY!QEEIPDY

R AVIVA SPASAKRAQSFAERLRLGIAV!HGEAQDAES*DLVD

IRF2BP2 (SEQ ID NO' 35)

MAAAVAVAAASRRQSCYLCDI^RMPWAMIWDFTEPVCRGCVNYEGADRVEFVIETARQLK

R.A HGCFPEGRSPPG AA AS A A AKPPPLSAKD !LLQQQQQ LGBGGP EAAPRA PQA 1-BRYPLA A AAERPPRLGS FGSSRPAASIAQPPTPQPPPVNGIL VPNGFSKLEEPPELNRQSPNPRR GHAVPPTLVPLMNGSATPLPTALGLGGRAAASLAAVSG AAASLGSAQPTDLGAH RPAS VSSSAAVEHEOREAAAKBKQPPPPAIJRGPADSLSTAAGAAELSAEOAG SRGSGEQDWVN RPKTVRDTLLALHQHGHSGPFESKF EPALTAGRU.GFEANGANGSKAVARTAR RKPS PEPEGEVGPP INGEAQPWlS STEGLKffMTP SSFVSPPPPTASPHSNRlTPPEAAQN GQSP AAULVADNAGGSHASKDANQVHSTTRRNSNSPPSPSS NQRRXGPREVGGOGAG

NTGGLEPVHPAS*LPD

HCLS1 (SEQ ID.NO:36)

MW SWGHDVSVSVETQGDDWDTDPDFWDISE EQRWGAKTIEGSGRTEHlNIilQLRNK

VSEEHDVLRK EMESGPKAS1IGYGGR GVERDR DKSAVGHEYVAEVEKHSS*QTD

RAD23B (SEQ ID NO:37)

MQ VTLI I LQQQTFK iDIDPEETVKALKEKIESEKGKD AF P V AGQ LiYAGK ILNDDT ALK EY lDE NFVVVMVTKPKAVS PAPATTQQSAPASTTAVTSSTrTTVAQAPT VPALAPT STPASrrPASATASSEPAPASAA QE PAE PAETPVATSPT*ATD

SMARCA4 (SEQ ID NO:3S)

MSTPDPPLGGTPRPGPSPGPGPSPGA LGPSPGPSPGSAHS MGPSPGPPSAGHPIPTQG

PGGYPQDN^'MHQ HKPMESMHEKGMSDDPR NQMKGMGMRSGGHAG GPPPSPMDQHSQGY

PSPLGGSEHASSPVPASGPSSGPQMSSGPGGAPLDGADPQALGQQNRGPTPF QNQLHQl,

RAQi¥^Y LARGQ)^,l.PDHLQMAVQG Ri^>MPG QQQ,MPTLPPPSVSATGPGPGPGPGPGP

GPGPAPPNYSRPHG GGPNMPPPGPSGVPPG PGQPPGGPP PWPEGPMANAAAPTSTPQ

LiPPQPrGRPSPAPPAVPPAASPVMPPQTQSPGQPAQPAPMVPLHQKQSR!TPiQKPRG iTJPVEiLQEREYRl/^RiAHRlQELEKLPGSLAGDLRTKATIELK^'ALRll.NFQRQLRQEV

VVCMRiDTALETALNAKAY RSKRQSLREARITEKLEKQQKIEQER RRQKHQEYENSIL

QHAKDFKEYHRSVTGKrQ L KAVATYHANTEREQ ENERIEKER RRLMAEDEEGYR

LIDQ KDKRI-AYLLQQTDBYVANLTELVRQHKAAQVAKE KKKKK KKAENAEGQTPAIG

PDGEPLOETSQMSDLPVKVmVESGKlLTGlDAP AGQLEAVVLEMNPGYEVAPRSDSEES

GSEEEEEEEEEEQPQAAQPPTLPVEEKKKIPDPDSDDV§*EVD

Ci2orft! (SEQ ID NO: 39).

MVVLFYWNNHESQVLKSLVlCLVAMEEREFLHV SSSRSlLEDPPSlSEGCGGRVTDYRl

TVVPLASVTVKESLTEEDVLNCQKTrVNLVDMERKNDPLPlSTVGTRGKGPKRDEQYRIM NELETLVRAHiNNSEKHQRVl^CLMACRSKPPEF ERK RGRKREDKED SE AVKDYE

QEKSWQDSERLKG!LERGKHELAEAEli DS*PD CL (SEQ ID O:40)

MV'KLAKAGVC QGDP MAPPP EVEEDSEDEFLVlSEDEEDDSSGEEVViPQ KG K/VAATS

A VVVSPTK VAVATPAKKAAVTPGKKAAATPA TVTPAKAVl^'l^'PG GATPGKALVA

TPGK GAAiPA OAKNGKNA KEDSDEEEDDOSEEDEEDDEDEDEDEDEIEPAAM AAAA

APASEDEDDEDDEDDEDDDDDEEDDSEEEA ETTPAKGKKAAKVVPVKAKXVAEDEDEEE

DDEDBDDDDDEDDEDDRDEDDEEEEEEEEEE V EAPG R KEMAKQKAAPEAKKQKVEG

TEPTTAPNLFVGNLNF ¾SAPELKTGISDVFA NDLAVVDVRiGMTRKFGYVi)PESAEDI_J

EKALELTGL WG^I LE PKGKDS KERDARTLLA NXPY VTQDELKEVFEDAAEI

LVS OGKSKGIAYIEPKTEADAEKTFEEKQGTEIDGRSISLYYTGE GQNQPYUGGKNST

WSGES TLVl^XLSYSATEETLQEVEEKATFI VPQNQNGKS GYAFlEEASFEOA EAi. SCN REiEGRAIRLELQGPRGSPNARSQPSKTLPV GLSEDTTEF.TL ES*FD

MYH9 (SEQIDNO:41)

MAQQAADKYLYVD F!NNPLAQADWAAKKLVWVPSD SGFEPASL EEVGEEAIVELVE

NG V V DDlQKM^TP FS VEDMAEiTCLNEASVL.HNiKERYYSGLIVTYSGI.FCVV lNPY NiPfYSEEIVEMYKG RHEMPPH iAITDTAYRSMMQDREDQSILCTGESGAG

TENT KVlQYLAYVASSH SKKDQGELERQLl-QANPiLEAFOlA TVi-NDNSSRFGKPlR

1NFDVNGYIVGANIE7YI E SRA1KQAKEERTFHJFYYLI.SGAGEHJ- TDJL.LLEPYN Y

RFl^NGHV ffGQQDKDMFQET EAMRIMGIPEEEQMGLLRVISGVLQEGNlVF KERNT

DQAS MPDN TAAQ VSH! .1.0 IN VTD FTRGIITPR ! VGRD YVQKAQTREQ ADFAIEA LAKA

TYERMi¾WLVLRm ALDKTKRQGASFlG!LDIAGFE!FDLN8FF;QI.CiNYT EKLQQlF

^HI FrLEQEEYQREGjLEV^NFlDFGEDLQPCIDEiEKPAGPPGILALLDEECWFPi ATDK

SFVEKVTv'iQEQGTHPKFQ PKOEKDKADFCnHYAG VDYKADEWLMK MDPLNDNlATLI.

