WO2013192298A1 - Biomarqueurs de l'apoptose - Google Patents

Biomarqueurs de l'apoptose Download PDF

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Publication number
WO2013192298A1
WO2013192298A1 PCT/US2013/046554 US2013046554W WO2013192298A1 WO 2013192298 A1 WO2013192298 A1 WO 2013192298A1 US 2013046554 W US2013046554 W US 2013046554W WO 2013192298 A1 WO2013192298 A1 WO 2013192298A1
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Prior art keywords
caspase
terminus
apoptosis
polypeptide
seq
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PCT/US2013/046554
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English (en)
Inventor
Melissa DIX
Gabriel M. SIMON
Benjamin F. Cravatt
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The Scripps Research Institute
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96402—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals
    • G01N2333/96405—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general
    • G01N2333/96408—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general with EC number
    • G01N2333/96413—Cysteine endopeptidases (3.4.22)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • 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.
  • caspases cysteine proteases
  • 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.
  • apoptosis 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.
  • 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.
  • the caspase cleaved terminus in these polypeptides is generated by a caspase after an aspartate residue.
  • 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.
  • 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.
  • 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.
  • the polypeptides can have a second terminus that is generated by trypsin cleavage.
  • 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.
  • 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,
  • 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.
  • 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.
  • the DC! to be examined is present in a biological sample obtained from a subject.
  • 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.
  • the novel apoptosis biomarkers are identified by proteomic analysis of caspase cleavage and phosphorylation of proteins in the cell.
  • the proteomic analysis is performed via the qP-PROTQMAP method described herein,
  • FIG. 1 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
  • 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).
  • 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
  • FIGS 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.
  • 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.
  • FIG. 4A-4C show in vitro substrate assays indicating linearity of product formation over the tested range of substrate concentrations.
  • SP3B2 peptide substrates EQQAQVEKBDFSDMVAEHAAK (SEQ ID NO:4) and
  • HCLS1 peptide substrates SAVGHEYVAEVEKHSSQTDAAK (SEQ ID NO:7) and
  • FIGS 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.
  • Argl 77 in caspase-8 interacts with the
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • a reference molecule e.g., an apoptosis biomarker
  • 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
  • caspases 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
  • CASP3, CASP6, and CASP7 cleave other protein substrates within the cell, to trigger the apoptotic process.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • 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.
  • a . "derivative" of a reference molecule 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.
  • recombinant host ceil 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.
  • 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.
  • 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.
  • 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.
  • 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.ejon 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • the peptides or polypeptides comprise from about 10 amino acid residues to about 200 amino acid residues in length.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • orthoiogs or “homologs” refers to polypeptides that share substantia! sequence identity and have the same or similar function from different species or organisms.
  • a specific apoptosis biomarker protein e.g., SF3B2
  • SF3B2 apoptosis biomarker protein from human, rabbit, rat, mouse and many other animal species are orthologs due to the similarities in their sequences and functions.
  • 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
  • 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).
  • 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.
  • 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.
  • 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.
  • the inventors uncovered several ways that phosphorylation and proteolytic pathways intersect in apoptotic ceils.
  • caspase recognition sites e.g., P3 position
  • 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.
  • 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.
  • 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.
  • DNA-PK DNA-PK
  • 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 caspase cleaved terminus can be either the N-terminus or the C -terminus of the polypeptide.
  • the polypeptide comprises the N-terminal fragment of a caspase cieaved protein, which accordingly has a caspase cieaved C-terminus.
  • the polypeptide comprises the C-terminal fragment of the cleaved protein, which therefore has a caspase cleaved N-terminus.
  • 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.
  • 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.
  • 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.
  • the apoptosis biomarkers of the invention can comprise a caspase cleaved C -terminus, a caspase cleaved N-terminus or both.
  • the polypeptide will usually have an aspartate residue (PI ) at its C-terminus.
  • PI aspartate residue
  • 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.
  • the phosphorylated residue is present at P2, P3, P4, P5, P6, P7, P8, P9 or P10 position.
  • 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).
  • the phosphorylated residue can be the residue located at any position from ⁇ to P20' or P25 ⁇
  • 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 ⁇ ⁇ ', P7 ⁇ P8 ⁇ P9' or P! O' position.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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).
  • polypeptides with a sequence shown in SEQ ID NOs: 77-1 12 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).
  • 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).
  • 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.
  • 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
  • apopiosis biomarkers described here can be readily used for detecting and monitoring apoptotic activities in ceils.
  • 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
  • the biological sample to be examined can be a biological fluid
  • the biological sample can also be an isolated ceil (e.g., in culture) or a collection of cells
  • tissue sample such as in a tissue sample or histology sample.
  • the sample can be suspended in a tissue sample or histology sample.
