US20150051093A1 - Reagents And Methods for Detecting Protein Lysine 3-Hydroxybutyrylation - Google Patents

Reagents And Methods for Detecting Protein Lysine 3-Hydroxybutyrylation Download PDF

Info

Publication number
US20150051093A1
US20150051093A1 US14/121,311 US201414121311A US2015051093A1 US 20150051093 A1 US20150051093 A1 US 20150051093A1 US 201414121311 A US201414121311 A US 201414121311A US 2015051093 A1 US2015051093 A1 US 2015051093A1
Authority
US
United States
Prior art keywords
lysine
hydroxybutyrylation
peptide
protein
3ohbu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US14/121,311
Other languages
English (en)
Inventor
Yingming Zhao
Zhongyu Xie
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ptm Bio LLC
Original Assignee
PTM Biolabs Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by PTM Biolabs Inc filed Critical PTM Biolabs Inc
Priority to US14/121,311 priority Critical patent/US20150051093A1/en
Publication of US20150051093A1 publication Critical patent/US20150051093A1/en
Assigned to PTM BIO LLC reassignment PTM BIO LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: PTM BIOLABS INC.
Assigned to PTM BIO LLC reassignment PTM BIO LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: JINGJIE PTM BIOLAB (HANGZHOU) CO., LTD.
Priority to US16/454,686 priority patent/US11385240B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • C07K14/43545—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes from Caenorhabditis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids
    • 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
    • G01N2440/10—Post-translational modifications [PTMs] in chemical analysis of biological material acylation, e.g. acetylation, formylation, lipoylation, myristoylation, palmitoylation
    • 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
    • G01N2440/24—Post-translational modifications [PTMs] in chemical analysis of biological material hydroxylation