HQSSDKFVSELWKDVDRI!GLDOVAGMSETALPGAFKTRKG FRTVGQEY EQEAKLMAT

LR TNPNf VRCIIPNFIEKKAGKEDPHEVLDQLRCNGVLEGIRICRQGFPNRVVFQEFUQR

YEn.TPNSiP GFMDGKQACVLMlKALELDSNLYRiGQS VFFRAGVI^HlEEERDLKrr

DVIiGFQACCRGYlARXAFA RQQQLTAMKVLQR CAAYXKLR WQWWRl.F^'rKVKPLLQV

SRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQEQEQLQAE^'I^'El.CAEAE

ELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEESARQ KLQLEKVTTcAKl l iEEQIlUjDQKCKLAKE LLEDRWEKnNLrEEEE SKSLAK

LKN HEA rrDLEERLRREEKQRQElEKTRRKLEGE>STDLSDQiAELQAQlAELK QLAK EEEL.QAAI.ARVEEEAAQ N ALKKIREEESQiSELQEDLESERASRNKAEKQ RDL⁽jEE

LEALKTELB.DT⁴'LD HSRP (SEQ ID NO:42)

MSDYSTGGPPPGPPPPAGGGGGAGGAGGGPPPGPPGAGDRGGGGPGGGGPGGGSAGGPSQPPGGGGPGIRK DAFADAVQRAROiAA lGGDAAITV NST^PD

EASE! (SEQ ID ΝΌ:43)

PNCARCG rVYPTEKVNCU)KFVraKACFHCErCKMTLNM NyKGYEKKPYCNAHypKQ SFTMVADTPBNUU-KQQSELQSQVRYlCEEFEK GKGFSVVAiyrPELQRl TQDQlSNI

KYHEEFEKSRMGPSGGEG EPERRI)g*QD

SPTAN! (SEQ TO NO:44)

DPSGVKVLE A.EDIQERRQQVL^'DRYHRFKELSTLRRQ^

LQ HQAFEAEVQANSGAiVKLDETGNLMISEGHFASETIRTRL^

V DWl DKAPlVi^'SEELGQDJJEHVBV^_^

LKGLALORQG EFGAAEVORFNRDVDETISWI E EQLMASDr>FC.RDLASVQALLRK}lEGLEROEAALED VKALCAE

ADRLQQSHPLSATQiOVKREELlTNVv QlRTLAA

ALLDRFIQEHKGEIDAHEDSFKSADESGQALLAAGHYASDEVREK^

EQVDNWMSKQFAPU.NEDLGDSLDSVBAUX^

NAJJHERAMRjRRAQlADSFHWJQFFRDSDEUCSWW

AGQ ElDVNHYAKbEV.<\/\R NEVlSLWK LLEATELKC KLREANQQQQF R^^'EDIELWLYEVEGHLASDDYG DLT VQNLQKKH AI .1 MAD VAAHQDRIDG FFIQ ARQ FQD AGHFDAENIKKKQEALV AR YEAI.KEPMVARKQKLADS LRLQQLF RDVEDEEWlREKEP-AAST RG DUGVQNLL KiiQALQAE-AGHEPW AVT¾

QKWEAL A ASQRRQDEEDSl ^AQOYFADANEAESVVMRE EPI\¾STDYGKDHDSAEALL KH¾lAiSDL.SAYGSSIQ ALREQAQSCRQQVAPTDDETGKELVLALYDYQEKSPRCT PAAYVKKUDPAQSASRJ^I EEQGSULRQEQmNQ^

FREANELQQWr EKEAALTSEEVGAl¾JiQVEVLQ KFD^^

yGMMPRDETDSKTASPW SARLMVFriA'ATFNSlKELNERWRSLQQLAEERSQLLGSAHEVQRFrTRDADETKEWIEE

Aί. ΊΌNYGH^ LASVQALQRKHEGFERDLAALGDK ^ΓNSLGErA RL^QSHPESAEDI ^EKCFΈLNQAVVSSLG RADQRKA

KLGDSHDLQRFLSDPRDLMSWiNGIRGf_^VSSDEI^KDVTGAEALLERHQEHRTEiDARAGTFQAPEQFGQQLLAHGHYAS

PfiI Q LDlLDQERADLEKAWVQRRM LDQCUELQLFHRDCEQAENVV^AREAFL TF;DKGDg*I.D

VPRBP (SEQ ID O:45)

MTTVVWVDSKAELTTLLEQWBKEHGSGQDMVPtLTRMSQLIE ETEEYRKGDPDPFDDR

HPGRADPECMLGHLLRILFKNDDFMNALVNAYVMTSREPPLNTAACRLLLDIMPGLETAV

VFQEKEGIYENLF1CWAREADQPLRTYS GLLGGAMENQD½AN YRDE SQLVAIVi.RRLR

ELQLQEVALRQENKRPSPRKUSSEPLLPLDEE,\VDMDYGD AVDWDGDQEEASGD EIS

FHLDSGHKTSSRVNS^'FTKPEDGGEKKN SA QGDRENFR AKQ LGFSSSDPDRMFVELS SSWSEMSPWVlGTNY^'rLYPM TAiEQRL .QYF:fPf-X^'iEYQEIFi^>5F QLGSREXM FYI