  • liquid medium or fixed onto a solid support such as a microscope slide for detection of an apoptosis biomarker as 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
  • 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 different time point can be, e.g., various points during
  • 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.
  • 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.
  • monitoring efficacy of therapeutic treatments can be performed via measuring level of one or more apoptosis biomarkers disclosed herein in combination with any known monitoring means or diagnostic tests.
  • 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),
  • FDG- PET FDG positron emission tomography
  • MRJ magnetic resonance imaging
  • FLI fluorescence imaging
  • 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).
  • metastatic cancer e.g., metastatic breast, colorectal tumor, and prostate tumor.
  • 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 edminister 2003)).
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • signals from phosphopeptides are generally weaker, as they are negatively charged and poorly ionized by electrospray MS, which is performed in the positive mode.
  • subjects undergoing treatment of diseases or conditions associated with or mediated by abnormal apoptotic activities can be monitored with one or more apoptosis biomarkers of the invention.
  • apoptosis biomarkers of the invention A great number of diseases and conditions are amenable to monitoring with methods and compositions of the present invention.
  • 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 DCi tumors); cancers of the nervous system (e.g., of the skull, meninges, brain, and spinal cord); gynecological cancers (e.g., uterus,
  • 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
  • cancers of the adrenal glands e.g., neuroblastoma.
  • 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.
  • diseases states other than cancer include restenosis, autoimmune disease, arthritis, graft rejection, inflammatory bowel disease, proliferation induced after medical procedures such as surgery, angioplasty, and the like.
  • 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.
  • TTP thrombotic thrombocytopenic purpura
  • HUS hemolytic-uremic syndrome
  • 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.
  • 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.
  • the cell undergoing (or induced to undergo) apoptosis and a control non-apoptotic cell are both subject to qP-PROTOMAP analysis.
  • 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.
  • 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.
  • 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.
  • 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
  • the hematologic system i.e., blood ceils
  • endocrine system e.g., adrenal glands.
  • Example 1 Analysis of phosphorylation, and proteolysis by qP-PROTOMAP
  • proteomic measurement of dynamic post-transiationai modifications requires quantification of individuai peptides, and we therefore sought to combine PROTOMAP with stable isotopic labeling methods for this purpose.
  • 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
  • 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.
  • 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.
  • 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.
  • control-specific or "apoptosis-specific”, respectively.
  • apoptosis-specific 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.
  • RB 1 retinoblastoma protein
  • 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.
  • the pSer347 event 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).
  • KiNativ Patriceili et al confuse Chemistry & biology 18, 699-710, 20 ⁇ ; see Example 8 for details.
  • the majorit of kinases showed reduced KiNativ signals in apoptotic cells ( Figure 1 C), likely reflecting inhibition by STS.
  • 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.
  • 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).
  • [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.
  • ATM and ATR 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.
  • the 1 ⁇ 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/.
  • Jurkat cells were grown at 37 °C under 5% C0 2 in RPMi 1640 media supplemented with 10% fetal calf serum (FCS) and 2 mM ghitamine.
  • FCS fetal calf serum
  • SILAC metabolic labeling
  • 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.
  • CGK 733 (10 ⁇ final), KU55933 ( 10 ⁇ final), NU-7441 ( 1 ⁇ final), and NU-7026 (10 ⁇ final)] were purchased from Tocris, with the exception of CGK 733, which was purchased from Calbiochem.
  • Ceils were then sonicated to lyse and centrifuged at 100,000 x g for 45 min. The supernatant was collected as the soluble fraction.
  • samples were prepared according to manufacturers instructions (NE-PER Nuclear and Cytosolic Extraction Kit Pierce).
  • 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 ⁇ of trypsin at 10 ng/ ⁇ .
  • TCEP tris(2-carboxyethyl)phosphine
  • 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 ⁇ buffer A (95% I LO. 5% .acetonitriie, 0.1 % formic acid) and loaded via autosampier onto a 100 ⁇ (inner diameter) fused silica capillary coiumrs with a 5 ⁇ tip that was packed with 1 0 cm of CI.8 resin (aqua 5 ⁇ . ⁇ , 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% H 2 0, 95% acetonitriie, 0.1% formic acid). The flow rate through the column was 0.25 ⁇ /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).
  • 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)..
  • 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.
  • 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).
  • 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'.
  • 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,
  • the GRAP2 antibody was from R&D Systems (AF4640).
  • 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 ⁇ / ⁇ ! puromycin. 7 days post-selection, cells were collected and nuclear fractions were prepared. DNA-PK knockdown efficiency was measured by western blot.
  • 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).
  • iNativ Profiling of Active Kinases Jurkat cells were plated at 1 x 10 6 cells/ml and treated with ⁇ ⁇ 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 ⁇ [EMD/Calbiochem, #524625]), and sonicated. Lysates were filtered and probe reactions were performed at room temperature with a final probe concentration of 5 ⁇ . Samples were labeled with both Biotin-Hex-Acyl-ATP and Biotin-Hex-Acyi-ADP probes, as previously described
  • 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 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.
  • 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).
  • tetrapeptide analogues were used as the starting template for modeling.
  • STPD unphosphorylated substrate peptide sequences
  • ESMD ESMD
  • caspase-3 the unphosphorylated substrate peptide sequences
  • 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.
  • SAPS3 (or PP6R3) (SEQ ID NO:27)
  • EA ERV AQ VQA LGi A EAQPMA V V QS VPG AMPVPVYA FSI GPS YGl- DVS TTT AQKRKCS
  • ANP32B (SEQ ID O'30)
  • VQTQESVRGKJDVFUQl SKI>WlTlMEU.lMVYACKTSCA SUGVtPYFPYSKQCKMR RGSIVSRLLASM CKAGLTHUT3 ⁇ 44DLHQ EiQGFF IPVDNLRASPFLLQY!QEEIPDY
  • Ci2orft (SEQ ID NO: 39).
  • VPRBP (SEQ ID O:45)
  • ARMC10 SEQ ID N0:4S
  • TRSM2S (SEQ ID NO;59)
  • FSRDVDEIEAWISF LQTASDESYKDniiiQlJPSSFS HQKHQAFEAULHAN 3 ⁇ 4R5RGViDMGNSiIERGACAGSEDAVKA
  • RBM39 (SEQ fD NO:76)

