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WO2013108126A2 - Méthyltransférases et leurs utilisations - Google Patents

Méthyltransférases et leurs utilisations Download PDF

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Publication number
WO2013108126A2
WO2013108126A2 PCT/IB2013/000420 IB2013000420W WO2013108126A2 WO 2013108126 A2 WO2013108126 A2 WO 2013108126A2 IB 2013000420 W IB2013000420 W IB 2013000420W WO 2013108126 A2 WO2013108126 A2 WO 2013108126A2
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Prior art keywords
vcp
kmt
substrate
methylated
enzyme
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PCT/IB2013/000420
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English (en)
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WO2013108126A3 (fr
Inventor
Pal Oystein FALNES
Stefan KERNSTOCK
Arne Klungland
Markus FUSSER
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University Of Oslo
Oslo Universitetssykehus Hf
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Publication of WO2013108126A2 publication Critical patent/WO2013108126A2/fr
Publication of WO2013108126A3 publication Critical patent/WO2013108126A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • G01N2333/91—Transferases (2.)
    • G01N2333/91005—Transferases (2.) transferring one-carbon groups (2.1)
    • G01N2333/91011—Methyltransferases (general) (2.1.1.)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/12—Post-translational modifications [PTMs] in chemical analysis of biological material alkylation, e.g. methylation, (iso-)prenylation, farnesylation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention relates to compositions and methods for biomarker screening and research.
  • the present invention relates to VCP-KMT methyltransferases, antibodies to methylated VCP, and targets of VCP-KMT for screening and research applications.
  • cancers Malignant tumors (cancers) are the second leading cause of death in the United
  • Cancer is characterized by the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites via a process called metastasis.
  • a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.
  • polypeptides associated with metastasis In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify polypeptides associated with metastasis. In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify (1) non-membrane-associated polypeptides that are specifically produced by one or more particular type(s) of cancer cell(s) as compared to by one or more particular type(s) of non-cancerous normal cell(s), (2) polypeptides that are produced by cancer cells at an expression level that is significantly higher than that of one or more normal non-cancerous cell(s), or (3) polypeptides whose expression is specifically limited to only a single (or very limited number of different) tissue type(s) in both the cancerous and non-cancerous state (e.g., normal prostate and prostate tumor tissue).
  • tissue type(s) e.g., normal prostate and prostate tumor tissue
  • polypeptides may remain intracellularly located or may be secreted by the cancer cell. Moreover, such polypeptides may be expressed not by the cancer cell itself, but rather by cells which produce and/or secrete polypeptides having a potentiating or growth-enhancing effect on cancer cells.
  • secreted polypeptides are often proteins that provide cancer cells with a growth advantage over normal cells and include such things as, for example, angiogenic factors, cellular adhesion factors, growth factors, and the like.
  • the present invention relates to compositions and methods for biomarker screening and research.
  • the present invention relates to VCP-KMT methyltransferases, antibodies to methylated VCP, and targets of VCP-KMT for screening and research applications.
  • Embodiments of the present invention provide compositions, kits, and methods useful in the detection of VCP-KMT methyltransferase activity. Such compositions and methods find use in research, screening (e.g., drug screening), and diagnostic (e.g., screening for the presence of cancer) applications.
  • the present invention provides methods of screening compounds for modulation of the methyltransferase activity of a VCP lysine-specific MTase (VCP-KMT) enzyme, comprising: (a) contacting said VCP lysine-specific MTase (VCP-KMT) enzyme with a VCP substrate and a test compound; and (b) detecting the level of methylation of said VCP substrate in the presence and absence of said test compound, wherein the level of methylation of said VCP substrate is indicative of methyltransferase activity of said VCP-KMT enzyme.
  • the VCP substrate is non-methylated prior to contact with the VCP-KMT enzyme in step (a) and methylation of the previously non-methylated substrate is detected.
  • the VCP-KMT enzyme is human VCP-KMT. In some embodiments, the VCP-KMT enzyme is C. elegans C42C1.13. In some embodiments, the VCP-KMT enzyme is an ortholog of human VCP-KMT. In some embodiments, the VCP substrate is selected from the group consisting of VCP and VCP variants. In some embodiments, the VCP variant is VCPAD2. In some embodiments, the test compound is a drug. In some embodiments, the drug is a cancer drug.
  • the detecting further comprises providing an antigen binding protein that specifically binds to a methylated VCP substrate but not a non- methylated VCP substrate and exposing said VCP substrate to said antigen binding protein to detect methylated VCP substrate.
  • the methylated substrate is VCP or VCPAD2 trimethylated at lysine residue K315.
  • the detecting further comprises providing a labeled methyl donor and detecting VCP substrate comprising a labeled methyl group donated from said labeled methyl donor.
  • the labeled methyl donor is selected from the group consisting of S-[methyl- 14 C]-SAM and S-[methyl- 3 H]-SAM.
  • the methods further comprise screening a library of test compounds. In some embodiments, the methods further comprise selecting test compounds from said library that modulate methyltransferase activity of said VCP-KMT enzyme. In some embodiments, the methods further comprise clinically testing at least one selected test compound. In some embodiments, the methods further comprise synthesizing a lead compound utilizing at least one selected test compound as a template. In some embodiments, the methods further comprise clinically testing said lead compound. In some embodiments, the methods further comprise providing the test compound or the lead compound for administration to a subject.
  • the present invention provides a test compound or lead compound identified by the foregoing methods.
  • the present invention provides for the use of a VCP lysine-specific MTase (VCP-KMT) enzyme and a VCP substrate in an assay for identification of one or more compounds that alter the methyltransferase activity of said VCP lysine- specific MTase (VCP-KMT) enzyme.
  • VCP-KMT VCP lysine-specific MTase
  • the present invention provides for the use of a VCP substrate in an assay to identify one or more compounds that alter the methyltransferase activity of said VCP lysine-specific MTase (VCP-KMT) enzyme.
  • VCP-KMT VCP lysine-specific MTase
  • the present invention provides a kit, comprising: a) a VCP- KMT enzyme; b) a VCP-KMT substrate; and c) reagents for detection of methylated VCP-KMT substrate.
  • the VCP-KMT enzyme is human VCP- KMT.
  • the VCP-KMT enzyme is C. elegans C42C1.13.
  • the VCP-KMT enzyme is an orthologue of human VCP-KMT.
  • the substrate is VCP or a VCP variant.
  • the VCP variant is VCPAD2.
  • the reagents comprise an antibody that specifically binds to a methylated VCP substrate but not a non-methylated VCP substrate.
  • the methylated substrate is VCPAD2 trimethylated at K315.
  • the reagents comprise a labeled methyl donor.
  • the present invention provides an antigen binding protein or antibody, scFV, or fragment thereof that specifically binds to a methylated VCP substrate, preferably methylated (e.g., trimethylated) at a lysine residue corresponding to amino acid residue K315 of wild type human VCP, but not a non-methylated VCP substrate.
  • the VCP substrate may be a wild type VCP, a portion of VCP comprising a lysine residue corresponding to amino acid residue K315 of wild type human VCP, or a variant thereof.
  • the methylated substrate is a VCP or VCPAD2 trimethylated at K315.
  • the present invention provides a prognostic or diagnostic method comprising contacting a patient sample with the foregoing antigen binding protein or antibody, scFV, or fragment thereof to determine the level of methylation of VCP.
  • the methods further comprise correlating said level of methylation with a disease, condition, prognosis or outcome for said patient.
  • the present invention provides for the use of VCP or VCPAD2 methylated at K315 or a portion thereof comprising said methylated K315 residue for generating an antibody that specifically binds to methylated VCP or VCPAD2.
  • METTL21D is a lysine specific Class I MTase.
  • METTL21D protein sequences H. sapiens, NP_078834; A. pisum, XP 001945214.1; C. elegans, NP_001 122759.1; N. vectensis, XP 001636505.1 ; A. thaliana, NP_973791.1 ; C. reinhardtii, XP_001692568.1).
  • VCP-KMT interacts with and methylates VCP in vitro
  • a Structure of hexameric VCP.
  • Three of the protomers have been individually color-labeled and darker color indicates the selected interaction domain (SID) involved in METTL21D interaction.
  • SID selected interaction domain
  • the structure was rendered using symmetry expansion on a published VCP structure [8].
  • b Domain structure of VCP showing the N- terminal, Dl ATPase and D2 ATPase domains, and indicating the SID and VCPAD2.
  • c VCP-KMT methylates VCP and VCPAD2.
  • VCP-KMT disrupts the hexamer and forms a complex with VCPAD2.
  • Hexameric VCP or VCPAD2 were incubated with a twofold molar excess of VCP-KMT, followed by SEC.
  • Elution profiles (absorbance at 280 nm) of protein samples and size standards.
  • VCP-KMT Zinc finger nuclease mediated disruption of the VCP-KMT gene in human cell lines.
  • boxes indicate the coding (grey) and non-coding (light grey) parts of exons, while solid black lines indicate introns (not to scale).
  • a blow-up of the sequence at the ZFN target site is shown.
  • ZFN cleavage is expected to occur between the two ZFN binding sites (uppercase).
  • the genotypes of selected clones harboring frame shift mutations are shown.
  • U87-MG cells contain only a single copy of the VCP-KMT gene, c, MS-analysis of VCP isolated from wild-type and VCP-KMT-deficient cell lines.