DLKQTODVLLTFEAL J-ILASLLLHN FATEFVAHGGVQKLLEIPRPS AATGVS CLYYL

S YNQDA ERVCMH PHN VLSDVVN YTLWL ECSH ASGCCHATMFFS JCFS FRA V LELFDRY DGLRRLV LiSTLEii.Nl£DQGALLSDDEIFASRQ HTCMALRfi.YFEAHLAiKLEQVK QSLQRTEGGiLVJIPQPPYKACSYTHEQIVE MEFLIEVGPAQLYWKPAEVFL LSCVQLL

LQL1S1ACNWKTYYARNDTVRFALDVLAE,TVVP IQLQLAESVDVLDEAGSTVSTV0IS lH..GVAEGEFFIHDAEIQ SALQlIlNCVCGPDSRiSSiGKflSG^'t^'PRRKl.PQ PKSSEK

TLAKMAV VVQSNNGr VLLSLLSIKMPiTDADQil^ALACKALVGLS SS^'PVRQiiS LPL

FSSCQlQQLM EPVLQD RSDHVKFCKYAAELiERVSG PLUGTDVSLARFQKADVVAQ

SRISFPEKElXLLiRKHLlS GLGETATVLT EADLPMTAASHSSAFrPVTAAASPVSEP

Rl?RLA,NGLAI7LGSS-iAAVGASAPSAP ^'AHPQPRPPQGPIALPGPSYAGNSPLiGRiSFI

RERPSPCNGR lRVLRQ SDHGAYSQSPAlK QLDRHEPSPPrLDSinEYLREQHARC

NPVATCPPFSLFTPHQCPEP QRRQAPINFTSRLNRRASFP YGOVDGGCFDRHLIFSR

RPISVFREANEDESGFTCCAPSARERFL LGTCTGQLKLYNVFSGQEEASYNCHNSAITH

LEPSRDGSLI TSATWSQPLSALWG S WDM HSFTEDHYVEFS HSQDRViG GrilA

HIYDIQTGN LLTLFNPDtANNY RNCATFNPTDDLVLNlXiVLWDVRSAQAlH FDKFNM

N!SGVFHPNGLEVIINTErWDLRTFHLUlTVPALDQCRVVFNHTGTVMYGAMLQADOEDD iAlEERMKSPFGSSFRTFNATDYKPIATlDVKRNIFDLCTDTKDCYl-AVHSNQGS DALNM

DTVCRl.YKVGRQRtAEDEDEEEDQEEEEQEEH)DDEDDDDI*»

NO:46

MATEFiPEPP AELQLPPPPPPGilYGAWAAQELQA LAEiGAPlOG REELVER-LQSYTRQ

TGWI, RPVlJGEDGDKAAPfTMSAQLPGIP PPPPLGI.PPLQPPPPPPPPPPGLGLGFP

MAHPPNLGPPPPLRVGEPVALSEEERlJ IJkQQQAAlXMQQBERAKQQGDHSt EHEU-EQ

Q RAAVLL-EQEROQEiAK GTPVPRPPQDMGQIGVRTPLGPRVAAPVGPVGFrP VLP G

APVPRP GPPPPPGDENREMDDPSVGP iPQALE lLQLKESRQEEMNSQOEEEEMETDA

RSSLGQSASETEEDTVSVSKKEK R RRNR KK. PQRVRGVSSESSGDREKDSTRSRGS

DSPAADVEiEYVTEEPEiYEPNFiFF RJFEAF LTDDV EKE EPEKLDKLENSAAPK

KKGFEEEHKDSDDDSSDDEQE KPEAP LSK KLRR NRFTVAELKQLVARPDVVE HDV

TAQDPKELVHL ATP_^ SVPVPRFWeF RKYLQGKRGiEKPPFELPDFI RTGIQE REAl,

QEKEEQ TM SKMREKVRPKLMGKTDiDYQKLHDAf FKWQ7T P LT1HGD!.YYEG EFETR

L E PGDLSDELRiSLGMPVGPNAHKVPPPWLIA QRYGPPPSYPNL iPGLNSPlPES

CSPGYHAGGWGKPPVDETGKPLYGDVFGTNAAEFQTKTEEEEIDRTPWGELEPSDEESSE

EEEEEESDEOKTOE GFFfPADSGLtTPGGFSSyPAG E PELiELR KIEEA DGSET

PQLFTVEPEKR ATVGGAMMGSTHlYDMS VMSRKGPAPELQGVEVAtAPEELELDPMA

TQKYEEHVREQQ AQ VEKEDFS* D NCL (SEQ !D ^" O: 7)

S*EEEA ETTPA GKKAAKVVPVXAKNVAEDEDEEE

DDEDEDDDDDEDDBDDDDEDDEEEEEEEEEEPVKEAPG RKKENIAKQ AAPBA KQKVBG

TEPTTAFN FVGNLNFX SAPEL TGISDVFA NDLAVVDVRIGMTR GYVDFESAEDL

E ALELTGLKVFGNEI LEKPKGKDS KE DARTLLAKNLPYKV QDEL EVFEDAAEIR

LVSKDCrKSKGIAYlEFKTEADAEKTFEEKQGTElDGRSlSLYYTGE GQNQDYRGGKNST

VVSGES TLVLS LSYSATEETLQEVFE ATFI VPQNQNGKSKGYAFJEFA.SFEDAii.EAL SCN RElEG AIRIELQGPRGSPNARSQPS 'iLFV GLSEDTTEKTL ESFDGSVRARl

VTD ETGSS GFGFVDFNSEEDAKAA EAMEDGEIDGKKVTLDWAKPKGEGGEGGRGGGR

GGFGGRGGGRGGRGGFGGRGRGGFGGRGGFRGGRGGGGDH PQG TKFE

ARMC10 (SEQ ID N0:4S)

GS*YDOVLNAEQLQKLLYLLESTEDPVUERAUT

LG N'AAFSVNQAllRELGGIPiVA Ki HS QSiKE ALNALNNLSVNVENQIKIKiYIS

QVCEDVFSGPLNSAVQI^GLTiXTO TVTODHQH EHSyiTDEFQVI-LTGNGNTKVQVLK 1-LLNESENPA TEGU.RAQVDSSFLSLYDSHVAKEILl.RVLTEFQNtKNCLKlEGHLAVQ PTFTEGSLFFLLHGEECAQ IRALVDroiDAEV EKVVTilPKI