Definitions

  • This invention relates to reagents and methods for detecting proteins having post-translational modifications. More particularly, it relates to peptides comprising a 3-hydroxybutyrylated lysine, and their uses to develop reagents and methods useful for detecting protein lysine 3-hydroxybutyrylation.
  • Enzymes regulating histone protein post-translational modifications use high energy co-substrates, such as acetyl-CoA and S-adenosylmethionine, for protein PTM reactions.
  • high energy co-substrates such as acetyl-CoA and S-adenosylmethionine
  • the intracellular concentrations of these cofactors may change, in turn affecting the status of histone marks.
  • the activity of histone PTM enzymes can be modulated by cellular metabolites, such as NAD and 2-hydroxyglutarate.
  • Histone PTMs such as lysine acetylation
  • Histone PTMs are also abundantly present in other proteins, and have diverse DNA-independent functions, including effects on metabolism 11 .
  • the recent discovery of new histone marks and the high complexity of cellular metabolisms imply the possibility of undescribed histone marks and PTM pathways which are modulated by metabolic signals.
  • 3-Hydroxybutyrate is a component of ketone bodies and an important energy source for tissues during starvation. It regulates gene expression and exhibits neuroprotective effects in diverse chronic neurological diseases. However, the molecular mechanisms underlying these effects remain unclear.
  • the present invention relates to the use of peptides comprising a 3-hydroxybutyrylated lysine (K 3ohbu ) to develop reagents and methods for detecting protein lysine 3-hydroxybutyrylation, especially site specific lysine 3-hydroxybutyrylation.
  • K 3ohbu 3-hydroxybutyrylated lysine
  • the isolated peptide comprising a 3-hydroxybutyrylated lysine is provided.
  • the isolated peptide may be derived from a histone protein or a fragment thereof.
  • the histone protein may be derived from an organism selected from the group consisting of human, mouse, S. cerevisiae, Tetrahymena thermophila, D. melanogaster , and C. elegans .
  • the isolated peptide may comprise an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-102.
  • the isolated peptide may comprise an amino acid sequence selected from SEQ ID NOs: 29-102.
  • the isolated peptide may comprise at least 2 amino acid residues on each of the N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine.
  • An isolated lysine 3-hydroxybutyrylation specific affinity reagent is also provided. It is capable of binding specifically to a peptide comprising a 3-hydroxybutyrylated lysine.
  • the peptide may comprise an amino acid sequence selected from SEQ ID NOs: 29-102.
  • the binding may be dependent on the presence of the 3-hydroxybutyrylated lysine but not a surrounding peptide sequence thereof in the peptide.
  • the binding may be dependent on the presence of the 3-hydroxybutyrylated lysine and a surrounding peptide sequence thereof in the peptide.
  • the lysine 3-hydroxybutyrylation specific affinity reagent may be a protein or an antibody.
  • a method for producing a lysine 3-hydroxybutyrylation specific affinity reagent that is a protein comprises screening a protein library using a peptide comprising a 3-hydroxybutyrylated lysine and at least two amino acid residues on each of the N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine.
  • the protein library may be selected from the group consisting of a phage display library, a yeast display library, a bacterial display library, and a ribosome display library.
  • a method for producing a lysine 3-hydroxybutyrylation specific affinity reagent that is an antibody comprises immunizing a host with a peptide comprising a 3-hydroxybutyrylated lysine and at least two amino acid residues on each of the N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine.
  • a method for detecting a 3-hydroxybutyrylated lysine in a protein or a fragment thereof comprises contacting the protein or a fragment thereof with the isolated lysine 3-hydroxybutyrylation specific affinity reagent capable of binding specifically to a peptide comprising a 3-hydroxybutyrylated lysine.
  • the lysine 3-hydroxybutyrylation specific affinity reagent and the protein or a fragment thereof forms a binding complex.
  • the method further comprises detecting the binding complex.
  • the presence of the binding complex indicates the presence of a 3-hydroxybutyrylated lysine in the protein or a fragment thereof.
  • the lysine 3-hydroxybutyrylation specific affinity reagent may be a protein or an antibody.
  • a method for determining the level of protein lysine 3-hydroxybutyrylation in a sample comprises detecting a 3-hydroxybutyrylated lysine in the sample.
  • kits for detecting a 3-hydroxybutyrylated lysine in a protein of a fragment thereof comprises an isolated lysine 3-hydroxybutyrylation specific affinity reagent capable of binding specifically to a peptide comprising a 3-hydroxybutyrylated lysine.
  • kits for isolating a peptide containing a 3-hydroxybutyrylated lysine comprises an isolated lysine 3-hydroxybutyrylation specific affinity reagent capable of binding specifically to a peptide comprising a 3-hydroxybutyrylated lysine.
  • FIG. 1 shows chemical structures of 3-hydroxybutyryllysine isomers and biosynthetic pathways.
  • Each isomer causes a predicted mass shift of +86.0368 Da.
  • Biosynthetic pathways for 3-hydroxybutyrate and 3-hydroxybutyryl-CoA are also listed.
  • the three ketone bodies 3-hydroxybutyrate, acetoacetate, and acetone.
  • FIG. 2 shows identification and confirmation of a Lys 3-hydroxybutyrylated peptide.
  • FIG. 3 shows detection of Lys 3-hydroxybutyrylation in cells.
  • Western blot analysis using a pan anti-K 3ohbu antibody, of (a) whole cell lysates from E. coli, S. cerevisiae, D. melanogaster S2 cells, MEF cells, and HEK293 cells, (b) liver whole-cell lysates from either control or starved (48 hours) male mice, and (c) liver whole-cell lysates from either control or STZ-treated female mice. Uniformity of sample loading was checked by staining the membrane with Ponceau S after protein transfer but prior to incubation with antibody. Concentrations of blood glucose and 3-hydroxybutyrate in (d) starved and (e) STZ-treated mice relative to controls. **P ⁇ 0.01, ***P ⁇ 0.001, Error bars show SD.
  • FIG. 4 shows proteomic screening of K 3ohbu substrates.
  • K 3ohbu sites identified on histones from HEK293 cells red diamonds
  • mouse livers green diamonds
  • the modified Lys residues are highlighted in red, and sites known to be lysine-acetylated in human and mouse proteins are marked with blue squares.
  • Cellular compartment analysis of the K 3ohbu proteome showing the enrichment (b) and subcellular distribution (c) of the K 3ohbu substrates.
  • the present invention is based on the discovery of a new type of histone marks, lysine 3-hydroxybutyrylation.
  • lysine 3-hydroxybutyrylation (K 3ohbu ) have been identified and verified as a new, evolutionarily conserved protein post-translational modification (PTM).
  • PTM protein post-translational modification
  • 3-Hydroxybutyrate can label and stimulate K 3ohbu , presumably via conversion of 3-hydroxybutyrate to 3-hydroxybutyryl-CoA.
  • K 3ohbu is a pervasive and dynamic PTM that is influenced by physiological conditions and cell status.
  • K 3ohbu sites in histones of HEK293 and mouse liver cells 45 non-redundant K 3ohbu sites in histones of HEK293 and mouse liver cells, and 3008 K 3ohbu sites in HEK293 cells have been identified.
  • the present invention provide evidence to link ketone metabolism to chromatin structure, and opens up a new avenue to study the pharmacological functions and diverse roles of 3-hydroxybutyrate in pathophysiological processes.
  • peptide refers to a linear chain of two or more amino acids linked by peptide bonds.
  • a peptide may have about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 100, 200 or more amino acids.
  • the amino acids of a peptide may be modified, deleted, added or substituted.