  • VCP-KMT-treated CDC48AD2 positive control for detection of methylated peptides
  • c VCP-KMT activity of C. elegans protein C42C1.13.
  • Figure 5a, 5b, 5c, 5d, 5e, 5f, 5g, and 5h Effects of VCP-KMT disruption on cellular phenotypes and VCP function
  • a Representative growth curves of HeLa (left), 293 T-REx Flp-In (middle) and U87-MG cells (right).
  • An exponential fit to the data is shown and the deduced doubling time ⁇ is indicated
  • b Doubling time of wild-type and VCP-KMT-deficient cell lines.
  • Data are represented as means +/- s.e.m., n is indicated.
  • VCP was partially purified from wild-type or VCP- KMT deficient HeLa cells by anion exchange chromatography and SEC, and the ATPase activity of SEC fractions was measured, h, Coomassie stained SDS-PAGE of VCP - containing SEC fractions A-D. Co-purified Valyl-tRNA synthetase complex subunits and VCP were identified by MS (arrows).
  • METTL21C(31-47) is detected upon incubation of METTL21C with SAM.
  • the masses of detected b- and y-ions are indicated
  • Figure 7a and 7b shows the nucleotide (SEQ ID NO: 1) and polypeptide (SEQ ID NO:2) sequence of human VCP -KMT (GenBank Acc. No. NM_024558).
  • VCP refers to Valosin-containing protein or p97.
  • a number of Valosin-containing protein orthologues have been identified including, but not limited to: Human VCP (GenBank Accession No. NP 009057.1 GL6005942;
  • VCP includes proteins that share at least 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity with the human, mouse or rat VCP.
  • VCP lysine-specific MTase or "VCP-KMT” refers to a methyltransferase that methylates, and preferably trimethylates, VCP at position K315 of the wild type human VCP.
  • the VCP-KMT enzyme is the Homo sapiens VCP-KMT.
  • the enzyme is a VCP-KMT orthologue from another organism (e.g., a eukaryotic or prokaryotic organism).
  • the VCP-KMT shares at least 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:2, human VCP-KMT.
  • VCP lysine-specific MTase (VCP-KMT) substrate refers to a VCP homolog or fragment thereof that is capable of being methylated, and preferably trimethylated, at a lysine residue corresponding to position K315 of wild type human VCP.
  • the VCP-KMT substrate may be a full-length, wild type VCP, a variant of VCP (e.g., a variant sharing at least 70%, 80%, 90%, 95%, 97%, 98% or 99% sequence identity with human, mouse or rat VCP), or a fragment, such as a truncation mutant, of human, mouse or rat VCP (e.g., a VCP truncation mutant lacking the D2 ATPase domain, wherein the truncated mutant shares at least 70%, 80%, 90%, 95%, 97%, 98% or 99% sequence identity with the corresponding portion of human, mouse or rat VCP).
  • a variant of VCP e.g., a variant sharing at least 70%, 80%, 90%, 95%, 97%, 98% or 99% sequence identity with human, mouse or rat VCP
  • a fragment such as a truncation mutant, of human, mouse or rat VCP (e.g., a VCP trun
  • detect may describe either the general act of discovering or discerning or the specific observation of a detectably labeled composition.
  • the term "subject” refers to any organisms that are screened using the diagnostic methods described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans.
  • mammals e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like
  • diagnosis refers to the recognition of a disease by its signs and symptoms, or genetic analysis, pathological analysis, histological analysis, and the like.
  • the term "purified” or “to purify” refers to the removal of components (e.g., contaminants) from a sample.
  • components e.g., contaminants
  • antibodies are purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin that does not bind to the target molecule.
  • the removal of non-immunoglobulin proteins and/or the removal of immunoglobulins that do not bind to the target molecule results in an increase in the percent of target-reactive
  • recombinant polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.
  • sample is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Such examples are not however to be construed as limiting the sample types applicable to the present invention.
  • biomarker refers to a specific biochemical in the body that has a particular molecular feature to make it useful for diagnosing and measuring the progress of disease or the effects of treatment.
  • Major classes of cancer biomarkers based on clinical utility and application include the following: (1) “diagnostic biomarkers” that are used to: (i) determine if the patient has cancer, and (2) define the type of cancer of the patient. Diagnostic biomarkers can also be used to detect and define recurrent disease after primary therapy. (2) "Prognostic biomarkers” are used to indicate a likely course of the disease.
  • Prognostic biomarkers can reflect, for example, the metastatic state or potential and/or the likely growth rate of the tumor, and are used to estimate patient outcome without consideration of the treatment given.
  • "Predictive biomarkers” are used to identify subpopulations of patients who are most likely to respond to a given therapy.
  • "Pharmacodynamic” or “pharmacological” biomarkers (sometimes referred to as PD biomarkers) can help identify which drug dose to use for an individual.
  • biomarkers can also be used to monitor a patient's response to treatment. Once a patient begins treatment with a drug, the biomarkers of the present invention can be used to monitor the patient's response, and if necessary, the treatment regiment (drug or dose) can be modified.
  • the biomarkers of the present invention can be used in any of these forms.
  • the present invention specifically encompasses K315 methylated VCP biomarkers as well as VCP-KMT biomarkers.
  • immunohistochemistry also known as immunohistochemistry
  • immunocytochemistry when applied to cells refers to a tool in diagnostic pathology, wherein panels of antibodies (e.g., monoclonal antibodies) can be used in the differential diagnosis of undifferentiated neoplasms (e.g., to distinguish lymphomas, carcinomas, and sarcomas) to reveal markers specific for certain tumor types and other diseases, to diagnose and phenotype malignant lymphomas and to demonstrate the presence of viral antigens, oncoproteins, hormone receptors, and proliferation-associated nuclear proteins.
  • panels of antibodies e.g., monoclonal antibodies
  • neoplasms e.g., to distinguish lymphomas, carcinomas, and sarcomas
  • antigen binding protein refers to proteins which bind to a specific antigen.
  • Antigen binding proteins include, but are not limited to, immunoglobulins, including polyclonal, monoclonal, chimeric, single chain, and humanized antibodies, Fab fragments, F(ab')2 fragments, and Fab expression libraries.
  • immunoglobulins including polyclonal, monoclonal, chimeric, single chain, and humanized antibodies, Fab fragments, F(ab')2 fragments, and Fab expression libraries.
  • Fab fragments fragments, F(ab')2 fragments, and Fab expression libraries.
  • Various procedures known in the art are used for the production of polyclonal antibodies.
  • various host animals can be immunized by injection with the peptide corresponding to the desired epitope including, but not limited to, rabbits, mice, rats, sheep, goats, etc.
  • the peptide is conjugated to an immunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin (KLH)).
  • an immunogenic carrier e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin (KLH)
  • Various adjuvants are used to increase the immunological response, depending on the host species, including, but not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacillus Calmette-Guerin
  • Corynebacterium parvum
  • any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used (See e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
  • Antibody fragments that contain the idiotype (antigen binding region) of the antibody molecule can be generated by known techniques.
  • fragments include, but are not limited to: the F(ab')2 fragment that can be produced by pepsin digestion of an antibody molecule; the Fab' fragments that can be generated by reducing the disulfide bridges of an F(ab')2 fragment, and the Fab fragments that can be generated by treating an antibody molecule with papain and a reducing agent.
  • Genes encoding antigen-binding proteins can be isolated by methods known in the art. In the production of antibodies, screening for the desired antibody can be
  • radioimmunoassay e.g., radioimmunoassay, ELISA (enzyme- linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), Western Blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and Immunoelectrophoresis assays, etc.) etc.
  • ELISA enzyme- linked immunosorbant assay
  • sandwich immunoassays immunoradiometric assays
  • gel diffusion precipitin reactions e.g., gel agglutination assays, hemagglutination assays, etc.
  • antibody is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they bind specifically to a target antigen.
  • primary antibody refers to an antibody which binds specifically to the target protein antigen in a tissue sample.
  • a primary antibody is generally the first antibody used in an immunohistochemical procedure. In one embodiment, the primary antibody is the only antibody used in an IHC procedure.
  • a primary antibody is typically directed to a label, wherein said label (e.g., hapten, etc.) is incorporated into an oligonucleotide probe that is directed to a target sequence. For example, if an oligonucleotide probe is labeled with DIG the primary antibody is an anti- DIG antibody that recognizes and binds the DIG hapten of the oligonucleotide.
  • a primary antibody used in ISH is typically conjugated to a detection reagent or enzyme which provides a detection means for detecting hybridization events.
  • secondary antibody herein refers to an antibody which binds specifically to a primary antibody, thereby forming a bridge between the primary antibody and a subsequent reagent, if any.
  • the secondary antibody is generally the second antibody used in an immunohistochemical procedure.
  • label is a detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule.
  • Specific, non- limiting examples of labels include fluorescent and fluorogenic moieties, chromogenic moieties, haptens, affinity tags, and radioactive isotopes.
  • the label can be directly detectable (e.g., optically detectable) or indirectly detectable (for example, via interaction with one or more additional molecules that are in turn detectable). Exemplary labels in the context of the probes disclosed herein are described below.
  • kit or "testing kit” denotes combinations of reagents and adjuvants required for an analysis. Although a test kit consists in most cases of several units, one- piece analysis elements are also available, which must likewise be regarded as testing kits.
  • Frcent as used herein may mean a portion of a reference peptide or polypeptide or nucleic acid sequence.