KUP93 (SEQID G:49)

FT*QESEPS Y1S D VGPPGR SSLDN fE¾t4YARQIYiYNE l\¾GHLQP LVDLCASVAELDDKStSD?vIWT VKQ TDVLLTPATD ALKNRSSVEVRMEFVRQAEAYLEQSY NYTLVTVFG EHQAQLGGVPGTYQLVRSFLNiK LPAPLPGLQDGEVEGHPVWALIVYCMRCGDLLAASQVVNRAQHQLGEFKTWFQEYMNSKD RRLSPATENKLRLHYRRALRNNT0PYKRAVYCIIGRCDVTDNQSEVADKTEDYEW1J LNQ VCFODDGTSSPQDRLTLSQFQKQELEDYGESHFTVNQQPFLYFQVLFLTAQFBAAVAf-LP RMERLRCFIAVHVALVLFEL LLL SSGQSAQLLSHEPGOPPCLRRL FVRLL LYTRKFE STDPR^LQYFYPLRDEKDSQGENMFLRCVSELVIESREFDMJLGKLENDGSR PGVID FTSDTKPi VASVAEN GLFEEAA LYDLA AD Vl.ELMNKI.L.SPVA'PQlSAPQSN E^,RL N ALSLAERYRAQGISANKFVDSTFYUXDLITFFDEYHSGHiDRAFDlIF.RL F.V PENQESVEERVAAFRNFSDEiraNL.3EVLI.A7MNILFTQF RLKGTSPSSSSiU^,QRVIED RDSQLRSQ ARTLf! FAG IPYR rSGDTNA RL VQ E V LMN FNBPi (SEQ I^'D NO:50)

GSY*TEEQSQESEMKVLATDFDDEFDD EEPLPA SGTCK ALYTFEGQNEGTIS VVEGE'f LY VJEEDKGDGWTRIRRN EDEEGY VPTS Y VEVCLD AKGAKTY1 C i (SEQ ID 0:5i)

SAS*PADDSFVDPGERL YDLN MP A Y VKFN YMA E REDE)..SLi GT VIVME CSDGW\VRGSYNGQVG FPSNYV^'reECDSPI..GDHVGSi.,SE LA AVV NtNT( )VLHVVQALWreSSNDEBLNFE GDVKTOVIE PENDPE CRKl GMVG LVP NYVTVMQNNPLTSGLEPS PQCDYIRPSLTGKFAGNPWYY'GKVTRHQAEMAL ERG HEGD JROSESSPNDFSVSLKAQG HFKVQL ETVYCIGQRKFST EELVEHYK AP IF TSEQGP.KL Y LVKHLS

EIF3B (SEQ IE> O:52)

DVS*EE£LLGDVLK-DRPQEA DGlDSVWVDNWQVGPDREEKLKNVIHKjFS FGKrrNDFYPEEPG TKGYlFLEYASP AIlAVDAVK ADGY LD QHTFRWLFTrJFD YMTiSDEWDlPEKQPF DLGNERYWLEEA ECRDQYSViFESGDRTSIFWNDVKDPVSiEERARWTETYVRWSPKGTYLATFHQRGiAE GGE F QIQRFSHQGVQLlDFSPCERYLVTFSPLMDTQDDPQAIlfWDlLTGHKKRGFHC ESSAHWPlFKWSHDGKFPARMTLDTl_^SIYErPS GLLDK SL ISGIKDFS SPGGNilA FWVPED DJPARVTLMQUPTRQEIRVRNLFNVVDCKLHWQ NGDYLCVKVDRTP GTQGV VTNFEJFRMRE QVPVD EMKETUAFAWEPNGSKFAVLHGEAPRISVSFYHVKNNGKl ELIKMFD QQA TIFV SPQGQFVVLAGLRSMNGALAFVDTSDCTV N'LAEHYMASDVEWD FrGRYVVTSVSWWSHKVDNAYWLWTFQGRLLQKNNKDRFCQLEWRPRPP LLSQBQl Ql K DLKKYSKO'^Q DRLSQSKAS EI 'ERF^TMMEDFR YRKMAQELYMEQKNERLEERG GTOTOELDSNVDDWEEETreFTVTEEHPLGIRSOLEHCAQPCVI.WSRGRPAGSRVTPAS SECSLALDCDCAWJLPLRH!FVPFSPWCLQWGI

CHD4 (SEQ ID NO:53)

GYE *DHQDYCEVCQQGGEIiLCDTCPRAYH VCLDPDMEKAPEGKWSCPHCEKEGIQ WEA EDNSEGEE .EEVGGDLEEEDDMHMEFCRVC DGGELLCCDTCPSSYHTHCLNPPL PEiPNfGEWLCPRCTCPALKGKVQKlLW WGQPPSPTPVPRPPDADPNTPSPKPLEGRPE

RQFFV QGMSYWHCSWVSBLQDJLHCQVMFRNYQRKNDMDEPPSGDFGGDEE SR R N

KDPKFAEM F.RFYRYGIKPEW MiHRILNHSVD KGHVHYLIKWRDLPYTXJASWESED^'Yt^"