  • a peptide may be obtained using conventional techniques known in the art. For example, a peptide may be synthesized or obtained from a native or recombinant protein by enzymatic digestion.
  • polypeptide used herein refers to a peptide having at least 4 amino acids, preferably at least about 20 amino acids, regardless of post-translational modification.
  • protein used herein refers to a biological molecule consisting of one or more polypeptides, regardless of post-translational modification. Each polypeptide in a protein may be a subunit.
  • the polypeptide or protein may be in a native or modified form, and may exhibit a biological function or characteristics.
  • a fragment of a polypeptide or protein refers to a portion of the polypeptide or protein having an amino acid sequence that is the same as a part, but not all, of the amino acid sequence of the polypeptide or protein.
  • a fragment of a polypeptide or protein exhibits a biological function or characteristics identical or similar to that of the polypeptide or protein.
  • a biological molecule such as a peptide (e.g., a polypeptide or protein) may be derived from an original molecule, becoming identical to the original molecule or a variant of the original molecule.
  • a peptide derived from an original peptide may have an amino acid sequence identical or similar to the amino acid sequence of its original peptide, with at least one amino acid modified, deleted, inserted, or substituted.
  • a derived peptide may have an amino acid sequence at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, preferably at least about 50%, more preferably at least about 80%, most preferably at least about 90%, identical to the amino acid sequence of its original peptide, regardless of post-translational modification.
  • a derived biological molecule e.g., a peptide
  • antibody used herein includes whole antibodies, and antigen binding fragments (or antigen-binding portions) and single chains thereof.
  • a whole antibody can be either one of the two types.
  • the first type refers to a glycoprotein typically having two heavy chains and two light chains, and includes an antigen binding portion.
  • the antibody may be a polyclonal or monoclonal antibody.
  • the term “antigen binding portion” of an antibody used herein refers to one or more fragments of the antibody that retain the ability of specifically binding to an antigen.
  • the second type refers to a heavy-chain antibody occurring in camelids that is also called Nanobody.
  • single-chain variable fragment of an antibody used herein refers to a fusion protein of the variable regions of the heavy and light chains of the antibody, connected with a short linker peptide, for example, of about 20-25 amino acids, that retains the ability of specifically binding to an antigen.
  • 3-hydroxybutyrylated lysine refers to a lysine residue that is modified by a 3-hydroxybutyryl group at its epsilon-amine group. It may be in R-form or S-form, preferably R-form.
  • lysine 3-hydroxybutyrylation site used herein refers to a lysine residue in a peptide, polypeptide or protein that may be 3-hydroxybutyrylated on the epsilon-amine group of the lysine residue.
  • lysine 3-hydroxybutyrylation refers to 3-hydroxysobutyrylation on the epsilon-amine group of a lysine residue that generates a 3-hydroxysobutyryl lysine residue or 3-hydroxybutyrylated lysine.
  • the peptide of the present invention may have at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 or 200 amino acids.
  • the peptide may have about 3-25 amino acids, preferably 5-20 amino acids, more preferably 6-14 amino acids.
  • the peptide of the present invention may be prepared using conventional techniques known in the art.
  • the peptide may be derived from a protein, for example, a histone protein, or a fragment thereof, having a lysine 3-hydroxybutyrylation site.
  • the histone protein may be derived from a eukaryotic cell.
  • a eukaryotic cell include cells from a yeast (e.g., S. cerevisiae ), an C. elegans , a Drosophila (e.g., D. melanogaster (S2)), a Tetrahymena (e.g., Tetrahymena thermophila ), a mouse (e.g., M. musculus (MEF)), or a human.
  • the eukaryotic cell is a mammalian cell, for example, a human, primate, mouse, rat, horse, cow, pig, sheep, goat, chicken, dog or cat cell. More preferably, the eukaryotic cell is a human cell.
  • the histone protein may be a histone linker protein or a histone core protein.
  • a histone linker protein may be selected from the members of the H1 family, including the H1F subfamily (e.g., H1F0, H1FNT, H1FOO, and H1FX) and the H1H1 subfamily (e.g., HIST1H1A, HIST1H1B, HIST1H1C, HIST1H1D, HIST1H1E and HIST1H1T).
  • H1F subfamily e.g., H1F0, H1FNT, H1FOO, and H1FX
  • H1H1 subfamily e.g., HIST1H1A, HIST1H1B, HIST1H1C, HIST1H1D, HIST1H1E and HIST1H1T.
  • a histone core protein may a member of the H2A, H2B, H3 or H4 family.
  • a histone core protein in the H2A family may be a member of the H2AF subfamily (e.g., H2AFB1, H2AFB2, H2AFB3, H2AFJ, H2AFV, H2AFX, H2AFY, H2AFY2, and H2AFZ), the H2A1 subfamily (e.g., HIST1H2AA, HIST1H2AB, HIST1H2AC, HIST1H2AD, HIST1H2AE, HIST1H2AG, HIST1H2AH, HIST1H2AI, HIST1H2AJ, HIST1H2AK, HIST1H2AL, and HIST1H2AM), or the H2A2 subfamily (e.g., HIST2H2AA3, HIST2H2AA4, HIST2H2AB, and HIST2H2AC).
  • a histone core protein in the H2B family may be a member of the H2BF subfamily (e.g., H2BFM and H2BFWT), the H2B1 subfamily (e.g., HIST1H2BA, HIST1H2BB, HIST1H2BC, HIST1H2BD, HIST1H2BE, HIST1H2BF, HIST1H2BG, HIST1H2BH, HIST1H2BI, HIST1H2BJ, HIST1H2BK, HIST1H2BL, HIST1H2BM, HIST1H2BN, and HIST1H2BO), or the H2B2 subfamily (e.g., HIST2H2BE and HIST2H2BF).
  • H2BF subfamily e.g., H2BFM and H2BFWT
  • H2B1 subfamily e.g., HIST1H2BA, HIST1H2BB, HIST1H2BC, HIST1H2BD,
  • a histone core protein in the H3 family may be a member of the H3A1 subfamily (e.g., HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, and HIST1H3J), the H3A2 subfamily (e.g., HIST2H3A, HIST2H3C, and HIST2H3D), or the H3A3 subfamily (e.g., HIST3H3), the H3A3 subfamily (e.g., H3F3A, H3F3B, and H3F3C).
  • H3A1 subfamily e.g., HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H
  • a histone core protein in the H4 family may be a member of the H41 subfamily (e.g., HIST1H4A, HIST1H4B, HIST1H4C, HIST1H4D, HIST1H4E, HIST1H4F, HIST1H4G, HIST1H4H, HIST1H4I, HIST1H4J, HIST1H4K, and HIST1H4L), or the H44 subfamily (e.g., HIST4H4).
  • H41 subfamily e.g., HIST1H4A, HIST1H4B, HIST1H4C, HIST1H4D, HIST1H4E, HIST1H4F, HIST1H4G, HIST1H4H, HIST1H4I, HIST1H4J, HIST1H4K, and HIST1H4L
  • H44 subfamily e.g., HIST4H4
  • histone protein sequences of human, mouse, S. cerevisiae, Tetrahymena, D. melanogaster , and C. elegans can be found in GenBank database Accession Nos. GenBank database Accession No.
  • P16403 H1.2_HUMAN (SEQ ID NO: 1), P0C0S8 (H2A.1_HUMAN) (SEQ ID NO: 2), P33778 (H2B.1B_HUMAN) (SEQ ID NO: 3), P84243 (H33_HUMAN) (SEQ ID NO: 4), and P62805 (H4_HUMAN) (SEQ ID NO: 5); P15864 (H12_MOUSE) (SEQ ID NO: 6), P22752 (H2A1_MOUSE) (SEQ ID NO: 7), P10853 (H2B1F/G/L_MOUSE) (SEQ ID NO: 8), P84244 (H33_MOUSE) (SEQ ID NO: 9), and P62806 (H4_MOUSE) (SEQ ID NO: 10); P04911 (H2A.1 — S.
  • a fragment of a histone protein may have an amino acid sequence that is the same as a part, not all, of the amino acid sequence of the histone protein comprising at least one lysine 3-hydroxybutyrylation site.
  • the histone protein fragment may have at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 or 200 amino acids.
  • the histone fragment may have about 3-25 contiguous amino acids, preferably about 5-20 contiguous amino acids, more preferably about 6-14 contiguous amino acids, of the histone protein covering at least one lysine 3-hydroxybutyrylation site in the histone protein.
  • the histone protein or fragment may have a 3-hydroxybutyrylated lysine at a lysine 3-hydroxybutyrylation site.
  • the lysine 3-hydroxybutyrylation site may be any one of the lysine 3-hydroxybutyrylation sites in exemplary histone proteins of human (Table 1) and mouse (Table 2).