  • Identity as used herein in the context of two or more polypeptide or nucleotide sequences, may mean that the sequences have a specified percentage of residues or nucleotides that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation.
  • variant as used herein in the context of a nucleic acid may mean a substantially identical or substantially complementary sequence.
  • a variant in reference to a nucleic acid may further mean a nucleic acid that may contain one or more substitutions, additions, deletions, insertions, or may be fragments thereof.
  • a variant may also be a nucleic acid capable of hybridizing under moderately stringent conditions and specifically binding to a nucleic acid encoding the agent. Hybridization techniques are well known in the art and may be conducted under moderately stringent conditions.
  • a variant in reference to a peptide may further mean differing from a native peptide in one or more substitutions, deletions, additions and/or insertions, or a sequence substantially identical to the native peptide sequence.
  • the ability of a variant to react with antigen-specific antisera may be enhanced or unchanged, relative to the native protein, or may be diminished by less than 50%, or less than 20%, relative to the native peptide.
  • Such variants may generally be identified by modifying one of the peptide sequences encoding an agent and evaluating the reactivity of the modified peptide with antigen- specific antibodies or antisera as described herein.
  • Variants may include those in which one or more portions have been removed such as an N-terminal leader sequence or transmembrane domain.
  • variants may include variants in which a small portion (e.g., 1-30 amino acids, or 5-15 amino acids) has been removed from the N- and/or C- terminal of the mature protein.
  • a variant in reference to a peptide may contain conservative substitutions.
  • a "conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry may expect the secondary structure and hydrophobic nature of the polypeptide to be substantially unchanged.
  • Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
  • variants may also contain nonconservative changes.
  • Variant peptides may differ from a native sequence by substitution, deletion or addition of amino acids. Variants may also be modified by deletion or addition of amino acids, which have minimal influence on the immunogenicity, secondary structure, hydropathic, and hydrophobic nature of the polypeptide.
  • anticancer agent As used herein, the terms “anticancer agent,” “conventional anticancer agent,” or “cancer therapeutic drug” refer to any therapeutic agents (e.g. , chemotherapeutic compounds and/or molecular therapeutic compounds), radiation therapies, or surgical interventions, used in the treatment of cancer (e.g., in mammals, in primates, in humans, etc.).
  • therapeutic agents e.g. , chemotherapeutic compounds and/or molecular therapeutic compounds
  • radiation therapies e.g., radiation therapies, or surgical interventions, used in the treatment of cancer (e.g., in mammals, in primates, in humans, etc.).
  • drug and “chemotherapeutic agent” refer to pharmacologically active molecules that are used to diagnose, treat, or prevent diseases or pathological conditions in a physiological system (e.g., a subject, or in vivo, in vitro, or ex vivo cells, tissues, and organs). Drugs act by altering the physiology of a living organism, tissue, cell, or in vitro system to which the drug has been administered. It is intended that the terms “drug” and “chemotherapeutic agent” encompass anti-hyperproliferative and antineoplastic compounds as well as other biologically therapeutic compounds. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention relates to compositions and methods for biomarker screening and research.
  • the present invention relates to VCP-KMT methyltransferases, antibodies to methylated VCP, and targets of VCP-KMT for screening and research applications.
  • VCP (also called p97) is an essential and highly conserved ATP-dependent chaperone implicated in a wide range of cellular processes in eukaryotes, and mild VCP mutations can cause severe neurodegenerative disease.
  • the present invention is an essential and highly conserved ATP-dependent chaperone implicated in a wide range of cellular processes in eukaryotes, and mild VCP mutations can cause severe neurodegenerative disease.
  • VCP-KMT VCP lysine methyltransferase
  • Protein methyltransferases modify a wide range of cellular proteins, with lysine and arginine being the predominant acceptor sites [1]. Protein methylation can be dynamic and serve regulatory purposes, or it can be static and function as an expansion of the amino acid repertoire. Most MTases use S-adenosyl methionine (SAM) as methyl donor, and the human genome encodes over 200 putative SAM-dependent MTases, most of which remain uncharacterized [2]. The majority of these MTases belongs to either the SET domain family or to the seven ⁇ -strand superfamily, also designated "Class I MTases" [2,3].
  • SAM S-adenosyl methionine
  • lysine-specific MTases are SET domain proteins acting primarily on histones, but a few lysine-specific protein MTases have also been found in Class I. In humans, these are the histone-specific MTase DOT1L [3] and the calmodulin-specific MTase CaMKMT [4]; in addition, human METTL [10] is a likely ortholog of yeast Seel, which methylates elongation factor 1A5. The MTases responsible for many lysine methylations remain elusive [6], and some are likely to be found among the numerous uncharacterized Class I MTases.
  • VCP is an abundant, essential and highly conserved AAA+ protein (ATPase associated with various cellular activities) found in all eukaryotes [7]. It contains two ATPase domains, denoted Dl and D2, which form two stacked rings around a central pore in the homohexameric quaternary structure [8]. VCP is involved in a wide range of biological processes, such as cell cycle regulation, membrane fusion, autophagy, and ubiquitin mediated protein degradation, which may be explained by its ability to act as an ATP-dependent chaperone [7,9].
  • VCP-KMT the putative human Class I MTase METTL21D specifically catalyzes the trimethylation of Lys315 in VCP in vitro, and the enzyme was thus named VCP-KMT.
  • VCP-KMT the putative human Class I MTase METTL21D specifically catalyzes the trimethylation of Lys315 in VCP in vitro, and the enzyme was thus named VCP-KMT.
  • VCP-KMT was in a recent study found to be a promoter of tumor metastasis [1 1], and accordingly, VCP-KMT - deficient cells showed reduced proliferation, migration and invasive potential.
  • biochemical and bioinformatics analyses indicates that VCP-KMT belongs to a novel human protein MTase family with ten members, of which the majority seems to be lysine specific.
  • KMTs lysine-specific protein MTases
  • NVM-1 novel metastasis-promoting gene 1
  • VCP-KMT vanel metastasis-promoting gene 1
  • VCP-KMT expression may promote cancer cell migration and metastasis by two alternative mechanisms.
  • high expression of VCP-KMT in rapidly growing cancer cells might ensure optimal VCP function through efficient methylation of newly synthesized VCP.
  • elevated VCP expression leads to increased cell proliferation and is correlated with metastasis and cancer [25].
  • VCP-KMT may drive cancer metastasis through methylation of proteins other than VCP, i.e. through the methylation of a yet unidentified bona fide substrate, or through aberrant methylation of other proteins.
  • VCP is a highly conserved and essential protein in eukaryotes and affects many cellular processes through its central function as a molecular chaperone.
  • putative VCP-KMT orthologs are only found in certain eukaryotes, including vertebrates, and we found VCP to be unmethylated in the yeast S. cerevisiae, which lacks such an ortholog.
  • mutation at Lys315 did not strongly affect VCP function [26].
  • VCP-KMT-deficient human cells to be viable, showing no detectable impairment of the ubiquitin-proteasome system or autophagy, and
  • VCP isolated from such cells displayed ATPase activity similar to that of VCP isolated from wild-type cells. Taken together, this indicates that trimethylation at Lys315 is not required for basic VCP function, but may serve a more specialized purpose in VCP-KMT containing organisms, supported by the observed effects of VCP-KMT knockout on cell growth and migration.
  • VCP disease Various mutations in VCP have been reported to cause the so-called "VCP disease” IBMPFD, which is the phenotypic overlap of three distinct diseases, namely inclusion body myopathy, Paget's disease of bone and/or frontotemporal dementia.
  • IBMPFD is a late-onset, dominant disease and the disease-causing mutations are primarily localized in the N-terminal part of VCP, which serves as a binding platform for adaptor proteins, but some mutations are also found in the Dl domain, the region which is subject to methylation.
  • IBMPFD-causing mutant proteins have normal or elevated ATPase activity, and some have been reported to associate more avidly with protein co-factors.
  • VCP methylation may play a role in similar phenotypes.
  • VCP-KMT belongs to Family 16 of MTases, which is almost identical to what was denoted "Group J" in a recent bioinformatics study, the major difference being that the FAM86 proteins are part of Family 16 but excluded from Group J [2]. Two out of the ten human members of this family have previously been implicated in protein
  • compositions, kits, systems and methods for utilizing VCP, VCP substrates, and VCP-KMT in screening and research applications provide compositions, kits, systems and methods for utilizing VCP, VCP substrates, and VCP-KMT in screening and research applications.
  • compositions and methods utilize H.
  • VCP-KMT sapiens VCP-KMT, as described herein.
  • an ortholog of VCP-KMT is utilized (e.g., from an eukaryotic or prokaryotic organism).
  • the present invention provides compositions and methods for identifying inhibitors of VCP-KMT methyltransferase activity.
  • drug screening applications utilize VCP-KMT and VCP or VCP variants (e.g., VCPAD2).
  • the present invention further provides antibodies that recognize methylated VCP and variants thereof (e.g., VCPAD2).
  • VCPAD2 methylated VCP and variants thereof
  • antibodies recognize tri-methylated VCPAD2.
  • neurodegenerative disease e.g., IBMPFD.
  • the present invention provides isolated antibodies.
  • the present invention provides monoclonal or polyclonal antibodies that specifically bind to an isolated polypeptide comprised of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15, 17, 18, 19, or 20 amino acid residues of the methylated form (e.g., trimethylated) VCPAD2 or to wild type VCP, wherein the polypeptide comprises amino acids flanking the K315 position of wild type human VCP.