IQDYPU^? QSYAVNHR£LMRG EGRPGKKLKKV LRK.1.ERPPETPTVDP1^'VXYERQPEY^'J_JD

.^TGGTLHPYQMEGLNWLRKSWAO^TDT!LADEJvlGLGKTVQTAVFLYSLYKEGHS GPFLV

SAPI^IINWEREFE TWAPDMYVV7YVGDKDSRAI]RE EFSFEDNA1RGGKKASRMKKE

ASVKFFiVi jrSYELniD lfliLGSIDVv'ACLn'DEAHRLKN QSKFFRVI.NGYSLQHKLLL

TGTPLQ NLEE ¾LLNFLTPERFH LEGFLEEFADL¾KEDQI IHDMLGPFIMLRRL A

DVFK MPSKTr iVRVEESP Q YYKYlL RNFEALNARGGG QVSLtKVVMDL iaX

MPYLFPVAAMEAPK fP G YDGSALffiASG i XJ..Q MLKNf. £GGMRV1JFSQMT MLD

LLEDFLEHEGYKYERIDGGlTGNMRQEAIDRFNAPGAQQFeFLLSTRAGGLGJNLATADT

VIiYDSDWNPH DIQAFSRAEiRiGQNK VMiYRFVTR.ASVEERiTQVAK M l,THLVVR

PGLGS ^'rGSMSKQELDDiL FG ^'EELF DEATDGGGD KEGEDSSVlHYDDKAIERLLDR

NQrF;rEDTELQGMNEYLSSFKVAQYVWEEE GEEEEVERF;il QEF;SVDPD\'WE LLRH

HYEQQQEDIARNI^KGKRIR QVNYNDGSQEDRGVCGRPRPPP GRSTRAVGPAHLPSLP

PD QDDQSDNQSDYSVASEEGDEDFDERSEAPRRPSRKGLRNDKDKPIiPPULARVGGNlE

VLGF ARQRKAFLNAIMRYG PPQDAFTi JWtVRDLRGKSEKEF AYVSLFMRHl^EPGA

DGAETFADGVPREGlJSRQHVLTRlGVMSLIRKKVQEreHVNGRWS PELAEVEENKK SQ

PGSPSPKTPTPSTPGDTQPNTPAPVPPAEDGi lEENSL EEESIEGEKEVKSTAPETAI

ECTQAPAPASEDEKVVVEPPEGEE VEKAEVKERTEEPMETEPKGAADVEKVEEKSAIDi.

TPfVVEDKEE EEEEKKEV tQNGETP DLNDEKQ KNI QRFMFNIADGGFTELHSLW

Q EERAATVT ^' YEIWHRRKDYWLLAGII HGYARWQDIQNDPRYAILNEPFKGEMNRG

NFLEi N FLARRFKLLEQATA'IEEQLRRAAYLNMSEDPSHPSM\i HKFAEVECLAESH

QHI^ ESMAGN PANAVLil VLKQLEELLSDMKADVTRlJPA IARlPPVAVTfll/iMSERNl

LSRLANRAPEPTPQQVAQQQ

NO:S4}

GRAT*PSENLVPSSARVDKPPSVLPYFNRPPSALPLMGLPPP-

PffPPPPLSSSFGVPPPPPGmYQHL PPPPRLPPHLAVPPPGAiPPALHLNPAFFPPPN

AWGPPPDTYMKASAPYNHHGSRDSGPPPS^'JVSEAEFEDIMKRKRAISSSAISKAVSGAS

AGDYSDAIETLIJAlAVIKQSRVANDERCRVLISSi OCLHGlEA SYSVGASGSSSRKR HRSRERSPSRSRESSRRHRDLLHNEDRHDDYFQERNREHERHRDRERDRHH PLERHC! (SEQ LO NO.SS)

AALS*ffl,EnXEG

K^'fSTII iDiTSIPEL DYIKW P LTLKGVKQYWCTFKDTSISCYKSKEESSGTPAHQ

MNLRGCEVTPDraiSGQKFK^KLI^PVAEG NEm'LRCDNEKQYAHW^iAACRt.ASKGK™

Ai SSY¾LKVQNii.S}'I.KMQHLNPDPQLiPE¾rr-i^'DJTPECLVSPRYLKKY NKQiTARIL

EAHQNVAQMSUEA MRFiQAWQSli>EJ¾iTflFIARFQGG EELiGiAY RLIRMDAST

GDA1KTWRFSN MKGWN VNWEIK VTVEFADEVRLS F ICTEVDCKVVKEFIGGYIFLSTRA

KDQNESL0EEMFYKJLTSGWV

HTATSFl (SEQ ID NG:56)

AGGEPDS*LGQQPTDTPYEWDI.D KAV¥F

P ITEDHA^'^QANYGFSNDGASSSTANVEDVHARTAEEPPQB APEP DAR KGEKRKA SGWWVEEDR TOVYVSOLPPDn-Vt⁾EFlQl.MSKFG«MR0POTEEF V LY DNQGNL GDGLCCYLKRESVELALKLLDEDEiRGYKLHVEVAKFQL GEYDASKKKKKC PYKK t.

SMQQKQLDWRPERRAGPSRWHFJRWI NMFHP DFEDDPLVLNEIREDLRVECSKFGQ

IR LLLFDRHPDGVASVSFRDPEEADYCIQTLDGRWFGGRQlTAQAWOGTTDYQVEErSR

EMERLRGWEAFLNAPEA RGLRRSDSVSASERAGPSRARHFSEFiPSTSK NAQE^'i^'A^'rG

AFEEPJDE FEKTEDGGEPEEGASENNA ESSPE EAEEGCPEKESEEGCPKRGFEGSC

SQKESEEGNPVRGSEEDSP KESKK TLKNDCEENGLAKESEDDLNKESEEEVGPTKESE

EDDSEKESDEDGSEKQSEDGSEREFEE GLEKDLDEEGSEKELHENVLDKELEE DSENS

EFEDDGSBKVLDEEGSEREFDEDSDE EEEEDTYEKVF DESDE EDEEYADEKGLEAAD

K AEEODADEKi.FEESDD EDEDADGKEVE0ADEKl.FEDDDSNEKl.FDEEEDSSEKLFDD

SDERGTLGGFGSVEEGPLSTGSSFILSSDDDDDDI

THiL (SEQ JDNO:S7>

GGQQEDDS *GEGEDD AE V QQECLH F

STROYDviEPSIFN U RYFQAGGSPENVIQU^E YTAVAQTVNLWF^V QTGVEPVQV

QETVE HLK LLiKHFDPRRADSIFTEEGETPAWLEQ IAH^'FTVv'RDLFYKLAEAHPDCL tNFTV LlSDAGYQGErrSVSTACQQLEVFSRVLRTSLATlLDGGEENLEKNLPEFAK V

CHGEHTYLFAQAMMSVLAQEEQGGSAVRRIAQEVQRFAQEKGHDASQITLALGTAASYPR

ACQALGAMLSKGAiA'PADrrVLFK FI^'S DPPPVELSRVPAFLDLF QSLF PGARi^' QD HKHKYmiLAYAASVVETW NKRVSl KDEIJ STSKAVETVHNLCCNENKGASBLVAEL

SI YQCSRFP\^A GVL VVVDWTVSEPRYFQLQTDS-'.TPVHLALLDEiSTCHQLLHPQVLQ LtV LFE EHSQLDVMEQiEIJKKTUJJRMVHLLSRGyVLPVVSY!RKCLEKLDTDISUR