  • a histone protein may be obtained from a biological sample or prepared using recombinant techniques.
  • a histone protein fragment may be prepared by recombinant techniques, or by digesting the histone protein with an enzyme (e.g., trypsin).
  • the lysine 3-hydroxybutyrylation site in the histone protein or fragment may be lysine 3-hydroxybutyrylated naturally or artificially.
  • the presence of a 3-hydroxybutyrylated lysine may be confirmed by using conventional techniques known in the art, for example, mass spectrometry.
  • the peptide of the present invention may comprise an amino acid sequence having at least about 70%, 80%, 90%, 95% or 99%, preferably at least about 90%, more preferably 100%, identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-102.
  • the peptide may encompass any lysine 3-hydroxybutyrylation site with or without its surrounding sequences from a histone proteins.
  • the peptide may comprise more than one 3-hydroxybutyrylated lysine.
  • the peptide may also comprise a protein post-translational modification other than 3-hydroxybutyrylated lysine, such as acetylated lysine or methylated lysine.
  • the peptides may further comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues on either or both of N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine.
  • the peptide may comprise at least 2 amino acid residues on each of the N-terminal and C-terminal side of the 3-hydroxybutyrylated lysine.
  • Exemplary peptides of the present invention are shown in Tables 1 and 2.
  • lysine 3-hydroxybutyrylation specific affinity reagent refers to a molecule that is capable of binding to a peptide, polypeptide or protein having a lysine 3-hydroxybutyrylation site, which may be a histone protein or a peptide of the present invention.
  • the lysine 3-hydroxybutyrylation specific affinity reagent may be a protein, for example, an antibody.
  • the lysine 3-hydroxybutyrylation site may be any lysine 3-hydroxybutyrylation site in any histone protein from any species. Examples of the lysine 3-hydroxybutyrylation sites include those in human (Table 1) and mouse (Table 2), and homologous lysine sites in corresponding eukaryotic histone proteins.
  • the lysine 3-hydroxybutyrylation specific affinity reagent binds a peptide, polypeptide or protein having a lysine 3-hydroxybutyrylation site that is 3-hydroxybutyrylated, either in R-form or S-form, preferably in R-form, having an affinity that is at least about 10, 50, 100, 500, 1000 or 5000 times higher than that for its counterpart when the site is not 3-hydroxybutyrylated.
  • the lysine 3-hydroxybutyrylation specific affinity reagent binds a peptide, polypeptide or protein having a lysine 3-hydroxybutyrylation site that is not 3-hydroxybutyrylated, having an affinity that is at least about 10, 50, 100, 500, 1000 or 5000 times higher than that for its counterpart when the site is 3-hydroxybutyrylated, either in R-form or S-form, preferably in R-form.
  • the lysine 3-hydroxybutyrylation specific affinity reagent may be a peptide, polypeptide or protein, which may be an antibody.
  • the peptide is a peptide of the present invention.
  • the lysine 3-hydroxybutyrylation specific affinity reagent may be site specific, i.e., the binding is dependent on the presence of the 3-hydroxybutyrylated lysine, either in R-form or S-form, preferably in R-form, and its surrounding peptide sequence.
  • the surrounding peptide sequence may include at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues on either or both of N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine.
  • the binding depends on the presence of the 3-hydroxybutyrylated lysine and at least 2 amino acid residues on each of the N-terminal and C-terminal side of the 3-hydroxybutyrylated lysine.
  • the lysine 3-hydroxybutyrylation specific affinity reagent may not be site specific, i.e., the binding is dependent on the presence of the 3-hydroxybutyrylated lysine but not its surrounding peptide sequence.
  • One example is an anti-lysine-3-hydroxybutyrylation pan antibody.
  • a method for producing the lysine 3-hydroxybutyrylation specific affinity reagent of the present invention is further provided.
  • the protein may be produced by screening a protein library (also known as a display library or a degenerated protein library) using the peptide of the present invention.
  • the peptide may have at least two amino acid residues one each of the N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine.
  • the protein library may consist of many degenerated protein sequences, which may comprise two regions: one or more fixed peptide sequence regions and a plurality of degenerated amino acid sequences.
  • the protein library may be a phage protein library, a yeast protein library, bacterial protein library, ribosome protein library, or other synthetic protein library comprising peptides having randomized amino acid sequences.
  • the lysine 3-hydroxybutyrylation specific affinity reagent is an antibody
  • the antibody may be produced by different methods known in the art.
  • the production method may comprise immunizing a host with an antigenic peptide to produce the antibody.
  • the method may further comprise collecting antisera from the host.
  • the host may be a mammal suitable for producing antibodies.
  • the host may be a mouse, rabbit, goat, Camelidae family animal (such as Lama and camel), or cartilaginous fishes.
  • the generated antibody can contain either two chains (a heavy chain and a light chain) or one chain (or heavy chain-only antibody occurring in camelids) that is also called Nanobody.
  • the antigenic peptide may be derived from a histone protein or a fragment thereof comprising a lysine 3-hydroxybutyrylation site, which may be 3-hydroxybutyrylated or not.
  • the antigenic peptide may comprise a peptide of the present invention.
  • Examples of antigenic peptides having 3-hydroxybutyrylated lysine may comprise one or more of the peptides in Tables 1 and 2.
  • Examples of antigenic peptides not having 3-hydroxybutyrylated lysine may have an amino acid sequence identical to those in Tables 1 and 2, except that the lysine 3-hydroxybutyrylation site is not 3-hydroxybutyrylated.
  • the N-terminal or C-terminal end of any of these peptides may be extended by 1-20 residues.
  • the method may further comprise purifying the antibody from the antisera.
  • the method may further comprise utilizing spleen cells from the host to generate a monoclonal antibody.
  • the antibody specifically binds to a histone protein or fragment having a lysine 3-hydroxybutyrylation site when the site is 3-hydroxybutyrylated, but not when the site is not 3-hydroxybutyrylated.
  • the antibody specifically binds to a histone protein or fragment having a lysine 3-hydroxybutyrylation site when the site is not 3-hydroxybutyrylated, but not when the site is 3-hydroxybutyrylated.
  • the method may further comprise deduce the antibody sequences by high-performance liquid chromatography (HPLC)-mass spectrometry analysis of the isolated antibodies and followed by protein sequence database search against all the possible IgG protein sequences (derived from cDNA sequences) from bone marrow (or B cells) of the immunized host.
  • the IgG cDNA sequences can be obtained from conventional DNA sequencing technologies from IgG cDNAs that are generated by RT-PCR using the known art.
  • the derived heavy- and light-chain variable regions (VH and VL) can be further paired (in case the IgG is from a two-chain antibodies from a host like mice or rabbit). Such a pairing is not necessary for those IgG derived from heavy chain-only antibody (or Nonabody) from Lama.
  • the antibody can then be generated using the antibody sequence information using the known art.
  • a method for detecting a 3-hydroxybutyrylated lysine in a protein or its fragment is provided.
  • the 3-hydroxybutyrylated lysine may be R-3-hydroxybutyrylated lysine or S-3-hydroxybutyrylated lysine, preferably R-3-hydroxybutyrylated lysine.