  • antibodies of the present invention specifically bind to VCP or variants (e.g., VCPAD2) trimethylated at K315 and do not bind to VCP or variants thereof that lack methylation or trimethylation of position K315.
  • the term "specifically binds" refers to, with respect to an antigen such as the trimethylated form of VCP or VCPAD2, the preferential association of an antibody or other ligand, in whole or part, with the trimethylated form of VCP or a fragment thereof or a cell or tissue bearing that antigen and not to cells or tissues lacking that antigen (i.e., VCP or fragment thereof that is trimethylated at K315). It is recognized that a certain degree of non-specific interaction may occur between a molecule and a non- target protein, cell or tissue. Nevertheless, specific binding can be distinguished as mediated through specific recognition of the antigen. Although selectively reactive antibodies bind antigen, they can do so with low affinity.
  • specific binding results in a much stronger association between the antibody (or other ligand) and cells bearing the antigen than between the bound antibody (or other ligand) and cells lacking the antigen.
  • Specific binding typically results in greater than 2-fold, such as greater than 5 -fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody or other ligand (per unit time), for example to the trimethylated form of VCP as compared to VCP that is not trimethylated.
  • Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein.
  • a variety of immunoassay formats are appropriate for selecting antibodies or other ligands specifically immunoreactive with a particular protein. For example, solid- phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A
  • the present invention provides an antigen binding protein or reagent comprising one or complementary determining regions (CDRs) derived from an antibody that specifically binds to VCP or VCPAD2 trimethylated at K315 and not to wild type or otherwise methylated VCP.
  • Suitable antigen binding proteins include, antibodies and humanized antibodies, fragments thereof such as Fab, F(ab'), F(ab')2 and Fv fragments, dsFvs (disulfide-linked Fv), scFvs (single chain Fv).
  • An antibody against a protein of the present invention may be any monoclonal or polyclonal antibody, as long as it can recognize the protein.
  • Antibodies can be produced by using a protein of the present invention or a peptide fragment thereof as the antigen according to a conventional antibody or antiserum preparation process.
  • the present invention contemplates the use of both monoclonal and polyclonal antibodies. Any suitable method may be used to generate the antibodies used in the methods and compositions of the present invention, including but not limited to, those disclosed herein.
  • a suitable carrier or diluent is administered to an animal (e.g., a mammal) under conditions that permit the production of antibodies.
  • an adjuvant for example, complete or incomplete
  • Freund's adjuvant may be administered. Normally, the protein is administered once every 2 weeks to 6 weeks, in total, about 2 times to about 10 times. Animals suitable for use in such methods include, but are not limited to, primates, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, llama, chicken, etc.
  • an individual animal whose antibody titer has been confirmed e.g., a mouse
  • 2 days to 5 days after the final immunization, its spleen or lymph node is harvested and antibody-producing cells contained therein are fused with myeloma cells to prepare the desired monoclonal antibody producer hybridoma.
  • Measurement of the antibody titer in antiserum can be carried out, for example, by reacting the labeled protein and antiserum and then measuring the activity of the labeling agent bound to the antibody.
  • the cell fusion can be carried out according to known methods, for example, the method described by Koehler and Milstein (Nature 256:495 [1975]).
  • a fusion promoter for example, polyethylene glycol (PEG) or Sendai virus (HVJ), preferably PEG is used.
  • myeloma cells examples include NS-1, P3U1, SP2/0, AP-1 and the like.
  • the proportion of the number of antibody producer cells (spleen cells) and the number of myeloma cells to be used is preferably about 1 : 1 to about 20: 1.
  • PEG preferably PEG 1000-PEG 6000
  • Cell fusion can be carried out efficiently by incubating a mixture of both cells at about 20°C to about 40°C, preferably about 30°C to about 37°C for about 1 minute to 10 minutes.
  • a hybridoma producing the antibody e.g., against protein of the present invention
  • a supernatant of the hybridoma is added to a solid phase (e.g., microplate) to which antibody is adsorbed directly or together with a carrier and then an anti-immunoglobulin antibody (if mouse cells are used in cell fusion, anti-mouse immunoglobulin antibody is used) or Protein A labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase.
  • a solid phase e.g., microplate
  • an anti-immunoglobulin antibody if mouse cells are used in cell fusion, anti-mouse immunoglobulin antibody is used
  • Protein A labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase.
  • a supernatant of the hybridoma is added to a solid phase to which an anti-immunoglobulin antibody or Protein A is adsorbed and then the protein labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase.
  • Selection of the monoclonal antibody can be carried out according to any known method or its modification. Normally, a medium for animal cells to which HAT
  • RPMI 1640 medium containing 1% to 20%, preferably 10% to 20% fetal bovine serum, GIT medium containing 1% to 10% fetal bovine serum, a serum free medium for cultivation of a hybridoma (SFM-101, Nissui Seiyaku) and the like can be used.
  • the cultivation is carried out at 20°C to 40°C, preferably 37°C for about 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5% CO2 gas.
  • the antibody titer of the supernatant of a hybridoma culture can be measured according to the same manner as described above with respect to the antibody titer of the anti-protein in the antiserum.
  • Separation and purification of a monoclonal antibody can be carried out according to the same manner as those of conventional polyclonal antibodies such as separation and purification of
  • immunoglobulins for example, salting-out, alcoholic precipitation, isoelectric point precipitation, electrophoresis, adsorption and desorption with ion exchangers (e.g., DEAE), ultracentrifugation, gel filtration, or a specific purification method wherein only an antibody is collected with an active adsorbent such as an antigen-binding solid phase, Protein A or Protein G and dissociating the binding to obtain the antibody.
  • an active adsorbent such as an antigen-binding solid phase, Protein A or Protein G and dissociating the binding to obtain the antibody.
  • Polyclonal antibodies may be prepared by any known method or modifications of these methods including obtaining antibodies from patients.
  • a complex of an antigen and a carrier protein is prepared and an animal is immunized by the complex according to the same manner as that described with respect to the above monoclonal antibody preparation.
  • a material containing the antibody against the immunogen is recovered from the immunized animal and the antibody is separated and purified.
  • any carrier protein and any mixing proportion of the carrier and a hapten can be employed as long as an antibody against the hapten, which is crosslinked on the carrier and used for immunization, is produced efficiently.
  • bovine serum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. may be coupled to an hapten in a weight ratio of about 0.1 part to about 20 parts, preferably, about 1 part to about 5 parts per 1 part of the hapten.
  • various condensing agents can be used for coupling of a hapten and a carrier.
  • glutaraldehyde, carbodiimide, maleimide activated ester, activated ester reagents containing thiol group or dithiopyridyl group, and the like find use with the present invention.
  • the condensation product as such or together with a suitable carrier or diluent is administered to a site of an animal that permits the antibody production.
  • an adjuvant for example, complete or incomplete Freund's adjuvant may be administered. Normally, the protein is administered once every 2 weeks to 6 weeks, in total, about 3 times to about 10 times.
  • the polyclonal antibody is recovered from blood, egg yolk, ascites and the like, of an animal immunized by the above method.
  • the antibody titer in the antiserum can be measured according to the same manner as that described above with respect to the supernatant of the hybridoma culture. Separation and purification of the antibody can be carried out according to the same separation and purification method of immunoglobulin as that described with respect to the above monoclonal antibody.
  • the polypeptide used herein as the immunogen is not limited to any particular type of immunogen.
  • full length VCP or or immunogenic portions thereof can be used as the immunogen.
  • fragments of these proteins, methylated or unmethylated may be used. Fragments may be obtained by any methods including, but not limited to expressing a fragment of the gene, enzymatic processing of the protein, chemical synthesis, and the like.
  • the present invention provides drug screening assays (e.g., to screen for anticancer agents).
  • drug screening assays screen for drugs that inhibit or modulate the methyltransferase activity of VCP-KMT.
  • the present invention provides methods of screening for compounds that modulate (e.g., inhibit, decrease, block or increase) the expression or activity of VCP-KMT.
  • candidate compounds are antibodies or small molecules that specifically bind to or interact with VCP-KMT, VCP or a regulator thereof to modulate or inhibit VCP-KMT biological function, e.g., reducing
  • candidate compounds are antisense or interfering RNA agents (e.g., oligonucleotides) directed against VCP-KMT.
  • candidate compounds are evaluated for their ability to alter VCP-KMT expression by contacting a compound with a cell expressing a VCP- KMT and then assaying for the effect of the candidate compounds on expression.
  • the effect of candidate compounds on expression of VCP-KMT is assayed for by detecting the level of VCP-KMT expressed by the cell.
  • mRNA expression can be detected by any suitable method.
  • the present invention provides assays for screening compounds for modulation of VCP-KMT methyltransferase activity comprising contacting a VCP lysine-specific MTase (VCP-KMT) enzyme with a VCP substrate and a test compound; and detecting the level of methyltransferase activity of said VCP-KMT enzyme in the presence and absence of said test compound.
  • VCP-KMT VCP lysine-specific MTase
  • the VCP substrate is preferably a VCP homolog or fragment thereof that is capable of being methylated, and preferably trimethylated, at a lysine residue corresponding to position K315 of wild type human VCP.