Yl^;VTEVI^,DViiVPPYTSDf^;VQLFLPiLE DSIAGTra^"EGEHDPVTEFlAHCKSNFtMVN

ST 39 (SEQ ID NO;58)

MAEPSGSPVHVQLPQQAAPVTAAAAAAPAAATAAPAPAAPAAPAPAPAPAPAAQAYGWPi

CRDAYELQEVIGSGATAVVQAAi..C PRQERVAl RiNLE CQ^'{SMDEU,KElQAMSQCSH

PNVVTYYTSFVV DELWLVMKLLSGGSMLDlI YrV RGEHKNGVl.EEA51ATIUCEVUE

Gil Yi iRNGQmRDL AGNiLLGEDGSVQjADFGVSAFi.ATGGDVTRN VR TFVGTPCW

MAPEV EQVRGYDF ADMWSFGrrAIin^ATGAAPYH YPPM VLMLTLQNDPPTLETGVE

D EMMK .YGKSFRKLLSLCLQKDPS Ri'TAAELLKC EFQKA NREYLIEftl_^LTRTPDIA

QRA . VRRVPGSSGHLHKTEDGDWE'A'S*DDEMD

TRSM2S (SEQ ID NO;59)

MAASAAAASAAAASAASGSPGPGEGSAGGEIVRSTAPSAAASASASAAASSPAGGGAEALE LLEHCGVCRERLRPEREPRIXPCLHSACSACLGPAAPAAANSSGDGGAAGDGTVVDCPVC

KQQCFSKDlVENYFMRDSGS AATDAQDANQCCTSCEDNAPATSYCVECSEPlvCETCVEA

HQRV YIXDHTVRSTGPA SRDGERTVYCNVH HEPLVLFCESCDTI CRDCQL AHKDH QYQFLEDAVRNQR LLASEV RLGDKHATLQKS^'i^'KEVRSSIRQVSDVQKRVQVDV MAIL QlM ELNKRGRVLVNDAQ VTEGQQE LERQHWT T IQKlIQEHE.RPASWALESDNiNTA LLLS KLIYFQLHRAlJK IVDPVEPHG-iMKFQWDLNA T SAEAFGKrVAERPGTNSTGP

APMAPPRAPGPLSKQGSGSSQPMFJVQEGYGFGSGDDPYSSAEPi-SVSGV RSRSGEGEVSG Urf RVSLERLDLDLTADSOPPVFKVFPGSTTEDYNLIVIERGAAAAATGQPGTAPAG

TPGAPPLAGMAW EEE^'rEAAiGAPPTATEGPET PVLMALAEGPGAEGPRLASPSGSTS

SGLEVVAPEGTSAPGGGPGTLDDSAIICRVCQKPGDLV CNQCEFCFHLDCHLPALQDVP

GEEWSCSLCHVLPDL EEDGSLS"1.DGAD

HCLS1 (SEQ ID O:60)

MWKSVVGHDVSVSVETQGDDWDTDPDFVNDlSE EQRWGA IEGSGRTEill lHQLRNK

VSEEHDVLR EMESOP ASHGYGGRF GVERiJRMD SAVGHEYVAEVE HS*SQTD CASP3(SEQlDNO:6!>

MAKRNAE EL'rDRNWDQE EAEEVGTES ,\£EEVL RA! A RRNVGFESD^'fGGAF G F GEVVPSGGGRFSGFGSGAGG PLEGLSNGN llSAPPFASAkAAADPK VAFGSFAA G PTJL VDK VSNPKTN GDSQQPSS SOLAS SK AC VGN AYHKQL AAL CS VR.D I VKH V N T PL CDL^'iTiP D YE Y^'LA NIEQQHGN SGRN SESES ^"N VAAE TQSPS LFGS T .LQQE.STFLFHG NKTEDT*PD

RBI (SEQ ID NO 63)

PPK^'I R TAATAAAAAAEPP APPPPPPPEEDPEQDSGPEDLPL VRLEFEETEEPDFTAL CQ L JPDHVRERAWLTWE VSSVDGVLGOYlQKKKEUWOIGlFIAAVDLDEMSFfFTE!. Q NffiiSVHK3n¾LiJCEIDTSTKVDNA SIU.L KYDVLFALreKLERTCEimTQPSSS IS7¾INSAl.VL VS rrFlXAKCEVtQMEDIXVISFQLMLCVLDYFIKLSPPMLL EPYK TAVIPINGSPRTPRRGQNKSARIAKQLE DTRJIEVLCKEHECNIDEVKNYYF NF!PFM NSLGLVISNGLPEVENLS RYEElYL NKDLDARLFLDHD ^LQrDS^iD

EFE1B2 (SEQfD O:64)

MGFGDLKSPAGLQVL DYL.ADKSYiEGYVPSQADVAVFEAVSSPPPADLCHALRWYNHiK SYE EKASLPGV KALOKYOPADVEDTTGSGATDSKDDDDrDLFGSDDEEESEEAKRLRE

ERLAQYES KAKKPALVAKSSiLLi)V P DDE *D

RSRCi (SEQtDNO:65)

MGRRSSDTEEESRS RKKK!IRRRSSSSSSSDSRTYSRKKGGRKSRSKSRSWSRDLQPRSH

SYDRRBKHR3SSSSSYGSRRKRSRSRSRGRG SYRVQRSRSKSRTRRSRSRPRLRSHSRS

SERSSHRRTRSRSRDRBRRKGRPKE RE EKDKG DKELHNIKRGESGNIKAGLEHLPPA

EQAlvARLQLVLEAAAKADFALKAKEANEEEAKRR E_^EL^QATLA- ^V RVKEiEAIES^D

Η2ΑΡΎ (SEQ iD.NO:66)

S*TTEGTPA

DGFTVLSTK:SLFLGQRLNLiHSEIS LAGFEVEAirNPTNADtt⁾L DJjLGN^'iI.EKKGG E FVEAVl.F.LR K' GPLEVAGAAVSAGHGLPAKFVIHCNSPVWGAD CEELl.F.K^'i^'VKNCLAi. ADDK LKSJAFPSrGSGRNGFPKOTAAQLlIJ AlSSYFVST SSSI TVY VLPDSESlG

lYVQEMAKLDA

SPTANi (SEQ ID NO:67)