  • the method comprises (a) contacting the protein or its fragment with a lysine 3-hydroxybutyrylation specific affinity reagent of the present invention to form a binding complex, and (b) detecting the binding complex.
  • the presence of the binding complex indicates the presence of the 3-hydroxybutyrylated lysine in the protein or its fragment.
  • the binding complex may be detected by using various conventional methods in the art.
  • the protein may be a histone protein.
  • the method may further comprise quantifying the amount of the binding complex.
  • the amount of the binding complex may indicate the level of lysine 3-hydroxybutyrylation in the protein or its fragment.
  • kits For each detection method of the present invention, a kit is provided.
  • the kit comprises a lysine 3-hydroxybutyrylation specific affinity reagent of the present invention.
  • the lysine 3-hydroxybutyrylation specific affinity reagent may be R-lysine 3-hydroxybutyrylation specific affinity reagent or S-lysine 3-hydroxybutyrylation specific affinity reagent.
  • the kit may further comprise an instruction directing how to carry out the method.
  • a fusion protein reporter comprises a core flanked by a donor fluorescent moiety and an acceptor fluorescent moiety.
  • the core includes a peptide, which comprises a lysine 3-hydroxybutyrylation site and a lysine 3-hydroxybutyrylation binding domain.
  • lysine 3-hydroxybutyrylation binding domain used herein refers to a region in a protein sequence capable of specific binding to the lysine 3-hydroxybutyrylation site.
  • the fusion protein reporter of the present invention may be useful for determining protein lysine 3-hydroxybutyrylation level in a sample or screening for an agent that regulates protein lysine 3-hydroxybutyrylation by using the fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the FRET involves the transfer of photonic energy between fluorophores when in close proximity. Donor fluorescent moieties and acceptor fluorescent moieties suitable for FRET are known in the art.
  • the donor fluorescent moiety may be selected from the group consisting of cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), and A206K mutants thereof, and the acceptor fluorescent moiety may be selected from the group consisting of yellow fluorescent protein (YFP), enhanced yellow fluorescence protein (EYFP), Citrine, Venus, and A206K mutants thereof.
  • CFP cyan fluorescent protein
  • ECFP enhanced cyan fluorescent protein
  • A206K mutants thereof cyan fluorescent protein
  • the acceptor fluorescent moiety may be selected from the group consisting of yellow fluorescent protein (YFP), enhanced yellow fluorescence protein (EYFP), Citrine, Venus, and A206K mutants thereof.
  • the peptide in the fusion protein reporter may comprise a peptide of the present invention. It may be derived from a histone protein or fragment comprising a lysine 3-hydroxybutyrylation site, where the histone protein or fragment may be 3-hydroxybutyrylated or not at the lysine 3-hydroxybutyrylation site.
  • the lysine 3-hydroxybutyrylation site may be located in the N-terminus, C-terminus or the core region of a histone protein.
  • the N-terminus, C-terminus, and core regions of histone proteins e.g., human or mouse H1.2, H2A, H2B, H3 or H4 are known in the art.
  • the fusion protein reporter may comprise one or more lysine 3-hydroxybutyrylation binding domains.
  • a lysine 3-hydroxybutyrylation binding domain may be derived from a lysine 3-hydroxybutyrylation specific affinity reagent of the present invention.
  • the lysine 3-hydroxybutyrylation site in the peptide is not 3-hydroxybutyrylated, and the lysine 3-hydroxybutyrylation binding domain specifically binds to the lysine 3-hydroxybutyrylation site when the site is 3-hydroxybutyrylated, but not when the sites is not 3-hydroxybutyrylated.
  • the lysine 3-hydroxybutyrylation site in the peptide is 3-hydroxybutyrylated
  • the lysine 3-hydroxybutyrylation binding domain specifically binds to the lysine 3-hydroxybutyrylation site when the peptide is not lysine 3-hydroxybutyrylated, but not when the site is 3-hydroxybutyrylated.
  • the lysine 3-hydroxybutyrylation site may be conjugated to the lysine 3-hydroxybutyrylation binding domain with a linker molecule.
  • the linker molecule may be a peptide have any amino acid sequence, and may have about 1-50 amino acids, preferably 1-30 amino acids, more preferably 2-15. In some embodiments, the linker molecule may be -Gly-Gly-.
  • the length and contents of a linker molecule may be adjusted to optimize potential fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor fluorescent moiety when the lysine 3-hydroxybutyrylation site in the fusion protein reporter is 3-hydroxybutyrylated or not, and bound by the lysine 3-hydroxybutyrylating binding domain.
  • FRET fluorescence resonance energy transfer
  • the fusion protein reporter may further comprise a targeting polypeptide.
  • the targeting polypeptide may be selected from the group consisting of a receptor ligand, a nuclear localization sequence (NLS), a nuclear export signal (NES), a plasma membrane targeting signal, a histone binding protein, and a nuclear protein.
  • a method for determining the level of protein lysine 3-hydroxybutyrylation in a sample comprises detecting a 3-hydroxybutyrylated lysine in the sample.
  • the method may comprise (a) contacting the sample with a fusion protein reporter of the present invention, and (b) comparing the level of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor fluorescent moiety after contacting with that before contacting.
  • FRET fluorescence resonance energy transfer
  • the level of FRET indicates the level of protein lysine 3-hydroxybutyrylation in the sample.
  • the level of FRET may be increased or decreased after contacting.
  • a method for determining the level of protein de-lysine-3-hydroxybutyrylation in a sample comprises (a) contacting the sample with a fusion protein reporter of the present invention, and (b) comparing the level of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor fluorescent moiety after contacting with that before contacting.
  • the level of FRET indicates the level of protein de-lysine-3-hydroxybutyrylation in the sample.
  • the level of FRET may be increased or decreased after contacting.
  • a sample may be a biological sample (e.g., bodily fluid or serum).
  • the biological sample may comprise a cell, a tissue biopsy, or a clinical fluid.
  • the biological sample may be obtained from a subject (e.g., a mouse, rat, or human).
  • the subject is healthy.
  • the subject may have suffered from or may be predisposed to a protein lysine 3-hydroxybutyrylation or de-lysine-3-hydroxybutyrylation related disorder, which may be any disorder or disease linked to abnormal regulation of protein lysine 3-hydroxybutyrylation or de-lysine-3-hydroxybutyrylation, respectively.
  • disorders or disease may include cancer, neurodegenerative diseases, aging, metabolic disorder, and dysgenesis.
  • the determination method of the present invention may further comprise comparing the FRET level in the sample with a control FRET level.
  • the control FRET level may be the FRET level in a control sample obtained from a subject, who is healthy or has not suffered from or predisposed to a protein lysine 3-hydroxybutyrylation related disorder.
  • the FRET level in the sample may be higher or lower than the control FRET level.
  • the determination method of the present invention may further comprise adding an agent to the sample.
  • the agent is known to promote or inhibit protein lysine 3-hydroxybutyrylation.
  • the agent is a screening candidate for a regulator of protein lysine 3-hydroxybutyrylation.
  • the screening candidate may be a compound or a biological molecule.
  • kits For each determination method of the present invention, a kit is provided.
  • the kit comprises a fusion protein of the present invention.
  • the kit may further comprise an instruction directing how to carry out the method.
  • kits for isolating a peptide containing a 3-hydroxybutyrylated lysine comprises an isolated lysine 3-hydroxybutyrylation specific affinity reagent capable of binding specifically to a peptide comprising a 3-hydroxybutyrylated lysine.