  • the VCP substrate may be a full-length, wild type VCP, a variant of VCP (e.g., a variant sharing at least 70%, 80%, 90%, 95%, 97%, 98% or 99% sequence identity with human, mouse or rat VCP), or a fragment, such as a truncation mutant, of human, mouse or rat VCP (e.g., a VCP truncation mutant lacking the D2 ATPase domain, wherein the truncated mutant shares at least 70%, 80%, 90%, 95%, 97%, 98% or 99% sequence identity with the corresponding portion of human, mouse or rat VCP).
  • a variant of VCP e.g., a variant sharing at least 70%, 80%, 90%, 95%, 97%, 98% or 99% sequence identity with human, mouse or rat VCP
  • a fragment such as a truncation mutant, of human, mouse or rat VCP (e.g., a VCP truncation mutant
  • the VCP substrate is non-methylated prior to contact with the VCP-KMT enzyme in assays of the present ivnention and the extent of methylation of the previously non-methylated VCP substrate by VCP-KMT is detected.
  • the candidate compounds are screened for their ability to modulate, and most preferably at least partially inhibit, VCP-KMT methyltransferase activity.
  • the effect on VCP-KMT methyltransferase activity is assayed by detecting methylation at the K315 residue of the substrate. The methylation at the K315 substrate can be compared to methylation in the absence of a test compound using the same assay conditions (i.e., comparison to a control or baseline level of K315 methylation).
  • candidate compounds e.g., libraries of compounds
  • Examples of inhibition include decreasing VCP-KMT methyltransferase activity by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% as compared to a control reaction in the absence of the candidate compound.
  • assays utilize VCP-KMT, a VCP-KMT substrate (e.g., VCP or a VCP variant (e.g., VCPAD2), a candidate compound or library of compounds and reagents for detecting methylated substrates.
  • the assays of the present invention are used in high-throughput screening methods.
  • the assays of the present invention can be provided in a multiplate format suitable for high-throughput screening of modulators of VCP-KMT activity.
  • High-throughput formats include, but are not limited to, multi-well plates, capillary systems, beads, and flow cytometry, and in particularly preferred embodiments are automated through the use of robotics.
  • libraries of synthetic compounds or tissue extracts are screened for their ability to modulate VCP-KMT methyltransferase activity.
  • Candidate compounds may be obtained from any suitable source.
  • the test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone, which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckennann et al, J. Med. Chem.
  • assays for modulation of VCP-KMT activity utilize an antigen binding reagent (e.g., antibodies as described above) that specifically binds to methylated VCP or VCPAD2, and in particularly preferred embodiments VCP or VCPAD2 that is trimethylated at position K315.
  • an antigen binding reagent e.g., antibodies as described above
  • VCP or VCPAD2 that is trimethylated at position K315.
  • a variety of methods are known in the art for detecting antigen binding reagent binding to a target molecule such as VCP or VCPAD2 that is methylated, preferably trimethylated at position K315, including, but not limited to, Western blotting, protein detection chips, bead-based assays, lateral flow devices, and enzyme-linked immunosorbent assays (ELISAs).
  • the assays of the present invention use a detectably labeled antigen binding reagent.
  • the VCP antigen binding reagent is labeled with a detectable label.
  • the detectable label may be either directly or indirectly detectable.
  • a primary VCP antigen binding reagent is detected by indirect detection via use of a secondary antibody, such as an antimouse, antirabbit, or antihuman secondary antibody.
  • the secondary antibody may preferably comprise a detectable label that can be directly or indirectly detected.
  • Suitable labels for direct detection of the VCP antigen binding reagent or second antibody include enzymatic labels, radiolabels, luminescent labels, and fluorescent labels, which may be directly incorporated into or conjugated to the antigen binding reagent or antibody.
  • the detectable label may be an enzymatic label
  • the assay may be an enzyme-linked immunosorbent assays (ELISA), as is well known in the art.
  • Suitable enzymatic labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, ⁇ -galactosidase, ⁇ -glucuronidase or ⁇ -lactamase.
  • the detectable label includes an enzyme
  • a chromogen, fluorogenic compound, or luminogenic compound can be used in combination with the enzyme to generate a detectable signal (numerous of such compounds are commercially available, for example, from Invitrogen Corporation, Eugene Oreg.).
  • chromogenic compounds include diaminobenzidine (DAB), 4-nitrophenylphospate (pNPP), fast red, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), BCIP/NBT, fast red, AP Orange, AP blue, tetramethylbenzidine (TMB), 2,2'-azino-di-[3- ethylbenzothiazoline sulphonate] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN), nitrophenyl- -D-galactopyranoside (ONPG), o-phenylenediamine (OPD), 5-bromo-4- chloro-3-indolyl- -galactopyranoside (X-Gal), methylumbelliferyl-.beta.-D- galactopyranoside (MU-Gal), p-nitrophenyl-a-D-galact
  • the detectable label may be a radiolabel, and the assay termed a radioimmunoassay (RIA), as is well known in the art.
  • RIA radioimmunoassay
  • the radioisotope can be detected by a gamma counter, a scintillation counter or by autoradiography. Isotopes
  • the detectable label is a fluorophore or fluorochrome.
  • the fluorescently labeled antibody When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence of the fluorophore.
  • fluororochromes are known to those of skill in the art, and can be selected, for example from Invitrogen, e.g., see, The Handbook— A Guide to Fluorescent Probes and Labeling Technologies, Invitrogen Detection Technologies, Molecular Probes, Eugene, Oreg.).
  • fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule or protein such as an antigen binding molecule
  • a nucleic acid molecule or protein such as an antigen binding molecule
  • fluorophores include, but are not limited to, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'- aminoethyl)aminonaphthalene- 1 -sulfonic acid (EDANS), 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin
  • DBCYL 4-dimethylaminophenylazophenyl-4'-isothiocyanate
  • DBITC 4-dimethylaminophenylazophenyl-4'-isothiocyanate
  • eosin and derivatives such as eosin and eosin isothiocyanate
  • erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate
  • ethidium fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'- dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein
  • FITC isothiocyanate
  • QFITC XRITC
  • 2',7'-difluorofluorescein ORGON GREENTM
  • fluorescamine IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde
  • pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (CibacronTM Brilliant Red 3B-A);
  • rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6- carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod),
  • rosolic acid and terbium chelate derivatives include thiol- reactive europium chelates which emit at approximately 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, Lissamine.TM., diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof.
  • fluorophores known to those skilled in the art can also be used, for example those available from Invitrogen Detection Technologies, Molecular Probes (Eugene, Oreg.) and including the ALEXA FLUORTM series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos.
  • a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.; see also, U.S. Pat. Nos. 6,815,064, 6,682,596 and
  • Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the bandgap of the semiconductor material used in the semiconductor nanocrystal. This emission can be detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can be coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et. al. (1998) Science 281 :2013-6, Chan et al. (1998) Science 281 :2016-8, and U.S. Pat. No. 6,274,323.
  • semiconductor nanocrystals can be produced that are identifiable based on their different spectral characteristics.
  • semiconductor nanocrystals can be produced that emit light of different colors based on their composition, size or size and composition.
  • quantum dots that emit light at different wavelengths based on size (565 nm, 655 nm, 705 nm, or 800 nm emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Invitrogen.
  • the detectable label is a fluorescent protein.
  • Fluorescent proteins also can be used as a carrier, or can be coupled to a carrier, to facilitate visualization.
  • green fluorescent protein GFP
  • Chimeric GFP fusions can be expressed in situ by gene transfer into cells, and can be localized to particular sites within the cell by appropriate targeting signals.
  • Spectral variants with blue, cyan and yellowish- green emissions have been successfully generated from the Aequorea GFP, but none exhibit emission maxima longer than 529 nm.
  • GFP-like proteins have been isolated from Anthozoa (coral animals) that significantly expanded the range of colors available for biological applications.
  • Fluorescent proteins refers to proteins that can become spontaneously fluorescent through the autocatalytic synthesis of a chromophore. Proteins that fluoresce at red or far-red wavelengths (red fluorescent proteins or RFPs) are known. RFPs can be used in combination with other fluorescent proteins that fluoresce at shorter wavelengths for both multicolor labeling and fluorescence resonance energy transfer (FRET) experiments.
  • FRET fluorescence resonance energy transfer
  • Commercially available RFPs are derived from two wild-type GFP-like proteins. DsRed (drFP583) has excitation and emission maxima at 558 nm and 583 nm, respectively.
  • a far-red fluorescent protein was generated by mutagenesis of a chromoprotein that absorbs at 571 nm.
  • HcRedl (Clontech) has excitation and emission maxima at 588 nm and 618 nm, respectively.
  • the fluorescent protein that emits fluorescence at the longest wavelength is eqFP61 1, cloned from the sea anemone Entacmaea quadricolor. This protein absorbs at 559 nm and emits at 611 nm.
  • the detectable label is a chemiluminescent compound.
  • the presence of a chemiluminescent-tagged antibody or antigen is then determined by detecting the luminescence that arises during the course of a chemical reaction.
  • useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound such as a bioluminescent protein may be used to label the antibody reagent useful in the present invention. Binding is measured by detecting the luminescence.
  • Useful bioluminescent compounds include luciferin and luciferase.
  • the detectable label is indirectly detected.
  • the detectable label is preferably a hapten which may be subsequently detected by a detectably labeled anti-hapten antibody.
  • the detectable labels may be the same as described above.
  • Suitable haptens include, but are not limited to, pyrazoles, particularly nitropyrazoles; nitrophenyl compounds; benzofurazans;
  • triterpenes triterpenes; ureas and thioureas, particularly phenyl ureas, and even more particularly phenyl thioureas; rotenone and rotenone derivatives, also referred to herein as rotenoids; oxazole and thiazoles, particularly oxazole and thiazole sulfonamides; coumarin and coumarin derivatives; cyclolignans, exemplified by Podophyllotoxin and Podophyllotoxin derivatives; and combinations thereof.
  • haptens include, but are not limited to, 2,4-Dintropheyl (DNP), Biotin, Fluorescein derivatives (FITC, TAMRA, Texas Red, etc.), Digoxygenin (DIG), 5-Nitro-3-pyrozolecarbamide (nitropyrazole, NP), 4,5,-Dimethoxy-2-nitrocinnamide (nitrocinnamide, NCA), 2-(3,4-Dimethoxyphenyl)- quinoline-4-carbamide (phenylquinolone, DPQ), 2, l,3-Benzoxadiazole-5-carbamide (benzofurazan, BF), 3 -Hydroxy -2-quinoxalinecarbamide (hydroxyquinoxaline, HQ), 4- (Dimethylamino)azobenzene-4' -sulfonamide (DABSYL), Rotenone isoxazoline (Rot), (E)-2-(2-(2-oxo-2,3-dihydr
  • a labeled antigen binding molecule that binds to the hapten is used in the assay.
  • suitable antigen binding molecules include, but are not limited to, antibodies, immunoglobulins or immunoglobulin-like molecules (including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM), antibody fragments such as F(ab')2 fragments, Fab' fragments, Fab'-SH fragments and Fab fragments as are known in the art, recombinant antibody fragments (such as sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), disulfide stabilized Fv proteins ("dsFv”), diabodies, and triabodies (as are known in the art), and camelid antibodies (see, for example, U.S.
  • modulation of VCP-KMT activity is detected using other suitable methods.
  • Methods for detecting methylation of substrates are known in the art.
  • the assays of the present invention include a labeled methyl donor. It is contemplated that in the absence of an inhibiting candidate compound VCP-KMT will catalyze transfer of the labeled methyl group from the labeled methyl donor to the VCP- KMT substrate. The labeled substrate can then be detected by any suitable means.
  • Candidate inhibiting compounds are those compounds that cause a decrease in methylation of the VCP-KMT substrate as compared to methylation of the VCP-KMT substrate in the absence of the candidate compound.
  • the present invention is not limited to the use of any particular labeled methyl donor.
  • the labeled methyl donor comprises a radiolabeled methyl group. Examples include, but are not limited to, detection of a labeled methyl donor (e.g., S-[methyl- 14 C]-SAM, S-[methyl- 3 H]-SAM, etc.).
  • Detection of the detectably labeled reagent or labeled methyl group may be accomplished by a scintillation counter, for example, if the detectable label or group is a radioactive particle emitter, or by a fluorimeter, for example, if the label is a fluorophore.
  • the detection is accomplished by colorimetry to measure the colored product produced by conversion of a chromogenic substrate by the enzyme. Detection may also be accomplished by visual comparison of the colored product of the enzymatic reaction in comparison with appropriate standards or controls.
  • diagnostic assays screen for the presence of VCP-KMT activity associated with the presence of cancer (e.g., metastatic cancer) or neurodegenerative disease (e.g., IBMPFD) in a subject.
  • cancer e.g., metastatic cancer
  • neurodegenerative disease e.g., IBMPFD
  • assays are cellular or in vitro and provide information useful in research (e.g., cancer research or research of neurodegenerative disease).
  • VCP-KMT activity or lack thereof can be determined using any suitable method (e.g., those described above).
  • assays are preformed that detect the level methylation of VCP in a sample such as a test fluid or tissue sample, or that detect the level of VCP-KMT activity in a sample such as a fluid or tissue sample.
  • the present invention provides a biomarker for predicting, detecting, diagnosing or monitoring disease, e.g., cancer or neurodegenerative disease, in a human subject having a biomarker to determine a methylation level of VCP or activity of VCP-KMT, wherein a higher methylation level of VCP, particularly at position K315, or VCP-KMT activity, as compared to a control level is indicative of disease, e.g., cancer or neurodegenerative disease.
  • the biological samples are selected from the group consisting of a tissue sample, a fecal sample, a cell homogenate, a blood sample, one or more biological fluids, or any combinations thereof.
  • the VCP methylation level or activity of VCP-KMT is determined by, for example, an assay as described in detail above using, for example, VCP antigen binding reagents specific for trimethylated VCP or VCP substrates for detection of VCP- KMT methyltransferase activity.
  • the assays utilize VCP antigen binding reagents in suitable assays such as immunohistochemistry assays, ELISAs, RIAs or other assays as are known in the art.
  • the immunohistochemistry assays utilize a detectably labeled VCP antigen binding reagent that specifically binds to the trimethylated form of VCP as described in detail above.
  • the immunohistochemistry assays utilize a second (or third, fourth, etc.) detectably labeled antibody in conjunction with the VCP antigen binding reagent.
  • the present invention provides a method for selecting a therapy for a patient diagnosed with cancer or a neurodegenerative disease by determining a methylation level of VCP or activity of VCP-KMT in a biological samples of the subject; and selecting the therapy based on the determination of the presence of increased VCP methylation at position K315 or increased VCP-KMT activity.
  • the present invention also provides a method of performing a clinical trial to evaluate a candidate drug believed to be useful in treating a disease (e.g., cancer or neurodegenerative disease) associated with increased VCP methylation at position K315 or increased VCP-KMT activity by a) determining the VCP methylation level or VCP-KMT activity level in a biological sample from a the subject, wherein a higher methylation level of VCP, particularly at position K315, or VCP-KMT activity, as compared to a control level is indicative of disease; b) administering a candidate drug to a first subset of the patients, and a placebo to a second subset of the patients; c) repeating step a) after the administration of the candidate drug or the placebo; and d) monitoring a change in the overall VCP methylation level or VCP- KMT activity as compared to the level in the second subset of patients, wherein a statistically significant reduction indicates that the candidate drug is useful in treating said disease state.
  • a disease e
  • Yet another embodiment of the invention is a method of using a pharmacodynamic (PD) biomarker for determining a pharmacological response to a treatment of early-onset of colorectal cancer, the method comprising: determining an overall VCP methylation or VCP-KMT activity level in one or more cells obtained from a first biological sample of a subject, wherein a higher methylation level of VCP, particularly at position K315, or VCP-KMT activity, as compared to a control level is indicative of disease; administering a drug to the subject at a first time, repeating the step of determining an overall VCP methylation or VCP-KMT activity level in one or more cells obtained from a second biological sample from the subject at a second time; and comparing the VCP methylation or VCP-KMT activity level at the first and the second time, wherein a statistically significant reduction in LINE VCP methylation or VCP-KMT activity level indicates that the drug is useful in treating said disease state.
  • PD pharmacodynamic
  • the present invention provides compositions and kits including all components necessary, sufficient or useful for detecting VCP-KMT enzyme activity (e.g., reduced or increased activity).
  • the kits described herein find use in research, therapeutic, screening, and clinical applications.
  • kits include VCP-KMT enzymes, substrates
  • kits further comprise additional reagents for performing assays (e.g., controls, buffers, etc.), instructions, assay vessels, analysis software, etc.
  • Plasmid 12373 pBD-0001, plasmid 12281 : pBD-0005 [26], and plasmid 17229: pQE9-His-p97deltaD2 [27] were obtained from AddGene (URL: addgene.org).
  • Human VCP-KMT GenBank nucleotide core accession code NM_024558.2 coding for Q9H867- 4
  • Human METTL21A GenBank nucleotide core accession code NM 001127395.1 : 5750T, rs2551949, coding for Q8WXB1 T192I
  • METTL21B Human METTL21B (GenBank nucleotide core accession code NM_015433.2: rs923829, coding for Q96AZ1) and METTL21C (GenBank nucleotide core accession code NM_001010977.1 43G>C, rs2390760, coding for Q5VZV1 G15R) were cloned from A-704 cell cDNA.
  • the indicated SNPs represent the major alleles in the population cohorts participating in the NHLBI Exome Sequencing project.
  • a variant of human METTL23 (GenBank nucleotide core accession code BC045819.1 with an artificial start codon introduced, coding for AAH45819 ⁇ 1-69, L70M) was cloned from A-704 cell cDNA.
  • C. elegans C42C1.13 (GenBank nucleotide core accession code NM_001129287.1) was amplified from cDNA.
  • S. cerevisiae Y L024c (GenBank nucleotide core accession code NM_001 182863.1) was amplified from genomic DNA of strain BY4741.
  • VCP-KMT was cloned into MCS 1 of pETDuet-1 (Novagen), other amplificates were cloned into pET28a ( ovagen).
  • Site-directed mutagenesis was performed using Quikchange II site-directed mutagenesis kit
  • MTase reactions contained 10 ⁇ g of substrate, 100 pmol MTase, and 13 ⁇ of [ 3 H]-SAM (1.5 Ci mmol -1 ) in 50 ⁇ of 50 niM Tris-HCl, 50 niM KC1 and 5 mM MgCl 2 , unless otherwise indicated.
  • the pH value was 7.5 at 37 °C for methylation of VCP or 8.5 at 37 °C for automethylation and methylation of pseudosubstrates.
  • the reaction was terminated by adding ice-cold 10 % trichloroacetic acid followed by filtration on GF/C glass microfibre filters (Whatman).
  • Recombinant monomeric VCP-KMT, hexameric VCP and hexameric VCPAD2 were isolated by SEC in 10 mM Tris pH 8.0, 100 mM NaCl and 1 mM dithiothreitol (DTT) on a Superdex 200 10/300 GL column (GE Healthcare). Following concentration, VCP and VCPAD2 were mixed with a two-fold molar excess of VCP-KMT, incubated for 30 min at 4°C and then rerun over the Superdex 200 column.
  • Protein samples to be analyzed by MS were processed and analyzed by nanoflow on-line liquid chromatographic MS analysis as described [30], using either trypsin (Sigma-Aldrich), chymotrypsin (Roche Applied Science), or Arg-C (Roche Applied Science). Mass spectrometric data were analyzed with the in-house maintained human proteome and human VCP single protein database using SEQUESTTM. The mass tolerances of fragment and parent ions were set to 0.05 Da & 7 ppm for HCD, and 0.5 Da & 5 ppm for CID. The following variable (methionine oxidation, Kmel, Kme2, Kme3, K- Ac, Rmel, Rme2, R-Ac, Hmel, Nmel, and Qmel) or fixed (cysteine
  • Unmethylated peptide was normalized to the sample without enzyme addition and reaction products were normalized to the sum of all reaction products at the given enzyme concentration.
  • VCP-KMT-deflcient cell lines HeLa, U87-MG and 293 T-REx Flp-In cells (Invitrogen) were cultivated in DMEM high glucose medium (Lonza) supplemented with 10 % FBS, Glutamax I (Invitrogen) and penicillin/streptomycin.
  • the cells were transfected with mRNA encoding a zinc finger nuclease pair targeting exon 1 of human VCP-KMT (Sigma-Aldrich) using Trans-IT mRNA transfection kit (Minis BIO) and subsequently cloned by limiting dilution.
  • Genomic DNA was prepared and a 789 bp fragment containing the ZFN target site was PCR amplified with primers TGCTACGACTACAGCAGTATAGCTC (SEQ ID NO:27) and TGGTCCCTGGTGACTTTTATCTTC (SEQ ID NO:28).
  • the wild-type sequence targeted by the ZFN pair constitutes a Blpl-cleavage site, enabling primary screening by Blpl-digestion of the PCR product.
  • Uncut PCR product was sequenced to verify the genome editing and select clones with frameshift mutations.
  • Adherent cells and mouse tissues were lysed in buffer containing 1 % IGEPAL CA-630.
  • a S. cerevisiae strain BY4741 cell lysate was prepared with a MicroBeadBeater.
  • Immunoprecipitation of VCP or CDC48 was performed with mouse anti-VCP [5] (Abeam) as described [32].
  • mouse anti-VCP [5] (Abeam) and mouse anti-GAPDH (Ambion) were used according to manufacturer's suggestions.
  • Rabbit polyclonal anti-VCP trimethylated at lysine 315 (custom methylation-specific antibody to the modification site identified herein; the custom peptide synthesis and immunization was performed by New England Peptide) was used at 1 :500 to detect trimethylated VCP in cell lysates.
  • E. coli cells (Stratagene) transformed with pETDuet-1 or pET28a constructs, and TOP10F' cells (Invitrogen) transformed with pQE9 constructs were grown and induced as described [31].
  • the bacteria were lysed by freeze-thawing and sonication in lysis buffer (50 mM sodium phosphate buffer pH 7.5, 300 mM NaCl, 5 % (v/v) glycerol, 0.5 % IGEPAL CA-630, 3 mM imidazole, 3 mM 2-mercaptoethanol, lx complete EDTA-free protease inhibitors (Roche Applied Science)) and recombinant methyltransferases with N-terminal hexahistidine tag were purified using TALON immobilized metal affinity chromatography resin (Clontech) according to manufacturer's instructions and buffer exchanged to 20 mM Tris pH 7.3 at 4 °C, 100 mM NaCl, 1 mM DTT over a Vivaspin 20 ultrafiltration unit (MWCO 10,000) (Sartorius-Stedim).
  • lysis buffer 50 mM sodium phosphate buffer pH 7.5, 300 mM NaCl, 5 % (v/
  • the product was further purified by size exclusion chromatography on Superdex 200 10/300 GL (GE healthcare) in SEC buffer (10 mM Tris pH 8.0 at 4 °C, 100 mM NaCl and 1 mM DTT).
  • VCP and CDC48 variants were purified as described above with the following modifications:
  • the culture volume was 200 ml and expression induced with 500 ⁇ IPTG.
  • Cells were lysed as described[32] and recombinant proteins with N-terminal hexahistidine tag were bound on 5 ml HisTrap immobilized metal affinity
  • VCP and VCPAD2 were further purified by size exclusion chromatography on a Superdex 200 10/300 GL column (GE healthcare) in SEC buffer and hexameric protein was isolated.
  • Cells were analyzed in transwell chambers with or without Matrigel coating to distinguish between invasion and migration, respectively, as described previously [33].
  • the lower chamber contained 10% FBS to attract the cells that were plated in medium containing 1% FBS.
  • the assay was analyzed after 24 h.
  • a detergent-free cytosolic extract was prepared using hypotonic swelling, cell lysis using a Dounce-homogenizer and high speed centrifugation as described [34].
  • HeLa wt or VCP-KMT-deficient cells were grown to ca. 70% confluence and lysed with 0.5% IGEPAL CA-630 in 20 mM Bis-Tris, pH 7.0 at 1 1 °C, 100 mM NaCl, 2 mM DTT and lx complete protease inhibitor cocktail (Roche Applied Science).
  • the lysate from 10 dishes per cell line was combined and centrifuged for 1 h at 4,600 g. The cleared lysate was loaded on a RESOURCE Q 6 ml column (GE healthcare).
  • buffer A [20 mM Bis-Tris, pH 7.0 at 1 1 °C, 100 mM NaCl, 2 mM DTT, 1 mM ethylenediaminetetraacetic acid (EDTA) and 0.2 mM 4-(2- Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF)]
  • bound proteins were eluted over 20 column volumes with a linear gradient from 0 to 50% Buffer B (20 mM Bis-Tris, pH 7.0 at 11 °C, 1 M NaCl, 2 mM DTT, 1 mM EDTA and 0.2 mM AEBSF).
  • VCP containing fractions were identified by Western blotting.
  • VCP Vivaspin 20 ultrafiltration units (molecular weight cut off (MWCO) 100,000) (Sartorius- Stedim). The concentrate was applied to a Superose 6
  • VCP-KMT also called METTL21D
  • METTL21D Human VCP-KMT belongs to a small subfamily of four highly homologous (30-50 % sequence identity) uncharacterized putative
  • METTL21A-METTL21D methyltransferases
  • VCP-KMT Plausible orthologs of VCP-KMT are found in a wide range of multicellular eukaryotes, but show a rather scattered distribution. Vertebrates, plants, nematodes and green algae all have a VCP-KMT ortholog, whereas such proteins are absent in the majority of fungi and insects, like in the common model organisms budding yeast (5 * . cerevisiae) and fruit fly (D. melanogaster). Class I MTases contain a Rossmann-fold-like bundle of seven ⁇ -strands and display four characteristic conserved sequence motifs denoted I, Post I, II and III [12].
  • VCP-KMT orthologs indicates the presence of the characteristic core motifs, and secondary structure prediction further indicates a topology of an archetypical Class I MTase core domain with an added N-terminal elongation harboring three short ⁇ -strands (Fig. la).
  • This initial bioinformatics analysis consolidates VCP-KMT as a member of the seven ⁇ -strand MTase family, and indicates that this enzyme is present in a wide range of multicellular eukaryotes.
  • VCP-KMT When testing recombinant VCP-KMT for MTase activity, we were unable to detect any activity on DNA or RNA, but VCP-KMT displayed activity on recombinant histone proteins, in particular on H2B (Fig. lb).
  • To further analyze the protein MTase activity of VCP-KMT we tested homopolymers of the L-amino acids lysine (K), arginine (R), histidine (H), asparagine (N), aspartate (D), and glutamate (E), which have all been shown to be methylated in proteins.
  • VCP-KMT displayed MTase activity on poly-(L)-K, but not on any of the other homopolymers (Fig. lb).
  • Protein MTase activity was also observed on a homopolymer of the D-stereoisomer of lysine (not naturally found in proteins), but not on a randomized copolymer of D-glutamate and D-lysine at a ratio of 3:2 (poly-(D)-E-K). No activity could be detected on protamine, which is a highly positively charged chromatin protein like H2B, but contains no lysines.
  • VCP-KMT interacts with and methylates VCP in vitro
  • VCP is a substrate for recombinant VCP-KMT in an MTase assay.
  • the SID is located in the Dl ATPase domain and appears inaccessible in the VCP hexamer structure; therefore, a VCP deletion mutant (VCPAD2) which lacks the D2 ATPase domain and forms a less stable hexamer [13, 14] was also tested (Fig. 2b). Indeed, both VCP and VCPAD2 were methylated by VCP-KMT in vitro, but
  • VCPAD2 Fig. 2c
  • VCP-KMT VCP lysine-specific MTase
  • VCPAD2 was a considerably better substrate than VCP suggested that VCP-KMT might be able to disrupt the hexameric structure of VCPAD2 to exert its MTase activity, and that this could only occur efficiently with the less stable VCPAD2 mutant.