S*LDSVEAL!K HEDFD AniVQEE IAAlQAFADOElAAGHYA GDISSRaNEVEDRWRRi. AO IEKRS LG£SQTLQQ

FSRDVDEIEAWISF. LQTASDESYKDniiiQlJPSSFS HQKHQAFEAULHAN ¾R5RGViDMGNSiIERGACAGSEDAVKA

R \ALADQWQFLVQKSAEKSQ LKEA KQQNFNTG1KDFDFW3.SEVEAE!,ASEDYG IUASVN LL KHQELEADISAH

EDRDiDLNSQADSLMTSSAFD7SQVKD RD1 NGRFQK.HiSMAASRRA ENESHRLHQFFRDMDDBESWlK.EKKLLVGSE

DYGRDLTGVQNl.R HKRl L\ELAAHEPAIQGVLDlXr LSDDN ^'iG EE!OijRLAQFVEH KEL QLAAARGQRLEES

LEYQQFVANVBEEEAWFNEKMTLVASEDYGDTLAAIQ^

S MKGLNG VSDLE AAAQRKAJ DENSAFLQF WKADVVPI! IGEKENSLKTDDYGRDLSSVQTLUXQERFDAGLQ AFQOEGIANITALKDQLEAAKHVQSKAIEARHASI_^M RWSQLLANSAAR K LLEAQSHFRKVEDLFLTFAKKASAFNS FENAEEDLTDPVRCNSLBEIKALREAHDAFRSSLSSAQADFNQ^{^}^

KERELBI ⁾KEQRRQEE><DKLRQEFAQRA¾ FHQ

DLGAAMEEALILDN YTEHSTVGLAQQWUQLDQ ^

NHQEPXSCUISIGYDLPMVEEGEPDPEFEAHJSTVDPNTI^

KEELYQNLTREQAD YCVSifMKPYVDGKGRFXP^'iAFDYVEF^'PRSI..F^;VN

USP14(SEQ ID NO:68)

S*SSASAATPSK

SUDQFFGVEFET^'m CTESEEEEVlXG I-NQLQLSCFiNQEVKYLFTGL LRL.QEEri^'K

QSPTLORNALYI SSK{SRLPAYlriQMVRFFY EKESV AKVLKDV FPL LDMYELCf

PELQEKMVSFRS FKDLEDKKVSQQP TSDK SSPQKEV V^'EPFSfADDSGSN CGYYDL

QAVUHQGRSSSSGHYVSWVKRKQDEWIKFDDDKVS1VTPED1LRLSGGGDWHIAYVU.Y

GPRRVE1MEEES BQ

NCL (SEQ II>NO:69)

GS'VRARI

VTDRElOSS GFGFV FNSEEDA AAKEA EDGEfDGNKVTLDWA P GEGGFGGRGGGR GGFGGRGGGRGGRGGFGGRGRGGFGGRGGFRGGRGGGGDHKPQGKKTKFE

NUP93 (SEQ ID NO:?0

FT*QESEPSYiSDVGPPGRSSLDN lEMAYARQniY EK!VNGHLQPNLVDLCASVAEL DKSiSDMWT VKQ TDVLLTPATD AL NRSSVEVRMEFVRQALAYLEQSYKN'Yl^'LVTVFG TJIQAQLGGVPGTYQEVRSFLNiK tPAPL JI JDGEVEGHPVWALlYYCMRCGDliAASQVVNRAQHQLGEF TWFQEY NS D

RrLSPATENKLRLHYR8ALRNNTD?YK AVYClK^jRCDVTDNQSEVADKTEDYLWLKLN

VCFDDOGTSSPQDRLTLSQFQKQLLEDYGKSHFTV QQPFLYFQVEFi..TAQFEAAVAf^'LF

RMERLRCHA ¾VAEVLFELKtlJ_.KSSGQSAQLl.SFlF.PGDPPCLRrLNFVRl.L. t.Y^"I^'RKFE STDPREAU¾YFYFLRDE DSQGEN }¾.RCVSBl^ BSREFDMiLGKLE DGS5¾KPGViDK

FrSDT PIJN VASVAE^ GtFEEAAKLYDLAfC AD VLELMNKLLSPVVPQJSAPQSN

ERL MALSiAERYRAQGlSA FVDSTFYlELOLriFFDEYifSGHlDRAf^'DIiERL LV

PL QESVEERVAAFRNFSDE!RHNLSEVLl.ATMN LP^"[QFKRi.KGTSPSSSSRPQRV!ED

RDSQLRSQARTLITFAGMIPYRTSGD^'f^'NARIVQ EVLMFi

USP!4(SEQ ID N0.71)

SS*SASAATPSKRK

SLIDQFFXVEFETTM CTESEEEEWKG JjNQLQLSCFlNQEVKYLFTGX.Ki.Rl,QEEl K Q¾P1I.QR«ALY1KSS 1S11LPAYLT1QMVRFFYKE £SVNAKVL DVKFPLMLD YELC^'J^' PELQEK VSFRSKFKDLED VNQQPN SD SSPQ EVKYEPFSFADDiGSNNCGYYDL QAV7.THQGRSSSSGHYVSWVXRKQDEWIKFDDD VSTVTPF ⁾lLRLSGGGDWi-IIAYVLLY

GPRRVEIMEEESEQ

ARMCiO (SEQ ID NO:72)

GSY*DDVI..NA£Qi.Q LLYLLESTEDPViiE AUT LGNNAAFSV QAIIRELGGIPrVAN INHSNQSlKF AL AL NLSVNVENQIKlKIYIS QVCEDVFSGPL SAVQLAGLTLlTNMTVTNDiiQHMlHSYITDLFQVLL^'fG GNTKVQVLK LLLNl^ENPAMTEGLLRAQVDSSFLSLYDSHVAKEILLRVLTLFQNIKNCLKIEGHLAVQ PTFTEGSLFFLLHGEECAQKIRALVDHH0AEV EKVVTIIP I

SF3B2 (SEQ ID NO'73)

ARS'SI KJSASETEED-mVSKKE NR RRNRK KKKPQRVRGVSSESSGDREKDSTRSRGS

DSP/u\DVEffiYVTEEPEm'? FIfF RIFEAFKLTDDV .E EKEPEKLDKLE SAAPK KKGFEBEilKDSDDDSSDDEQE PEAP LSKKKLRRMNRFFVAEUKQLVARPDVVEMHDV