  • a method for treating or preventing a protein lysine 3-hydroxybutyrylation related disease in a subject in need thereof comprises administering to the subject an effective amount of a composition comprising an agent that regulates protein lysine 3-hydroxybutyrylation.
  • the agent may be a screen candidate identified by a determination method of the present invention.
  • the protein lysine-3-hydroxybutyrylation may be histone lysine-3-hydroxybutyrylation.
  • a method for treating or preventing a protein or de-lysine-3-hydroxybutyrylation related disease in a subject in need thereof comprises administering to the subject an effective amount of a composition comprising an agent that regulates protein de-lysine-3-hydroxybutyrylation.
  • the agent may be a screen candidate identified by a determination method of the present invention.
  • the protein de-lysine-3-hydroxybutyrylation may be histone de-lysine-3-hydroxybutyrylation.
  • HEK293 cells were grown in complete DMEM medium either not treated, or treated with chemicals at conditions specified elsewhere in the text.
  • C57BL/6 mice were either fed with standard chow diet, or fasted (with free access to water) for a specified number of hours as detailed in the text.
  • C57BKS/J db/db littermates (licensed by the Jackson Laboratory) were either given single-dose intraperitoneal injections of streptozotocin (STZ, 200 mg/kg body weight), or the sodium citrate buffer vehicle for 48 hours.
  • the liver tissues were collected for histone extraction and western blot analysis.
  • pan anti-K 3ohbu antibody was co-developed with PTM Biolabs, Inc. (Chicago, Ill.). Peptides were synthesized using racemic or enantiomeric modifier protected amino acid residues. Synthesis of Fmoc-protected amino acid residues is described in detail in Supplementary Methods. Modified sequencing-grade trypsin was purchased from Promega (Madison, Wis.). C18 ZipTips were bought from Millipore Corporation (Bedford, Mass.). Other chemicals were obtained from the following suppliers. Sigma-Aldrich (St.
  • HEK293 cells were grown to 90% confluence in complete DMEM medium at 37° C. in a humidified incubator supplemented with 5% CO 2 .
  • HEK293 cells were grown in complete DMEM medium containing 20 mM (R/S)-3-hydroxybutyrate [2,4- 13 C2] for 48 hrs until they reached 95% confluence.
  • HEK293 cells were grown in complete DMEM medium treated with or without sodium 3-hydroxybutyrate as specified elsewhere in the paper.
  • the cells were lysed in lysis buffer (100 mM NaCl, 20 mM Tris, 0.5 mM EDTA, 0.5% (v/v) NP40, 0.2 mM PMSF, 2 ⁇ g/ ⁇ L leupeptin, 10 ⁇ g/mL aprotinin, 5 mM sodium butyrate and 10 mM nicotinamide) at 4° C. for 20 min with constant rotation.
  • the sample was centrifuged for 10 min at 4° C. at 20,000 ⁇ g.
  • the insoluble pellet was resuspended in 10 volumes of lysis buffer followed by brief sonication at 4° C.
  • the protein lysate samples were combined and precipitated in 80% cold acetone (pre-chilled to ⁇ 20° C.) and 10% trichloroacetic acid solution at ⁇ 20° C. for 2 hrs.
  • the protein pellet was washed twice with cold acetone and the sample was digested with 50:1 sequencing grade modified trypsin (Promega) at 37° C. for 16 hrs.
  • the digestion was reduced with 5 mM dithiothreitol at 50° C. for 30 min, alkylated with 15 mM iodoacetamide at rt for 30 min, and blocked with 30 mM cysteine at rt for 30 min.
  • the sample was digested with 100:1 trypsin at 37° C. for an additional 4 hrs.
  • tryptic peptides were further separated by reversed phase chromatography as described below.
  • STZ streptozotocin
  • Extraction of core histones from HEK293 cells and mouse liver was carried out according to a previously described protocol with minor modifications.
  • Liver samples were homogenized using a glass Dounce homogenizer (20 strokes) in ice-cold lysis buffer. The homogenate was passed through two layers of cheese cloth and then centrifuged at 1,000 ⁇ g at 4° C. for 5 min. The pellet was briefly washed with lysis buffer and extracted with 0.4 NH 2 SO 4 at 4° C. overnight.
  • HEK293 cells were lysed in lysis buffer on ice for 10 min with gentle stirring. The lysate was removed and the pellet was washed once with the lysis buffer and then extracted with 0.4 N H 2 SO 4 at 4° C. overnight.
  • the suspension was centrifuged at 20,000 ⁇ g for 10 min at 4° C.
  • the histone-containing supernatants were precipitated with 20% trichloroacetic acid.
  • the precipitated histone pellets were washed twice with cold acetone and dried.
  • the histone samples were then digested with sequencing grade trypsin as described earlier.
  • Peptide fractionation by reversed-phase chromatography was performed on a Phenomenex Luna C18 column (10 mm ⁇ 250 mm, 5 ⁇ m particle, 100 ⁇ pore size) with a flow rate of 4 mL/min using the Shimadzu preparative HPLC system.
  • Buffer A consisted of 10 mM ammonium formate in water (pH 7.8) and buffer B consisted of 10 mM ammonium formate in 90% acetonitrile (pH 7.8).
  • Peptides were loaded onto the column in 2 mL of buffer A and eluted with a gradient of 2-30% B in 40 min and 30-90% B in 10 min. A total of 80 fractions were collected and concatenated into 20 fractions.
  • Acetonitrile was removed from each fraction using a Rotavapor evaporator connected to a water pump; the remaining samples were dried by lyophilisation. Immunoaffinity enrichment of K 3ohbu peptides was performed as previously described.
  • Tryptic peptides were dissolved in HPLC buffer A (0.1% formic acid in water) and loaded onto a self-packed C18 capillary column (10 cm in length, 75 ⁇ m ID) packed with Jupiter C12 resin (Phenomenex, 90 ⁇ , 4 ⁇ m in size) by Eksigent 1D-plus nano-flow HPLC.
  • Peptides were eluted with a linear gradient of 5%-30% B in 2 hrs with a constant flow rate of 200 nL/min.
  • Peptide ions were directly electrosprayed into a LTQ Velos Orbitrap mass spectrometer and analysed by either fragmenting the 20 most intense ions in a data-dependant mode, or fragmenting specified precursor ions for targeted analysis by collision-induced dissociation.
  • MS/MS data were analyzed by Maxquant (v1.3.0.5) with a built-in Andromeda search engine against an IPI human (v3.70) or IPI mouse (v3.74) database for protein and peptide identification. Lys acetylation, 3-hydroxybutyrylation, methionine oxidation, and protein N-terminal acetylation were specified as variable modifications. Mass tolerance was set to 6 ppm for precursor ions and 0.5 Da for fragment ions. Results were filtered at a 1% false discovery rate at protein, peptide and site levels. To reduce the number of low quality PTM identifications, we further remove all peptides with Maxquant peptide score below 60 or site localization probability below 0.9. We also removed all peptide identification with C-terminal Lys modifications and peptide identifications from known contaminant proteins.
  • Step 1 Racemic ethyl 2-hydroxybutyrate (15.1 mM, 1.78 g, 1.83 mL) was dissolved in 25 mL CH 2 Cl 2 . Then, 2 g of 99% H 3 PO 4 and 312 ⁇ L BF 3 .OEt 2 was added in sequential order. The resulting mixture was cooled in an ice-acetone bath and stirred. Then, 10 mL isobutylene measured in a 50 mL cylinder (pre-cooled in a dry ice acetone bath) was poured into the flask. The flask was sealed and the reaction was stirred under ⁇ 78° C.
  • Step 3 DCC (6.5 mM, 1.31 g) was added to a solution (80 mL) of 2-( t BuO) butyric acid (1.04 g, 6.5 mM) and N-hydroxysuccinimide — (6.5 mM, 748 mg) in CH 3 CN. The reaction was stirred at rt for 4 hrs. The resulting suspension was filtered and concentrated under vacuum. The residue was redissolved in 100 mL CH 2 Cl 2 . Et 3 N (13 mM, 1.81 mL) and Fmoc-Lys-OH (6.5 mM, 2.62 g) were sequentially added.
  • Fmoc-Lys((S)-3-( t BuO) isobutyryl)-OH, Fmoc-Lys((R)-3-( t BuO) isobutyryl)-OH, Fmoc-Lys((R)-3-( t BuO) butyryl)-OH and Fmoc-Lys((S)-3-( t BuO) butyryl)-OH were synthesized in a similar manner to Fmoc-Lys(( ⁇ )-2-( t BuO) butyryl)-OH, starting from different raw materials.