  • VCP-KMT The more stable full-length VCP hexamer was not disrupted by VCP-KMT (Fig. 2d), and while aggregated VCP contained in the VCP preparation could be methylated, no methylation of intact hexameric VCP was observed (Fig. 2e). In addition, no in vitro methylation of endogenous VCP in a cytosolic fraction from wild-type and VCP-KMT-deficient HeLa cells was detected (data not shown). This suggests that VCP-KMT mediated methylation of VCP occurs prior to hexamer assembly in vivo.
  • VCP-KMT methylates lysine 315 of VCP
  • VCP-KMT-mediated methylation was analyzed by MS. Amongst peptides covering 88% of VCPAD2, a single lysine residue, Lys315, was found to be trimethylated in the VCP-KMT -treated sample, but not in the untreated control sample, and no other methylated residues were detected (Fig. 2f). In agreement with these data, the VCP mutant K315L was not subject to METT2 ID-mediated methylation (Fig. 2g), strongly indicating that Lys315 is the only methylation site. Substrate and enzyme requirements for VCP-KMT activity
  • Protein lysine MTases recognize their substrates either through interaction with a short linear target sequence or through binding to a more complex three-dimensional structure.
  • VCP-KMT Protein lysine MTases recognize their substrates either through interaction with a short linear target sequence or through binding to a more complex three-dimensional structure.
  • GST glutathione S-transferase
  • VCPAD2 was incubated with different amounts of VCP-KMT and the methylation status of Lys315 was analyzed by MS (Fig. 2h). While nearly all VCP was trimethylated at high VCP-KMT
  • VCP-KMT-mediated methylation of K315 in VCP we observed highly efficient and specific VCP-KMT-mediated methylation of K315 in VCP in vitro, indicating that the corresponding methylation may also occur in vivo.
  • VCP was isolated by immunoprecipitation from protein extracts of mouse brain, heart, kidney, liver and testicle, and investigated the methylation status of K315 by MS analysis. In all three tissues, VCP was almost completely trimethylated, with only trace amounts of the dimethylated form present (Fig. 3 a).
  • VCP-KMT is the enzyme responsible for methylation in vivo
  • HeLa epidermal
  • U87-MG glioma
  • 293 T-REx Flp-In kidney
  • a pair of zinc finger nucleases was designed to specifically target exon 1 of the VCP-KMT gene upstream of motif I (aa 71-79) (Fig. 3b).
  • zinc finger nucleases cut a coding sequence, imperfect repair by non-homologous end-joining frequently leads to indel mutations, and a frameshift mutation at the targeted site in the VCP-KMT gene will lead to the loss of almost the entire MTase core domain. Clones harboring frameshift mutations in all VCP- KMT alleles were obtained for all three cell lines (Fig. 3b).
  • VCP-KMT-deficient cells We next analyzed the methylation status of VCP from wild-type and VCP-KMT - deficient cells. Similarly to the observations from mouse tissues, VCP was almost completely trimethylated in all three wild-type cell lines, while no methylation was detected in VCP-KMT-deficient cells (Fig. 3c). The methylation status was further confirmed by immunoprecipitation of VCP followed by immunoblotting with pan-Kme3 antibody (Fig. 3d). Taken together, these results show that VCP is almost completely trimethylated at position K315 in a variety of mammalian cell lines and tissues, and that VCP-KMT is the responsible enzyme. Organismal distribution of VCP methylation
  • VCP appears to be present in all eukaryotes, whereas VCP-KMT shows a far more limited distribution.
  • yeast 5 * . cerevisiae has an apparent homolog of the METTL21 proteins (YNL024c), but this protein is more similar to METTL21A than to VCP-KMT.
  • YNL024c a homolog of the METTL21 proteins
  • recombinant Y L024c failed to show MTase activity on human VCPAD2 or on the corresponding yeast protein CDC48AD2, whereas human VCP-KMT was active on both these substrates (Fig. 4a).
  • the peptide covering position K325 (corresponding to K315 in human VCP) was exclusively found in a non-methylated state in CDC48 isolated from yeast cells (Fig. 4b).
  • VCP-KMT ortholog from an organism distantly related to humans also displayed VCP-specific MTase activity
  • protein C42C1.13 from the nematode C elegans. Indeed, this protein showed even higher activity than human VCP-KMT on both human VCPAD2 and yeast CDC48AD2 (Fig. 4c).
  • the absence of VCP methylation in S. cerevisiae and the observed activity of the C elegans protein C42C1.13 on both yeast and human VCP indicate that VCP-methylation can occur in a wide range of eukaryotes but is absent in those that lack a VCP-KMT ortholog.
  • VCP-KMT-deficient cells Since VCP was non-methylated in VCP-KMT-deficient cells, we used these cells to address the biological significance of VCP methylation. No appreciable difference between VCP-KMT-proficient and -deficient cells was detected in degradation assays using reporters of the ubiquitin proteosome system or when monitoring autophagy by LC3B processing (data not shown). A recent study reported that VCP-KMT, there denoted NVM- 1 , was upregulated in a number of metastatic tumors, and that it was required for efficient cell migration and invasion [1 1]. First, we performed proliferation assays where the various VCP-KMT-deficient cell lines were compared to their wild-type counterparts. These experiments showed a significantly increased doubling time for VCP- KMT-deficient HeLa and 293 T-REx Flp-In cells, but not for the slower growing U87- MG cells (Fig. 5a,b).
  • VCP-KMT-deficient cells may have reduced migratory or invasive capacity.
  • a Boyden Chamber assay with uncoated and Matrigel- coated membranes allowed us to distinguish between migration and invasion, respectively (Fig. 5c). Wild-type U87-MG cells showed significantly higher migration and invasion potential than VCP-KMT-deficient U87-MG cells. Wild-type and VCP-KMT-deficient HeLa cells showed similar migratory capacity (Fig. 5c) and no invasive potential. 293 T- REx Flp-In cells were not analyzed due to their limited migration capability.
  • VCP was the predominant protein in the complex containing fractions, but a co-purifying complex consisting of the Valyl-tRNA synthetase (VARS) and the ⁇ , ⁇ and ⁇ subunits of translation elongation factor 1 (EF1) [18] was also detected (Fig. 5h).
  • VARS Valyl-tRNA synthetase
  • EF1 translation elongation factor 1
  • VCP-KMT belongs to a family of novel protein MTases
  • VCP-KMT belongs to Family 16 of MTases (InterPro: IPR019410; Pfam:
  • VCP-KMT orthologs contain a conserved DXXY motif located immediately c- terminal to Motif II (Fig. la). A more general expansion of this motif, (D/E)XX(Y/F), is present in all ten human Family 16 members (Fig. 6a), and in their orthologs from other organisms (not shown). A similar, so-called DPPY motif, with consensus
  • (D/N/S)PP(Y/F) is found at the corresponding position in DNA N 6 -adenine MTases, DNA N 4 -cytosine MTases, and the protein glutamine MTase HemK [20].
  • the first residue plays an important role in methyl transfer, and mutation at this position leads to a catalytically inactive enzyme.
  • mutation of the corresponding residue Asp 144 in VCP-KMT inactivated the enzyme (data not shown), suggesting that the (D/E)XX(Y/F) motif in the Family 16 proteins may play a similar role as the DPPY motif.
  • METTL21B, METTL21C and METTL23 form the distinct subfamily PTHR14614 in the PANTHER database21.
  • VCP-KMT protein MTases
  • METTL21A-C and METTL23 failed to show MTase activity towards histones, amino acid homopolymers, or VCP.
  • Some protein MTases display automethylation activity, and with an amino acid specificity identical to that observed for bona fide substrates [22-24]. Indeed, we detected significant automethylation in the case of METTL21A, METTL21C and VCP-KMT (Fig. 6b).
  • the observed automethylation activity was sensitive to the competitive inhibitor S-adenosyl homocysteine (SAH) when added at the beginning, but not at the end of the incubation period, indicating that the observed activity reflects bona fide methylation, rather than non-covalent association of [ 3 H]-SAM with the enzymes' SAM-binding sites.
  • SAH competitive inhibitor S-adenosyl homocysteine
  • the automethylation activity of VCPKMT, METTL21A and METTL21C was abrogated by mutations (D96A, D94A and D141A, respectively) of the conserved SAM-binding Asp residue in Motif Post 1, which in the case of VCP-KMT abrogated VCP methylation.
  • the level of automethylation was particularly high in the case of METTL21C, where substantial (-50%) monomethylation at a specific lysine residue, Lys35, was detected by MS (Fig. 6c,d).
  • methyltransferase is an evolutionarily conserved enzyme that trimethylates Lys-115 in calmodulin. Nat. Commun. 1, 43, (2010).

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Abstract

L'invention concerne des compositions et des méthodes pour le criblage et la recherche de biomarqueurs. En particulier, la présente invention concerne des VCP-KMT méthyltransférases, des anticorps pour des VCP méthylées et des cibles de VCP-KMT pour des applications de criblage et de recherche.
PCT/IB2013/000420 2012-01-16 2013-01-14 Méthyltransférases et leurs utilisations WO2013108126A2 (fr)

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