1¾QDPKLLVHl.KATRNSVPVPRHWCFKRKYLQGKRG!EKPPFELPDF[KR-rGlQEMREAL

QE EEQKl^ KS MRE VRP GKl IDYQKLtlDAFF QTKP L SlGDLYYEG EFETR

L EKKPGDLSDELRiSl MPVGPNAH VPPPWLiAMQRYGPPPSYPNLK!PGLNSPIPES

CSFGYHAGGWG PPVDETGKPLYGDVFG^'1^" AAEFQTKTEEEEIDR PWGELEPSDEESSE

EEEEEESDED PDETGMTPADSGLTrPGGFSSVPAGMETPEEIELRK KIEEAMDGSE

PQLFTVLPE RTATVGGAMMGSTHiYDMSTV SR GPAPELQGVEVALAPEELELDPMA

TQKYEEHVREQQAQVE EDFSD VAEilAAKQ QKKRKAQf'QDSRGGSKKYREF F US? 14 (SEQ iD NO-74)

SSS*ASAATPS KK.

SLIDQFFGVEFETTM C ESEEEEVTKGKE QLQL-SCFINQEV YLFTGLKiRLQEEIT QSPTLQRNALYIKJSSKISRLPAYUIQ VRFTYKE ESTO

PEI.QE MVSF S F DLED KVNQQP I^'SD KSSPQ EVKYEFFSFADDiGS NCGYYDi.. OAVLTFiQGRSSSSGHYVS VK KQDEWI FDDDKVSiVTPEDIERLSGGGDWHIAYVLi.Y GPRRVETMEEESEQ

NCL (SEQ LD NO:75)

DEEDDS*EEEAMETTPA G KAA WPV A VAEDEDKEE DDEDEDDDDDEDDEDDDOEDDEEEEEEEEEEPVKEAPG RKKE AKQ AAPEA QKVEG

TEFITAFNLFVGNLNFNKSAPELKTGiSDVFA N LAVVDVRIGMTR FGYVDFESAEDL

E ALELTGl VFG El LEKP GKDSKKERDAR-rii.AKNLPYKVTQDEi EVFEDAAE!R

LVS DGKS GIAYiEF TEADAEKTFEE OGTEIDGRSISLYyTGEKGQNQDYRGG NSr

WSGESKTLVLSNI.SYSATEETLQEVFEKATFiKVPQNQNGKS GYAFiEFASFEDA EAL

NSC KREIEGRAIRLELQGPRGSPNARSQPS. TEFVKGLSEDTTEETLKESFIGSVRAR1

V DRETGSSKGFGFVDniSEEDAKAA EAMEDGEiDGN VTLDWA P GEGGPGGRGGGR

GGFGGRGGGRGGRGGFGGRGRGGFGGRGGFRGGRGGGGDH PQG TKFE

RBM39 (SEQ fD NO:76)

ASSASSn-LDSDBLERTG!DLGTiGRLQL ARlAEGTGLQIPPAAQQALQMSGSLAFGAVAEFSFVfDLQTRLSQQ-rEASAlAAAASVQP LATQCFQLSNXiraPQTEEEVG DTEIKDDVlEECNKHGGVUlIYVDKNSAQGNVYVKCPS

!AAAiA A V AUiGRWFAGKMITA AYVPLPTYH NLFPDS MTATQU. SRR

Claims

WHAT IS CLAIMED IS:

L An isolated polypeptide comprising a caspase cleaved terminus and a phosphorylated residue within abou 15 amino acids of said terminus.

2. The polypeptide of claim I, wherein said caspase cleavage is after an aspartate residue.

3. The polypeptide of claim I, wherein the phosphorylated residue is within about 6, 5, 4 or 3 amino acids of said terminus.

4. The polypeptide of claim 1 , wherein said terminus is C-terminus of said polypeptide.

5. The polypeptide of claim 4, comprises a sequence that is the same as or substantially identical to a sequence selected from the group consisting of SEQ ID NOs:22-46 and 58-65.

6. The polypeptide of claim. 1 , wherein said terminus is N-terminus of said polypeptide. 7, The polypeptide of claim 6, comprises a sequence that is the same as or substantially identical to a sequence selected from the group consisting of SEQ ID NOs:47-57 and 66-76.

8. An isolated polypeptide, comprising (a) a caspase cleaved terminus, (b) a phosphorylated residue that is within about 15 amino acids of said caspase cleaved terminus, and (c) a second terminus that is generated by cleavage of a second protease.

9. The polypeptide of claim 8, wherein the second protease is trypsin.

10. The polypeptide of claim 8, wherein said caspase cleaved terminus is C-terminus, and said second terminus is N-terminus. Π, The polypeptide of claim 1.0, comprising a sequence that is the same as or substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 77-101.

12. The polypeptide of claim 8, wherein said caspase cleaved terminus is N-terminus, and said second terminus is C-terminus.

13. The polypeptide of claim 12, comprising a sequence that is the same as or substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 102-1 12.

14. A method for monitoring apoptotic activity of a cell, comprising detecting and quantifying an apoptosls biomarker in the ceil, wherein the apoptosis biomarker is a polypeptide comprising a caspase cleaved terminus and a

phosphorylated residue within about 1 5 amino acids of the terminus.

15. The method of claim 14, wherein the apoptosis biomarker is a polypeptide comprising a sequence that is substantially identical to a sequence selected from the group consisting of SEQ ID NQs: 22-1 12.

16. The method of claim 14, wherein the cell is present in a biological sample obtained from a subject.

17. The method of claim 16, wherein the subject suffers from a tumor,

38. A method for identifying an apoptosis biomarker in a cell, comprising (a) inducing apoptosis of the cell, (b) detecting in the cell a polypeptide comprising a caspase cleaved terminus and a phosphorylated residue within about 15 amino acids of the terminus, wherein absence of said polypeptide in a non-apoptoiic control cell identifies said polypeptide as an apoptosis biomarker.

19. The method of claim 1 8, wherein said polypeptide is identified by proteomic analysis of caspase cleavage and phosphorylation of proteins in the cell.

20. The method of claim 19, wherein the proteomic analysis is via q.P-

PROTGMAP.