  • Step 1 A mixture of 2.58 g (30 mM, 2.28 mL) of 4-butyrolactone and 1.2 g (30 mM) of sodium hydroxide in 30 mL of water was heated at 70° C. overnight. The clear solution was cooled and concentrated. The resulting white solid was suspended in toluene and concentrated further to remove the remaining trace amounts of water. An almost quantitative yield of sodium 4-hydroxybutyrate was obtained.
  • Step 2 Sodium 4-hydroxybutyrate (1.26 g, 10 mM) and trityl chloride (10 mM, 2.79 g) were dissolved in 30 mL pyridine for 3 days at 30° C. The solvent was evaporated and the residue was dissolved in ethyl ether. The ether solution was extracted with aqueous sodium hydroxide solution (4 g in 250 mL of H 2 O). The aqueous solution was acidified to pH 3.0 and extracted twice with ethyl acetate. The combined organic phases were washed with brine and dried over anhydrous MgSO 4 . The mixture was filtered and the filtration was evaporated to dryness give the solid product 4-(tritylO) butyric acid (1.29 g, 37%).
  • Step 3 DCC (3.7 mM, 760 mg) was added to a solution of 4-(tritylO) butyric acid (1.29 g, 3.7 mmol) and N-hydroxysuccinimide (3.7 mM, 425 mg) in 30 mL dioxane. The reaction was stirred at rt for 10 hrs. The solution was filtered and evaporated to dryness, and then the residue redissolved in 60 mL CH 2 Cl 2 . Et 3 N (8 mM, 1.2 mL) and Fmoc-Lys-OH (4 mM, 1.62 g) were sequentially added. The mixture was stirred at rt for 4 hrs.
  • R-3-hydroxybutyryl-CoA may also be the cofactor for the lysine 3-hydroxybutyrylation reaction.
  • R-3-hydroxybutyryl-CoA can be synthesized by several metabolic pathways ( FIG. 1 b ). Alternatively, it may be generated from cellular 3-hydroxybutyrate, possibly by 3-hydroxyacyl-Coenzyme A synthetase, in the same way that acetate and crotonate can be converted to their corresponding CoA derivatives. To test this hypothesis, we first treated HEK293 cells with 10 mM R-3-hydroxybutyrate and then examined lysine modifications by Western blot.
  • 3-hydroxybutyrate constitutes a major component of ketone bodies ( FIG. 1 b ) and its concentration can dramatically increase by more than 10-fold during starvation and over 20-fold in pathological conditions such as Type 1 diabetes (T1DM) and alcoholic liver damage (up to 20 mM).
  • T1DM Type 1 diabetes
  • alcoholic liver damage up to 20 mM
  • K 3ohbu levels may also change in response to an increased 3-hydroxybutyrate that may in turn enhance the concentration of 3-hydroxybutyrate CoA.
  • K 3ohbu abundance by Western blot analysis using the liver samples from C57BL6 mice either fed with a normal chow or fasted (supplied with water only). Our results showed that K 3ohbu was drastically up-regulated after 48 hours of fasting ( FIG. 3 b ) relative to the control.
  • Histone marks contribute to epigenetic mechanisms, playing a key role in diverse pathophysiological processes.
  • tryptic peptides of core histones from HEK293 cells treated with 10 mM R-3-hydroxybutyrate, and from liver cells of mice that were either fasted for 48 hours or treated with STZ.
  • histone K 3ohbu sites including 38 histone K 3ohbu sites from 3-hydroxybutyrate-treated HEK293 cells ( FIG. 4 a ), 21 histone K 3ohbu sites from starved mouse liver ( FIG. 4 a ), and 16 histone K 3ohbu sites from STZ-treated diabetic mouse liver ( FIG. 4 a ).
  • the 27 K 3ohbu sites identified in mouse liver cells, 11 of which were identified in both the STZ-treated diabetic mouse and starved mouse livers.
  • K 3ohbu substrates on a broad, cellular scale will reveal the scope of the modification and the pathways regulated by it. Additionally, development of a dataset of substrates will lay a foundation for studying non-chromatin functions of K 3ohbu , as the history of the study of lysine acetylation demonstrates. To this end, we carried out a systematic analysis to identify non-histone substrates bearing K 3ohbu in HEK293 cells. Ten milligrams of whole-cell lysate was digested by trypsin and separated into 20 fractions with basic reversed phase HPLC. K 3ohbu peptides were affinity-enriched from each fraction using an anti-K 3ohbu antibody and then analysed by HPLC-MS/MS.
  • K 3ohbu proteome is significantly enriched in the nucleus (7.2E-192), intracellular lumen (2.5E-174), cytosolic ribosome (1.8E-22) and mitochondrial matrix (2.4E-12) ( FIG. 4 , b and c ).
  • K 3ohbu is abundant in proteins involved in diverse processes related to transcription and metabolism, such as nucleic acid metabolism (1E-130), gene expression (1.0E-97), macromolecular complex organization (5.0E-41), chromatin modification (1.2E-40) and DNA repair (1.6E-31) (table 6b).
  • KEGG pathway enrichment analysis showed that the K 3ohbu proteome in HEK293 cells is significantly enriched in 16 complexes or pathways, including spliceosomes (7.2E-43), ribosomes (2.5E-19), RNA transport (2.0E-11), nucleotide excision repair (1.3E-8) and fatty acid elongation in mitochondria (2.3E-3).
  • the spliceosome and ribosome stand out as heavily Lys 3-hydroxybutyrylated complexes when a protein-protein interaction map is constructed among K 3ohbu proteins. Analysis of the sequences surrounding K 3ohbu sites shows preferences for serine and proline at the ⁇ 1 and +3 positions, respectively, and alanine and glycine at the +2 position ( FIG. 4 d ). This pattern is different from that seen for Lys acetylation.
  • 3-Hydroxybutyrate is generated mainly from oxidation of fatty acids in liver under physiological conditions such as starvation and during neonatal development when glucose is not sufficient 1 . Ketosis can also happen when the insulin signaling pathway is not well regulated, as in Type 1 diabetes. During starvation, ketone bodies are important for generating acetyl-CoA as an alternative energy source for the brain and other tissues (e.g., heart and skeletal muscle). Given the dynamic nature of 3-hydroxybutyrate and of K 3ohbu levels, K 3ohbu may serve as a mechanism for cells to adapt to changes in cellular energy sources (e.g., glucose versus lipids) by rewriting epigenetic programs and modulating the functions of cellular proteins. Emerging evidence suggests that some KDACs have very weak deacetylation activities or activities other than deacetylation. It would be interesting to determine whether any of the KDACs can catalyse removal of 3-hydroxybutyrylation, therefore modulating cellular metabolism.
  • 3-hydroxybutyrate has functions other than simply providing energy. 3-Hydroxybutyrate has been used successfully to treat epilepsy. It also shows potential for treating several neurological conditions, such as Alzheimer's disease, Parkinson's disease, traumatic brain injury, ischemia, and amyotrophic lateral sclerosis. At the cellular level, R-3-hydroxybutyrate was found to modulate sperm motility, receptor signaling pathways, and autophagy, and to regulate global gene expression profiles associated with cancer cell “stemness”. Nevertheless, the molecular mechanisms by which R-3-hydroxybutyrate exerts these functions remain unclear. Discovery of the lysine 3-hydroxybutyrylation pathway therefore illuminates a new direction in studying the diverse physiological functions of R-3-hydroxybutyrate and its pharmacological significance.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Insects & Arthropods (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Mycology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
US14/121,311 2013-08-16 2014-08-18 Reagents And Methods for Detecting Protein Lysine 3-Hydroxybutyrylation Abandoned US20150051093A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US14/121,311 US20150051093A1 (en) 2013-08-16 2014-08-18 Reagents And Methods for Detecting Protein Lysine 3-Hydroxybutyrylation
US16/454,686 US11385240B2 (en) 2013-08-16 2019-06-27 Reagents and methods for detecting protein lysine 3-hydroxybutyrylation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201361866725P 2013-08-16 2013-08-16
US14/121,311 US20150051093A1 (en) 2013-08-16 2014-08-18 Reagents And Methods for Detecting Protein Lysine 3-Hydroxybutyrylation