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Abstract

La présente invention concerne de nouveaux biomarqueurs pour l'apoptose. Les biomarqueurs de l'apoptose comportent un polypeptide ayant une extrémité terminale clivée par une caspase, ainsi qu'un résidu d'acide aminé phosphoacétylé localisé à proximité de l'extrémité terminale (par exemple à environ 15 résidus de celle-ci). L'invention concerne également des procédés d'utilisation de tels biomarqueurs, pour surveiller les activités apoptotiques, et des procédés d'identification de biomarqueurs de l'apoptose dans divers systèmes cellulaires.
PCT/US2013/046554 2012-06-22 2013-06-19 Biomarqueurs de l'apoptose WO2013192298A1 (fr)

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CN109563130A (zh) * 2016-04-07 2019-04-02 卡斯西部储备大学 用于治疗神经退行性疾病的tdp-43线粒体定位抑制剂

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WO2001046694A2 (fr) * 1999-12-22 2001-06-28 Biosignal Packard Inc. Molecule de fusion de transfert d'energie de resonance par bioluminescence et procede d'utilisation
WO2001096873A2 (fr) * 2000-06-16 2001-12-20 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procede d'identification de proteines a apoptose modifiee
WO2003014142A2 (fr) * 2001-08-03 2003-02-20 University Of Virginia Patent Foundation Histone h2b phosphorylee utilisee comme marqueur de l'apoptose

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WO2001046694A2 (fr) * 1999-12-22 2001-06-28 Biosignal Packard Inc. Molecule de fusion de transfert d'energie de resonance par bioluminescence et procede d'utilisation
WO2001096873A2 (fr) * 2000-06-16 2001-12-20 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procede d'identification de proteines a apoptose modifiee
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CN109563130A (zh) * 2016-04-07 2019-04-02 卡斯西部储备大学 用于治疗神经退行性疾病的tdp-43线粒体定位抑制剂

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