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US16/454,686 Division US11385240B2 (en) 2013-08-16 2019-06-27 Reagents and methods for detecting protein lysine 3-hydroxybutyrylation

Publications (1)

Publication Number Publication Date
US20150051093A1 true US20150051093A1 (en) 2015-02-19

Family

ID=52467242

Family Applications (2)

Application Number Title Priority Date Filing Date
US14/121,311 Abandoned US20150051093A1 (en) 2013-08-16 2014-08-18 Reagents And Methods for Detecting Protein Lysine 3-Hydroxybutyrylation
US16/454,686 Active 2035-01-09 US11385240B2 (en) 2013-08-16 2019-06-27 Reagents and methods for detecting protein lysine 3-hydroxybutyrylation

Family Applications After (1)

Application Number Title Priority Date Filing Date
US16/454,686 Active 2035-01-09 US11385240B2 (en) 2013-08-16 2019-06-27 Reagents and methods for detecting protein lysine 3-hydroxybutyrylation

Country Status (2)

Country Link
US (2) US20150051093A1 (zh)
CN (2) CN117567605A (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108017555B (zh) * 2017-11-28 2021-03-26 郑州海斯威生物技术有限公司 一种β-羟基丁酰-氨基酸化合物及制备方法和应用
CN112341542B (zh) * 2021-01-08 2021-05-14 清华大学 一种3-羟基丁酰化修饰蛋白质药物及其制备方法和应用
CN114702548B (zh) * 2022-03-11 2023-08-04 湖南源品细胞生物科技有限公司 一种促进间充质干细胞生长的多肽组合物及其培养基和应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012024612A1 (en) * 2010-08-20 2012-02-23 Pangea Biosciences, Inc. Cancer diagnostic and cancer therapeutic

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090075284A1 (en) 2006-09-19 2009-03-19 The Regents Of The University Of Michigan Metabolomic profiling of prostate cancer
US20080241862A1 (en) * 2007-01-29 2008-10-02 Yingming Zhao Propionyl and butyryl lysine modifications in proteins
US9625467B2 (en) 2010-05-27 2017-04-18 Ptm Bio Llc Agent and method for identifying lysine crotonylation in proteins

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012024612A1 (en) * 2010-08-20 2012-02-23 Pangea Biosciences, Inc. Cancer diagnostic and cancer therapeutic

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Linares, Nanostructured labels for enhanced paper based immunoassay, Retrieved from https://edoc.ub.uni-muenchen.de/19313/1/Moura_Linares_Elisangela.pdf on 07/25/2016, 2013, 148 *
Long et al., Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies, Arthritis Research & Therapy 2012, 14:R25, pages 1-14 *
Preuveneers et al., D-3-Hydroxybutyrate Dehydrogenase from Rhodopseudomonas spheroides, Biochem. J. (1973) 133, 133-157. *
Reusch et al., Posttranslational modification of E. coli histone-like protein H-NS and bovine histones by short-chain poly-(R)-3-hydroxybutyrate (cPHB), FEBS Letters 527 (2002) 319-322. *

Also Published As

Publication number Publication date
CN104592379A (zh) 2015-05-06
US20190331695A1 (en) 2019-10-31
CN117567605A (zh) 2024-02-20
US11385240B2 (en) 2022-07-12

Similar Documents

Publication Publication Date Title
US11385240B2 (en) Reagents and methods for detecting protein lysine 3-hydroxybutyrylation
Kebede et al. Histone propionylation is a mark of active chromatin
Brennan et al. The utility of N, N-biotinyl glutathione disulfide in the study of protein S-glutathiolation
Han et al. The novel caspase-3 substrate Gap43 is involved in AMPA receptor endocytosis and long-term depression
US20220091134A1 (en) Reagents and methods for detecting protein lysine 2-hydroxyisobutyrylation
Nagel et al. O-GlcNAc modification of the runt-related transcription factor 2 (Runx2) links osteogenesis and nutrient metabolism in bone marrow mesenchymal stem cells
US20110165606A1 (en) Methods for modifying, isolating, detecting, visualizing, and quantifying citrullinated and/or homocitrullinated peptides, polypeptides and proteins
Duan et al. Mitochondrial Drp1 recognizes and induces excessive mPTP opening after hypoxia through BAX-PiC and LRRK2-HK2
Kallemeijn et al. Proteome-wide analysis of protein lipidation using chemical probes: in-gel fluorescence visualization, identification and quantification of N-myristoylation, N-and S-acylation, O-cholesterylation, S-farnesylation and S-geranylgeranylation
Mazzini et al. Protease‐sensitive regions in amyloid light chains: what a common pattern of fragmentation across organs suggests about aggregation
Saleh et al. Dynamics of non-canonical amino acid-labeled intra-and extracellular proteins in the developing mouse
Cai et al. Proteome-wide mapping of endogenous SUMOylation sites in mouse testis
US10908169B2 (en) Reagents and methods for detecting protein crotonylation
Ramos et al. Native presynaptic metabotropic glutamate receptor 4 (mGluR4) interacts with exocytosis proteins in rat cerebellum
Qu et al. Quantitative proteomic approaches for analysis of protein S-nitrosylation
Hassani Nia et al. Targeting of δ-catenin to postsynaptic sites through interaction with the Shank3 N-terminus
Yuan et al. Quantitative comparison of sarcomeric phosphoproteomes of neonatal and adult rat hearts
EP2610266A1 (en) Reagents and methods for detecting protein crotonylation
Zheng et al. Histone monoaminylation dynamics are regulated by a single enzyme and promote neural rhythmicity
Solovyeva et al. Integrative proteogenomics for differential expression and splicing variation in a DM1 mouse model
Kofler et al. Proline-rich sequence recognition: I. Marking GYF and WW domain assembly sites in early spliceosomal complexes
Pobran et al. Detection and characterization of TDP-43 in human cells and tissues by multiple reaction monitoring mass spectrometry
Sileno et al. A possible role of transglutaminase 2 in the nucleus of INS-1E and of cells of human pancreatic islets
Hou et al. A terminal alkyne and disulfide functionalized agarose resin specifically enriches azidohomoalanine labeled nascent proteins
US20070134802A1 (en) Ionization modifier for mass spectrometry

Legal Events

Date Code Title Description
AS Assignment

Owner name: PTM BIO LLC, ILLINOIS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:PTM BIOLABS INC.;REEL/FRAME:039170/0068

Effective date: 20160701

AS Assignment

Owner name: PTM BIO LLC, ILLINOIS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:JINGJIE PTM BIOLAB (HANGZHOU) CO., LTD.;REEL/FRAME:039419/0144

Effective date: 20160812

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION