Methods for determining protein binding specificity using peptide libraries
09988665 ยท 2018-06-05
Assignee
Inventors
Cpc classification
C40B40/10
CHEMISTRY; METALLURGY
G01N33/6845
PHYSICS
International classification
Abstract
A method for determining protein binding specificity using a screen of a peptide library is provided. The method can be used to determine binding specificity for human NAD.sup.+-dependent deacetylase SIRT1, and to identify the most efficiently deacetylated peptide sequences. The method can be also used to screen a combinatorial H4 histone N-terminal tail peptide library to examine the binding preferences of a -phos (S1) H4 antibody toward all known possible H4 histone modification states.
Claims
1. A combinatorial library comprising a collection of peptides, wherein each peptide in the collection of peptides comprises at least 5 consecutive amino acid residues having an amino acid sequence located in the first 21 N-terminal residues of the structure of the polypeptide in
2. The combinatorial library of claim 1, wherein at least one of the covalent modifications comprises citrullination of an arginine in at least one peptide of the collection of peptides.
3. The combinatorial library of claim 1, wherein at least one of the covalent modifications comprises acetylation of a lysine in at least one peptide of the collection of peptides.
4. The combinatorial library of claim 1, wherein at least one of the covalent modifications comprises methylation of a lysine or arginine in at least one peptide of the collection of peptides.
5. The combinatorial library of claim 1, wherein at least one of the covalent modifications comprises phosphorylation of a serine, threonine or tyrosine in at least one peptide of the collection of peptides.
6. The combinatorial library of claim 1, wherein at least one of the covalent modifications comprises methylation of a lysine or arginine in at least one peptide of the collection of peptides, citrullination of an arginine in at least one peptide of the collection of peptides and acetylation of a lysine in at least one peptide of the collection of peptides.
7. The combinatorial library of claim 1, wherein at least one of the covalent modifications comprises ubiquitination of a lysine in at least one peptide of the collection of peptides.
8. The combinatorial library of claim 1, wherein at least one of the covalent modifications comprises sumoylation of a lysine in at least one peptide of the collection of peptides.
9. The combinatorial library of claim 1, wherein at least one of the covalent modifications comprises ADP-ribosylation of an arginine, aspartic acid or glutamic acid in at least one peptide of the collection of peptides.
10. The combinatorial library of claim 1, wherein the solid phase support is selected from beads, microarray, microplate or a chip.
11. A method of determining the binding specificity of a protein for a differentially covalently modified peptide comprising: a) contacting the combinatorial library of claim 1 with a protein to allow binding of the protein to the combinatorial library, wherein the protein is capable of binding to at least one of the covalently modified peptides; b) detecting the protein bound to the covalently modified peptides using a label; and c) determining the binding specificity of the protein for the covalently modified peptides.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(12) The practice of the present invention employs, unless otherwise indicated, conventional techniques of peptide synthesis, molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, immunology, protein kinetics, and mass spectroscopy, which are within the skill of art. Such techniques are explained fully in the literature, e.g. in Bodanszky et al., 1976, Peptide Synthesis, 2.sup.nd ed., John Wiley and Sons; Sambrook et al., 2000, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press; Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc.; Kriegler, 1990, Gene Transfer and Expression: A Laboratory Manual. Stockton Press, New York; Dieffenbach et al., 1995, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, each of which is incorporated herein by reference in its entirety.
(13) Generally, the nomenclature and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally, enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer's specifications. Procedures employing commercially available assay kits and reagents are typically used according to manufacturer-defined protocols unless otherwise noted.
(14) The terms a, an, the and the like, unless otherwise indicated, include plural forms.
(15) The term acetyl, sometimes called ethanoyl, is a functional group, the acyl of acetic acid, with chemical formula COCH.sub.3.
(16) A label is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include .sup.32P, fluorescent dyes, colorimetric labels, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or quantum dots. As used herein, the term label also includes indirect labeling of proteins using detectable labels bound to other molecules or complexes of molecules that bind to a protein of interest, including antibodies and proteins to which antisera or monoclonal antibodies specifically bind. As used herein, the term colorimetric label includes a label that is detected using an enzyme-linked assay.
(17) Antibodies as used herein includes polyclonal and monoclonal antibodies, chimeric, and single chain antibodies, as well as Fab fragments, including the products of a Fab or other immunoglobulin expression library. With respect to antibodies, the term, immunologically specific refers to antibodies that bind to one or more epitopes of a protein of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
(18) The terms isolated, purified, or biologically pure refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A peptide or protein that is the predominant species present in a preparation is substantially purified. The term purified denotes that a peptide or protein gives rise to essentially one band in an electrophoretic gel or HPLC spectrum. Particularly, it means that the peptide or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
(19) Two peptides or polypeptides are said to be identical if the sequence of amino acid residues in the two sequences is the same when aligned for maximum correspondence as described below.
(20) Peptide or protein sequence identities are evaluated using the Basic Local Alignment Search Tool (BLAST) which is well known in the art (Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268; Altschul et al., 1997, Nucl. Acids Res. 25: 3389-3402). The BLAST programs can be used with the default parameters or with modified parameters provided by the user.
(21) Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the peptide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid 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 window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
(22) The term substantial identity of amino acid sequences for purposes of this invention normally means peptide or polypeptide sequence identity of at least 40%. Preferred percent identity of peptides or polypeptides can be any integer from 40% to 100%. More preferred embodiments include at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.7%, or 99%.
(23) Peptides or polypeptides that are substantially similar share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.
(24) The term combinatorial library refers to a collection of compounds synthesized in parallel or as a collection of compounds synthesized with mixtures of reagents or employing a split-and-mix methodology from a set of defined building blocks and using these building blocks in many combinations to generate a complex library of novel compounds. One implementation of combinatorial synthesis is the generation of one-bead-one-compound (OBOC) libraries, where each novel compound is represented on a single bead. These libraries can be screened by performing a variety of chemical and biochemical assays to identify individual compounds eliciting a response. The identity of the compound on the support is either known by prior knowledge from direct parallel synthetic procedures, or is determined by direct analysis after detection using, e.g., micro-sequencing and mass spectrometry.
(25) In peptidic OBOC libraries, peptide from a group of selected amino-acid building blocks can be represented in an on-bead format in which many copies of only one sequence and of only one post-translationally modified state exist on each bead. Alternatively, peptides may be attached to other suitable types of support, including microarrays, microplates, chips, or other surfaces that are suitable for detecting protein binding events.
(26) The invention described here uses both modified and unmodified amino acids as building blocks to create combinatorial peptide libraries to evaluate the binding specificities of proteins. These peptide libraries can vary in amino acid sequence and in modification state, e.g. methylation, acetylation or phosphorylation. The chemical identity (amino-acid sequence and post-translational status) of these peptides can be determined by direct analysis, using, e.g., micro-sequencing and mass spectrometry. Identification of the peptide sequences and their post-translational status can be performed before a protein binding assay is conducted. Alternatively, identification of the peptide sequences and their post-translational status can be performed before a protein binding assay is conducted. As well, identification of the peptide sequences and their post-translational status can be performed both before and after a protein binding assay is conducted.
(27) The method includes screening a peptide library. In one example, the method provides for the use of a one-bead-one-compound (OBOC) peptide library, also known as OBOC combinatorial library. Examples of such libraries are described in Lam et al., 1991, Nature 354: 82-84, and in Furka et al., 1991, Int. J. Pept. Protein Res. 37: 487-493.
(28) The OBOC combinatorial library method synthesizes 10.sup.2-10.sup.8 of random compounds such that each bead displays only one compound. Bead libraries are screened, and positive beads are isolated for structure analysis. Peptide substrates and inhibitors of protein kinases, and peptide ligands for cell surface receptors can be identified using this method (Lam et al., 2003, Acc. Chem. Res. 36: 370-377).
(29) A peptide library can be synthesized on various types of solid supports using methods known in the art, for example those described in U.S. Pat. Nos. 7,122,323 and 5,510,240. Preferably, the library is synthesized using beads as solid phase support. In one example, the solid support can have the form of beaded resin (beads).
(30) The peptide library can be an OBOC acetyl-peptide library. One or more of the amino acids from the peptide can be deprotected using methods known in the art, e.g. trifluoroacetic acid. One or more of the amino acids from the peptide can be acetylated. In addition to acetylation of at least one amino acid, one or more of the amino acids can be modified in a variety of ways, for example through covalent modifications. Modifications can include post-translational modifications or introduction of non-classical amino acids, as described in U.S. Pat. No. 5,510,240.
(31) The length of the peptide chain can vary. Preferably, the OBOC acetyl-peptide library is generated using 5-mer peptides, i.e. peptides with 5 amino acid residues. One or more of these 5 amino acids from the peptide can be acetylated. Preferably, the central (third) amino acid is acetylated. More preferably, the central, acetylated amino acid is lysine. Preferably, unique sequences are constructed around a central epsilon-amino acetylated lysine.
(32) When the peptide library is an acetyl-peptide library, the method can be used for determining protein deacetylase substrate specificity. The protein deacetylase can belong to the sirtuin family. Preferably, the protein deacetylase is SIRT1.
(33) The peptide library can be a combinatorial peptide library based on N-terminal histone sequences. In this example, one or more of the peptide sequences include N-terminal histone sequences. Preferably, the combinatorial OBOC peptide library is based on N-terminal amino acid sequence of histone H4. The N-terminal histone sequences can have a different number of amino acid residues. Preferably, the N-terminal histone sequences are 21-mers, i.e. they have 21 amino acid residues. These sequences can be attached to solid support directly, or via one or more amino acids that act as linkers.
(34) The N-terminal histone sequences can be modified to include various post-translational modifications. The modifications can be covalent. The modifications can include, for example, methylation, acetylation, phosphorylation, ubiquitination, sumoylation, citrullination, or ADP ribosylation. Modifications can also include the introduction of non-classical amino acids. Each N-terminal histone sequence can include one or more of these modifications.
(35) When the combinatorial peptide library is based on N-terminal histone sequences, the invention provides methods for determining and evaluating histone-binding proteins or histone-modifying enzymes. Preferably, the histone-binding protein is an antibody. More preferably, the histone-binding protein is an -phos (S1) H4 antibody.
(36) Prior to library construction, peptide length may initially be considered to determine whether relatively short peptides would function as efficient protein substrates. For example, prior to library construction, peptide length may be considered to determine whether relatively short acetyl-peptides would function as efficient enzymatic substrates. Also, for example, prior to library construction, peptide length may be considered to determine the preferred length of the N-terminal amino acid chain of a histone and its efficiency as a protein substrate.
(37) The method provides for the use of a quantum dot tagging, i.e. labeling strategy. Quantum dots are nanoparticles that exhibit exquisite photochemical properties owing to their semiconductor cores and are emerging as ideal fluorophores for screening OBOC libraries (Falciani of al., 2005, Chem. Biol. 12: 417-426). These properties include robust photostability, high quantum yield, and a sharp emission with a broad range of excitation wavelengths. Coupled with a bead-sorting instrument, quantum dots allow the screening of hundreds of thousands of peptide sequences for protein binding and protein activity in a single day. Quantum dots can be used, for example, to label the protein that is in contact with a peptide that is attached to a bead.
(38) The method provides for sorting of beads. Bead sorting can be performed manually or it can be automated. Bead sorting is preferably performed based on beads that are labeled. Bead labeling can be performed using a variety of methods known in the art. Preferably, beads can be labeled with a fluorescent label or with any other type of label. When beads are labeled with a fluorescent label, then the method can include fluorescent bead-sorting. In one example, a protein that is assayed and that specifically binds to beads, can be labeled. In another example, beads with peptides deacetylated by a protein deacetylase can be labeled. For example, these beads can be first biotinylated and then tagged (labeled) with streptavidin-coated quantum dots.
(39) After bead-sorting, peptides sequences and their corresponding post-translationally-modified status can be extracted from individual beads in the library. The sequences and modification state of these peptides can then be identified. Identification of the peptides can be performed, for example, by mass spectrometry, or by micro-sequencing. Identification of the peptides can thus identify the particular sequences and modification state for which the assayed protein shows considerable binding preference.
(40) A peptide library that is already spatially-addressed, e.g. each sequence and modification state is known at each support (e.g. bead), can be screened by the methods described.
(41) The present invention provides a high-throughput method for determining substrate specificity of protein deacetylases using a one-bead, one-compound OBOC acetyl-peptide library with a quantum dot tagging strategy and automated bead-sorting. The OBOC acetyl-peptide library method allows context-specific identification of preferred peptide substrates. This is in contrast to the previously published approach that can only uncover globally-preferred amino-acids at each position. The OBOC acetyl-peptide library method can be applied to any histone/protein deacetylase from class I, II or III.
(42) Various applications of peptide libraries of this type can be envisioned. For example, the sequence information obtained from this library can be used to generate acetyl-peptide specific antibodies for Western blot analysis. This can provide in vivo validation of acetylation at protein acetylation sites discovered in BLAST searches and sequence comparisons. Such antibodies could also be employed in immunoprecipitation studies. Mass spectral analysis could then be performed to identify the acetylated proteins. Identification of enzymatic substrates, i.e. the sequence information obtained from the library, can also be used to generate acetyl-peptide specific inhibitors.
(43) Other uses of the library include the creation of super-substrates for the in vivo generation of O-acetyl-ADP-ribose (OAADPr) to elucidate its cellular roles. Co-crystal studies could be executed to uncover how Sir2 interacts with these optimal substrate sequences. Limited peptide substrates co-crystallized with Sir2 have shown interactions primarily with the peptide backbone (Avalos et al., 2002, Mol. Cell. 10: 523-535). However, the method of this invention provides for the use of side-chain interactions to bind and catalyze protein deacetylation.
(44) Hits from libraries of this type could serve as starting points for the design of peptidomimetics for a variety of applications, e.g. for use as potential therapeutics (Nefzi et al., 2004, J. Org. Chem. 69: 3603-3609; Falciani et al., 2005, Chem. Biol. 12: 417-426). Optimized substrates reflect higher binding affinity to a protein, e.g. enzyme. Modification of the peptide to prohibit enzymatic turnover and protease degradation could be implemented to generate a specific, tight-binding in vivo inhibitor.
(45) The original peptide sequences can be further modified to confer altered chemical and biological properties (Nefzi et al., 2004, J. Org. Chem. 69: 3603-3609; Falciani et al., 2005, Chem. Biol. 12: 417-426). This strategy has been used to tailor-make peptides into therapeutics that avoid the pitfalls of proteolytic cleavage, rapid clearance from the circulatory system, inability to pass through the blood brain barrier, and lack of oral activity (Nefzi at al., 2004).
(46) This invention provides a method for identification of the molecular recognition events involved in the histone code via OBOC combinatorial peptide libraries based on N-terminal histone sequences. This method provides for synthesis and evaluation of all possible permutations (at known modification sites) of the 21 N-terminal amino acids of histone H4. In a preferred embodiment, the evaluation of the binding specificity is performed using an antibody directed to serine phosphorylation and a quantum dot detection strategy (Garske and Denu, 2006, Biochemistry 45: 94-101; Kodadek et al., 2006, Mol. Biosyst. 2: 25-35). However, the general approach is applicable to interrogating the preferences of any histone-binding protein or any histone-modifying enzyme. As well, this approach may find use in histone-specific antibody screening.
(47) The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1. Use of a One-Bead-One-Compound Peptide Library to Identify Deacetylase Specificity
(48) General
(49) All amino acid derivatives and resins were purchased from Peptides International (Louisville, Ky.) or from Bachem (Bubendorf, Switzerland). Peptides used in the solution deacetylase assays were obtained from the University of Wisconsin-Madison Biotechnology Core Facility. Other chemical reagents were obtained from Sigma-Aldrich (St. Louis, Mo.), Acros (Geel, Belgium), Novabiochem (San Diego, Calif.), Amersham Biosciences (Buckinghampshire, England), or Quantum Dot (Hayward, Calif.). Reaction vessels for peptide library synthesis were purchased from Alltech Chromatography (Deerfield, Ill.).
(50) Analytical gradient HPLC was conducted on a Shimadzu series 2010C HPLC with a Vydac C18 column (10 m, 4.6250 mm). All runs used linear gradients of 0.05% aqueous TFA and 0.02% TFA in acetonitrile. MALDI-TOF MS was performed on a Bruker REFLEX II using -cyano-4-hydroxy-cinnamic acid as matrix. Fluorescent bead sorting was carried out on a COPAS Select (Union Biometrica, Holliston, Mass.) instrument. Fluorescence microscopy was done on an Olympus IX81 instrument (Tokyo, Japan) equipped with a Hamamatsu digital camera (Hamamatsu-City, Japan).
(51) SIRT1, SIRT2, and ySir2 were expressed and purified as previously described (Borra et al., 2004, Biochemistry 43: 9877-9887; Borra of al., 2005, J. Biol. Chem. 280: 17187-17195).
(52) Prior to kinetic analysis, peptide concentrations were established by amino acid analysis (AAA) or by a coupled assay in which NAD.sup.+ leftover from exhaustive deacetylation reactions (acetylated peptide was typically incubated with 5-10 M Sir2 and 80 M NAD.sup.+ for 20 min) was quantitatively converted to NADH with alcohol dehydrogenase and monitored spectrophotometrically in real-time at 340 nm. Peptide concentrations were obtained by subtracting the amount of NADH formed from the original amount of NAD.sup.+ used in the reaction.
(53) Solution Deacetylation Assays
(54) All solution phase Sir2 assays were carried out at 25 C. in 50 mM Tris.HCl. Reactions were done in 50-100 L with 0.1-1.5 M enzyme, 0.1-1.2 mM NAD.sup.+, 0.5-1000 M peptide and 1 mM DTT. Reaction mixtures were quenched with TFA to a final concentration of 1% after 5-10 min and nicotinamide levels were quantitated by HPLC at 264 nm.
(55) Alternatively, [.sup.32P]-NAD.sup.+ (10 mCi/mL) was used in assays and quenched reaction mixtures were spotted on a silica TLC plate and run in a chamber containing 60% ethanol and 40 2.5 mM ammonium acetate for 3-4 hours. Levels of .sup.32P-OAADPr and .sup.32P-NAD.sup.+ were then quantitated by phosphorimaging and the fraction turnover was calculated. Saturation curves were done at varying concentrations of peptide while holding that of NAD.sup.+ constant. Time points were chosen such that product formation never exceeded 20% conversion and data were plotted as rate (s.sup.1) vs. peptide concentration. Plots were fitted to the Michaelis-Menten equation, v=[(k.sub.cat/K.sub.m)[S]]/(1+[S]/K.sub.m) using Kaleidagraph software (Reading, Pa.) to extract K.sub.m and k.sub.cat/K.sub.m.
(56) Screening Methodology
(57) The screening strategy used the reaction of biotin N-hydroxy-succinimide ester with the newly generated -amino group formed upon SIRT1 deacetylation. Subsequent binding of the streptavidin conjugated quantum dots provided the fluorescent tag for screening.
(58) Initially, it was established that quantum dot labeling was proportional to the molar abundance of reacted biotin. Resin bearing free amino groups were aliquoted into five reaction vessels and labeled with 1, 0.5, 0.01, 0.001 and 0 equivalents of biotin N-hydroxy-succinimide ester. After differential labeling, the resin was pooled into reaction vessel and a streptavidin conjugated quantum dot (.sub.em=605 nm) solution was added. After draining the quantum dot solution and washing the resin, the resulting pooled beads displayed differential levels of associated quantum dots, correlating with the amount of covalently linked biotin.
(59) To provide a quantitative assessment of fluorescent quantum dot labeling, a complex object parametric analyzer and sorter (COPAS) instrument was utilized. COPAS sorts beads based on fluorescence intensity while also gathering data on bead size (time of flight). Using this instrument, beads labeled in the previously mentioned experiment were sorted with an excitation of 488 nm and an emission of 610 nm. The fluorescence distribution was plotted and distinct populations could be visualized (
(60) These populations resided in a fluorescence regime that encompassed more than two orders of magnitude. Moreover, the bead groupings corresponded to the differential levels of biotinylation (although the 0.001 and 0 biotin equivalents coalesced into a single cluster). When the emission wavelength was set to green light (.sub.ex=510 nm) corresponding to the intrinsic TentaGel autofluorescence, only a single population was observed. Quantum dot labeling was found to be quantitative in sub-stoichiometric amounts. It can be coupled to a sorting instrument such as the COPAS instrument for sorting beads on the basis of fluorescence intensity.
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(62) Library Design
(63) Prior to library construction, it was essential to determine whether relatively short acetyl-peptides would function as efficient substrates of Sir2 enzymes. To evaluate peptide length requirements, 10 acetyl-lysine containing peptides corresponding to the histone H3 sequence surrounding Lys-14 and of varying length were assayed using SIRT1 and a variety of other sirtuins (SIRT2; yeast Sir2, ySir2; and Trypanosoma brucei Sir2, TbSir2). Deacetylation assays were conducted at fixed NAD.sup.+ concentrations while peptide concentrations were varied to produce saturation curves. The resulting data were fitted to the Michaelis-Menten equation to yield catalytic efficiencies, as defined by the apparent second order rate constant (k.sub.cat/K.sub.m), which takes into consideration both binding and catalysis. All peptides used in these studies were N-terminally acetylated, but the N-terminus was not deacetylated by sirtuins in control assays.
(64) The results, shown in Table 1, are represented as relative k.sub.cat/K.sub.m values, with the longest peptide AcTGG(AcK)APRK (SEQ ID NO: 9) given a value of one. In these studies, all sirtuins surveyed showed no more than a 2 to 3-fold difference in k.sub.cat/K.sub.m for the various peptide substrates. Thus, the shortest peptide, a 5-mer, was similar in catalytic efficiency to the longest peptides in this preliminary set, regardless of the enzyme assayed. These observations suggest amino acids beyond the 2 and +2 positions are not necessary for efficient binding and catalysis by sirtuins. For library construction, balancing minimal peptide length with practical limitations of library complexity were important considerations. Consequently, a 5-mer library with an acetylated lysine residue in the central position was constructed.
(65) TABLE-US-00001 TABLE1 Summaryoftherelativecatalyticefficiencies(k.sub.cat/K.sub.m)ofvariousSir2 homologswithtenpeptidesubstrates Relativek.sub.cat/K.sub.m Peptide SIRT1 SIRT2 ySir2 TbSir2 SEQIDNO:3 AcGG(AcK)AP 1.72 0.48 0.56 0.07 1.51 0.69 1.27 0.16 SEQIDNO:4 AcTGG(AcK)AP 0.54 0.11 0.55 0.07 1.62 0.62 0.74 0.18 SEQIDNO:5 AcSTGG(AcK)AP 1.61 0.46 0.78 0.09 1.83 0.83 0.59 0.12 SEQIDNO:6 AcGG(AcK)APR 0.68 0.12 0.60 0.10 2.53 1.29 1.18 0.22 SEQIDNO:7 AcTGG(AcK)APR 0.64 0.10 0.72 0.09 1.72 0.81 0.89 0.23 SEQIDNO:8 AcSTGG(AcK)APR 0.67 0.10 0.93 0.20 1.60 0.79 1.39 0.16 SEQIDNO:9 AcGG(AcK)APRK 1.01 0.18 0.68 0.10 2.33 0.93 1.82 0.23 SEQIDNO:10 AcGG(AcK)APRKQ 0.80 0.21 0.66 0.09 2.09 0.79 1.43 0.20 SEQIDNO:11 AcKSTGG(AcK)AP 0.71 0.11 0.59 0.10 2.12 0.81 1.81 0.69 SEQIDNO:12 AcTGG(AcK)APRK 1.00 0.08 1.00 0.09 1.00 0.36 1.00 0.09
Library Construction
(66) After validating that quantitative quantum dot labeling could be used in conjunction with fluorescence-based bead sorting, an OBOC peptide library was constructed using the split-pool method (Lam et al., 1991, Nature 354: 82-84; Furka et al., 1991, Int. J. Pept. Protein Res. 37: 487-493). Eighteen variable amino acids were used at four positions centered around an acetylated lysine (two amino acids on each side of the acetylated lysine residue). All common natural amino acids excluding cysteine, lysine, methionine and arginine were used. To mimic charged residues, dimethyl arginine was substituted for lysine and arginine. To avoid unwanted cyanogen bromide cleavage points, isosteric norleucine was used in place of methionine. Lysine and cysteine were not included in the library because both residues would produce false hits (in addition to the problems posed by disulfide formation in the latter case) because the nucleophilicity of the amine and sulfhydryl groups respectively would result in their biotinylation and subsequent quantum dot labeling.
(67) The acetylated peptide library was constructed on TentaGel Macrobead NH.sub.2 resin (280-320 m, 0.21 mmol/g loading, 65,550 beads/g) using the split-pool approach. Fmoc/tBu methodology (Bodanszky, 1993, Principles of Peptide Synthesis, 2nd ed., Springer-Verlag, Germany) was used to carry out the library synthesis on 4.80 g of resin. Prior to randomization, a four amino acid linker, BBRM (B=-alanine) was synthesized. After deprotecting the N-terminus with 20% (v/v) piperidine in DMF for 15 min, the resin was split equally into eighteen separate reaction vessels (one for each amino acid in the library). Four equivalents of amino acid and coupling reagent in addition to 5% (mol/mol) capping reagent were added to each vessel for later sequencing.
(68) Capping reagents included phenylacetic acid and 4-pentenoic acid (
(69) In preliminary studies, incorporation of arginine residues beyond the linker position gave false positive signals in the on-bead assays, due to reaction with the biotin ester during the labeling step. This was an unfortunate result, as it precluded incorporation of arginine in the library. The same problems posed by the reactivity of arginine have prevented its incorporation in a previous library (Hu et al., 1999, Biochemistry 38: 643-650). To mimic positively charged residues, lysine and arginine, symmetrical dimethyl arginine was used. Thus, 18.sup.4=104,907 sequences were represented in the library. A threefold excess of beads was used to give 95% probability that all sequences were represented (Altschul et al., 1997, Nucl. Acids Res. 25: 3389-3402).
(70) After library synthesis, the on-bead SIRT1 deacetylation assay was carried out, as shown in
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(73) Determining the viability of quantitative quantum dot analysis was also performed. Five 10 mg portions of TentaGel S NH.sub.2 resin (90 m, 0.26 mmol/g loading, 2.8610.sup.6 beads/g) were divided out and swollen in DCM. After washing with DCM (31 mL) and DMF (31 mL), the beads were labeled with 1, 0.5, 0.01 and 0.001 molar equivalents of N-hydroxysuccinimidobiotin in 200 L portions of DMF. After an hour of rocking at room temperature, the solutions were drained and washed with DMF (31 mL). Approximately 5 mg of resin from each of the above reactions were combined and incubated with 1 mL BSA (1 mg/mL) in TBST buffer (25 mM Tris.HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween 20) for 1 hour. The resin was washed with TBST buffer (31 mL) and drained to the level of the resin bed. At this point, 500 L of 75 nM streptavidin coated Q-dot 605 in TBST buffer was poured over the resin and rocked for 2 hours, after which the solution was drained to the resin bed before washing with TBST (101 mL). Beads were photographed using a fluorescence microscope with a FITC filter and sorted on the basis of fluorescence (.sub.ex=488 nm, .sub.em=610 nm) with a COPAS Select sorting instrument. Sorting data were evaluated with FCS Express (De Novo Software, Thornhill, Ontario) in histogram and dot plot form.
(74) On-Bead Peptide Library Deacetylation by SIRT1
(75) The entire library was assayed in a 75-mL column equipped with a filter. Prior to the assay, the resin was sequentially washed with DCM (350 mL), DMF (350 mL) and deacetylation assay buffer (50 mM Tris, pH 7.5) (150 mL). The enzymatic reaction was initiated upon addition of 50 mL of deacetylation cocktail (0.35 M SIRT1, 1.5 mM -NAD.sup.+, 1 mM DTT). The reaction mixture was allowed to rock gently for 12 min. After draining, the resin was washed with doubly distilled water (550 mL) and DMF (550 mL). Afterwards, the resin was rocked with biotin N-hydroxy-succinimide ester in DMF (3.5 mM, 50 mL) for 20 min. The solution was drained and the resin was washed with DMF (650 mL) and TBST buffer (250 mL).
(76) To reduce nonspecific binding, the beads were incubated with 50 mL of BSA (2 mg/mL) in TBST buffer for 1.5 hours. After draining and washing with TBST buffer (150 mL), 50 mL of 4 nM streptavidin coated Q-Dot 605 in TBST buffer was added and the mixture was allowed to rock for 2 hours. Again, the solution was drained and washed with TBST buffer (1050 mL). The resin was then suspended in a minimal amount of TBST buffer and refrigerated at 4 C. overnight.
(77) Strategy for Sequencing Peptides on Beads
(78) In order to extract peptide sequences from individual beads in the library, a previously developed capping method was improved, in which sequence decoding is done by reading a mass spectral peptide ladder (Youngquist et al., 1995, J. Am. Chem. Soc. 117: 3900-3906). Instead of using the acetyl group for capping during peptide synthesis, two carboxylic acids were used: phenylacetic acid (1) and 4-pentenoic acid (2) (
(79) In each coupling step of a randomized residue, a small amount of capping reagent was added to terminate chain growth for later sequencing (
(80) Library Screening
(81) Beads were sorted on the basis of fluorescence (.sub.ex=488 nm, .sub.em=610 nm) using the COPAS instrument. Initially, the 300 most intensely fluorescent beads (0.1%) were collected, pooled and then sorted a second time to generate an enriched sample of the 45 brightest beads. After washing in a guanidinium hydrochloride solution, single beads were placed in separate microcentrifuge tubes and treated overnight with a cyanogen bromide cleavage cocktail. The cleavage products were subsequently subjected to MALDI-TOF MS for sequence analysis (
(82) Shown in Table 2 are peptide sequences of hits from the SIRT1 combinatorial library screen. Position 2 is the N-terminal end and Position +2 is the C-terminal end. Uncertainty in the order of N-terminal (and adjacent) amino acids is signified by the symbol /.
(83) TABLE-US-00002 TABLE 2 Peptide sequences of hits from the SIRT1 combinatorial library screen Sequence Identifiers Position 2 Position 1 Position 0 Position +1 Position +2 SEQ ID NO: Leu Asn AcLys Asp Gln 26 SEQ ID NO: Trp His AcLys Phe Gln 27 SEQ ID NO: Trp His AcLys Phe Glu 28 SEQ ID NO: Ser Tyr AcLys Gln Trp 29 SEQ ID NO: Gln Pro AcLys Gln Ile 30 SEQ ID NO: Val Gln AcLys Ile Ile 31 SEQ ID NO: His Me.sub.2Arg AcLys Nle Pro 32/33 SEQ ID NO: Ala Val AcLys Phe Nle 34 SEQ ID NO: Asn His AcLys Leu Leu 35 SEQ ID NO: Me.sub.2Arg Phe AcLys Pro Glu 36-37 SEQ ID NO: Nle Nle AcLys Gln Gln 38 SEQ ID NO: Trp Gly AcLys Ser Pro 39 SEQ ID NO: Phe Glu AcLys Tyr Me.sub.2Arg 40-41 SEQ ID NO: Trp Pro AcLys Trp Gln 42 SEQ ID NO: Me.sub.2Arg Ala AcLys Nle Asp 43-44 SEQ ID NO: Gly Thr AcLys Thr Gly 45 SEQ ID NO: Gly Tyr AcLys Pro Thr 46 SEQ ID NO: Ile Phe AcLys Thr Phe 47 SEQ ID NO: Thr Glu AcLys Gln Glu 48 SEQ ID NO: His Trp AcLys Thr His 49 SEQ ID NO: Asp Ser AcLys Gly Ala 50 SEQ ID NO: Ser Asp AcLys Tyr His 51 SEQ ID NO: Asn His AcLys Ile Ile 52 SEQ ID NO: Trp Trp AcLys His Gly 53 SEQ ID NO: Pro Ile AcLys Glu Gln 54 SEQ ID NO: Me.sub.2Arg Pro AcLys Gln Phe 55-56 SEQ ID NO: Asp Val AcLys Nle His 57 SEQ ID NO: Ile Tyr AcLys Asn Asp 58 SEQ ID NO: Thr Pro AcLys Asn Ala 59 SEQ ID NO: Pro Gly AcLys Leu Tyr 60 SEQ ID NO: Me.sub.2Arg/Trp Me.sub.2Arg/Trp AcLys Ile Thr 61-64 SEQ ID NO: Pro/Trp Pro/Trp AcLys Ile Thr 65-66 SEQ ID NO: Me.sub.2Arg/Pro Me.sub.2Arg/Pro AcLys Ser Ile 67-70
Hit Sequencing with MALDI-MS
(84) Beads from the enriched sample were pooled and washed with 8 M guanidinium hydrochloride (21 mL), doubly distilled water (101 mL) and DMF (31 mL). Individual beads were then deposited into separate microcentrifuge tubes containing 20 L of cleavage cocktail (70% TFA, 30% doubly distilled water and 20% cyanogen bromide by weight), as described by Hu et al., 1999). After incubation overnight in the dark, the samples were dried and resuspended in 5 L of 0.1% TFA. Each sample (1 L) was combined with saturated matrix solution (1 L) and dried on the target for MALDI-TOF MS analysis (positive ion mode).
(85) Library Validation
(86) To validate the results of the library screen, select hits and non-hits were resynthesized and subjected to in-solution kinetic analysis (Table 3). A radioactive TLC-based assay was employed with subsaturating levels of [.sup.32P]-NAD.sup.+ to determine the relative catalytic efficiencies (Jackson et al., 2003, J. Biol. Chem. 278: 50985-50998). In addition, two consensus peptides containing residues occurring with the highest and lowest frequency at each position, independent of context, were analyzed. For comparison, a 5-mer comprised of a sequence corresponding to a known site for p53 deacetylation by SIRT1 was assayed (Table 3).
(87) In Table 3, efficiencies (.sup.x=average) were obtained by fitting the data from [.sup.32P]-NAD.sup.+ assays to the modified Michaelis-Menten equation, v=[k.sub.cat/K.sub.m)[S]]/(1+[S]/K.sub.m). No definite catalytic efficiency for VQ(AcK)II SEQ ID NO: 31) was established due to problems with insolubility; a lower limit was established. Catalytic efficiencies of peptides containing the residues of the highest.sup.b/lowest.sup.c frequency at each position and the sequence relevant to p53 deacetylation in vivo.sup.d are shown for comparison.
(88) TABLE-US-00003 TABLE3 Peptidesequencesandcatalyticefficienciesofresynthesizedselect hitsandnon-hitsfromtheSIRT1peptidelibraryscreen Peptide k.sub.cat/K.sub.m(x10.sup.3M.sup.1S.sup.1) SelectHits SEQIDNO:13 QP(AcK)QI 27.2 4.2 SEQIDNO:14 Me.sub.2RP(AcK)QF 14.7 3.2 SEQIDNO:15 Me.sub.2RP(AcK)SI 8.36 0.57 SEQIDNO:16 NH(AcK)II 3.63 0.80 SEQIDNO:17 WH(AcK)FQ 3.29 0.43.sup.x SEQIDNO:18 VQ(AcK)II.sup.a 2.47 1.27.sup.x SelectNon-hits SEQIDNO:19 AY(AcK)EV 5.32 0.63 SEQIDNO:20 QNle(AcK)GF 2.37 0.14 SEQIDNO:21 LNle((AcK)VG 1.61 0.48.sup.x ForComparison SEQIDNO:22 WH(AcK)QQ.sup.b 7.23 1.12 SEQIDNO:23 WP(AcK)QQ.sup.b 1.54 1.06 SEQIDNO:24 EL(AcK)AS.sup.c 1.39 0.10 SEQIDNO:25 HK(AcK)LM.sup.d 3.11 0.45
(89) Hit sequences had significantly higher catalytic activity than non-hits. Some hits were near or greater than an order of magnitude more catalytically active than their non-hit counterparts. Hits correlated with increased catalytic activity by as much as 20-fold. Most hit sequences assayed in solution had significantly higher activity than the peptide sequence relevant to in vivo p53 deacetylation. One non-hit sequence AY(AcK)EV (SEQ ID NO: 19) had a catalytic activity comparable to those of a few of the hits.
(90) Although the apparent second order rate constant (k.sub.cat/K.sub.m) varied widely among the peptides tested, the turnover number (k.sub.cat) was relatively constant at 0.1 s.sup.1. Differences in k.sub.cat/K.sub.m reflect differences in peptide binding affinity.
(91) One of the main advantages of this OBOC library is its context-specific nature. In other words, there is no implicit assumption that residues in substrate sequences function independently of one another. While oriented peptide libraries can be useful in resolving globally-preferred consensus sequences (Songyang et al., 1994; Blander et al., 2005) they do not provide contextual information.
(92) The so-called consensus peptides WH(Ack)QQ (SEQ ID NO: 22) and WP(AcK)QQ (SEQ ID NO: 23) show a seven-fold difference in catalytic activity in favor of WH(AcK)QQ (SEQ ID NO: 22) (Table 3). Thus, in the context of WX(AcK)QQ (SEQ ID NO: 72), a histidine is greatly preferred at position 1. Within the XP(AcK)QX context (SEQ ID NO: 73), QP(AcK)QI (SEQ ID NO: 30) is favored over WP(AcK)QQ (SEQ ID NO: 23) by 18-fold. Thus, SIRT1 mediated deacetylation is stringently context dependent and that there is no best average sequence. Further support comes from the fact that although proline residues (at 1) are not well tolerated when adjacent to a tryptophan at 2, they appear to function well when adjacent to dimethyl arginine at 2. There are synergistic/anagonistic relationships among certain residues and that this plays a significant role in substrate recognition by SIRT1.
(93) BLAST searches of the SIRT1 hits (Table 2) within the mammalian proteome reveal correspondence to a number of proteins (Table 4), some of which are known to be acetylated in vivo.
(94) TABLE-US-00004 TABLE4 BLASTsearchesofSIRT1hitsequences Hit SequenceID Sequence Protein(name,accession,relevantsequence) SEQIDNO:26 LNKDQ Moesin,NP_002435,[Homosapiens] MSNprotein,AAH11827[Homosapiens] SEQIDNO:27 WHKFQ chondroitinsulfateproteoglycan,2NP_004376,[Homosapiens] SEQIDNO:28 WHKFE dualoxidase1precursor,NP_059130[Homosapiens] NADPHthyroidoxidase2,AAF73922,[Homosapiens] SEQIDNO:29 SYKQW fattyacidcoenzymeAligase5,BAA86054[Homosapiens] SEQIDNO:30 QPKQI Notchhomolog4(Drosophila),CAI17543[Homosapiens] Orphansodium-andchloride-dependentneurotransmitter transporter,Q9GZN6,[Homosapiens] SLC6A16protein,AAH34948[Homosapiens] TPA:classIIbHLHproteinscleraxis,DAA00239[Homosapiens] U5snRNP-specificprotein,AAH64370[Homosapiens] apoptosis-regulatedprotein1,AAS64748[Homosapiens] SEQIDNO:31 VQKII Wernersyndrome,AAR05448[Homosapiens] chaperonincontainingTCP1,subunit6B,NP_006575 [Homosapiens] MGC16733protein,AAH09995[Homosapiens] chemokine(C-X-Cmotif)ligand3,NP_002081[Homosapiens] MDN1protein,AAH14882[Homosapiens] LOC150159protein,AAH46636[Homosapiens] C9orf72protein,AAH68445[Homosapiens] SEQIDNO:32 HRKMP SCAM-1protein,AAH67260[Homosapiens] SH3-containingadaptormolecule-1,AAC09244[Homosapiens] KIAA1792protein,BAB47421[Homosapiens] RP5-1187M17.5,CAC32460[Homosapiens] MSTP060,AAO15306[Homosapiens] LAS1-like,NP_112483[Homosapiens] OTTHUMP00000021323,CAH70992[Homosapiens] SEQIDNO:33 HKKMP FLJ00158protein,BA884913[Homosapiens] SEQIDNO:34 AVKFM secretedfrizzled-relatedprotein5,CA114274[Homosapiens] RANbindingprotein17[Homosapiens] SEQIDNO:35 NHKLL protocadherin11,AAK13468[Homosapiens] engulfmentandcellmotility3,NP_078988[Homosapiens] PARP8protein,AAH37386[Homosapiens] complementcomponent3,AAR89906[Homosapiens] WDrepeatdomain35,isoform2,AAH36659[Homosapiens] SEQIDNO:36 RFKPE solutecarrierfamily30(zinctransporter),NP_037441member4 [Homosapiens] ubiquitinspecificprotease53,NP_061923XP_052597 [Homosapiens] membrane-associatedguanylatekinase-related3(MAGI-3), CAH70944[Homosapiens] zinctransporter4,AAB82561[Homosapiens] KIAA1350protein,BAA92588[Homosapiens] cytochromeP450,family2,subfamilyE,polypeptide1,NP_000764 [Homosapiens] SEQIDNO:37 KFKPE Pleckstrinhomologydomaincontaining,familyA(phosphoinositide bindingspecific)member3,AAH44567[Homosapiens] Phosphoinositol4-phosphateAdaptorProtein-1,AAG15199 [Homosapiens] SEQIDNO:38 MMKQQ golgiantigengcp372,BAA05025[Homosapiens] giantin,CAA53052[Homosapiens] SEQIDNO:39 WGKSP apolipoproteinL5,NP_085145[Homosapiens] NY-REN-55antigen,AAD42879[Homosapiens] NIMA(neverinmitosisgenea)-relatedkinase1,NP_036356 XP_291107[Homosapiens] KIAA1901protein,BAB67794[Homosapiens] UDP-N-acetylglucosamine:alpha-1,3-D-mannosidebeta-1,4-N- acetylglucosaminyitransferaseIV,NP_080519[Musmusculus] SEQIDNO:40 FEKYR protocadheringammaA11,AAD43765[Homosapiens] signal-inducedproliferation-associated1like1,NP_056371 [Homosapiens] high-riskhumanpapillomavirusesE6oncoproteinstargetedprotein E6TP1alpha;putativeGAPproteinalpha,AAD12543 [Homosapiens] KIAA0440,BAA23712[Homosapiens] spa-1-like;similartoAF026504(PID:g2555183),AAC83179 [Homosapiens] PRO0097,AAF24015[Homosapiens] SEQIDNO:41 FEKYK Nebulin,P20929[Homosapiens] SEQIDNO:42 WPKWQ Noidenticalsequencematch SEQIDNO:43 RAKMD largetumorsuppressor1.AAD16882[Homosapiens] LATS,largetumorsuppressor,homolog2,NP_055387 [Homosapiens] amyloidprecursor-likeprotein1,AAB50173[Homosapiens] potassiumchannel,subfamilyT,member1,NP_065873XP_029962 [Homosapiens] LOH12CR1,AAK71328[Homosapiens] olfactomedin1,AAP35810[Homosapiens] Zincfingerprotein541,AA101053[Homosapiens] p33,AAG11396[Homosapiens] nucleobindin1,AAP88830[Homosapiens] SEQIDNO:44 KAKMD meltransformingoncogene,NP_005361[Homosapiens] Tcellreceptorbetavariable21/OR9-2,CAH69869[Homosapiens] SEQIDNO:45 GTKTG WDrepeatdomain3,CAI22739[Homosapiens] cytoplasmiclinker2isoform1,NP_003379[Homosapiens] KIAA1858protein,BAB47487[Homosapiens] exophilin5,AAM44402[Homosapiens] SEQIDNO:46 GYKPT complementcomponent4bindingprotein,alpha,CAH70782 [Homosapiens] KCRM_HUMAN;M-CK,AAC62841[Homosapiens] creatinekinase,muscle,AAP35439[Homosapiens] SEQIDNO:47 IFKTF cullin4B,AAR13073[Homosapiens] KIAA0695protein,BAA31670[Homosapiens] SEQIDNO:48 TEKQE caspaserecruitmentdomainfamily,member11,EAL23962 [Homosapiens] CARD-containingMAGUKproteinCARMA1,AAL34460 [Homosapiens] oligophrenin-1likeprotein,AAd39482[Homosapiens] GTPaseregulatorassociatedwiththefocaladhesionkinasepp125, NP_055886[Homosapiens] myosinphosphatase-Rhointeractingprotein,AAQ63176 [Homosapiens] HLC-8,AAO25513[Homosapiens] cardiomyopathyassociatedprotein1,AAQ64003[Homosapiens] TRAFfamilymember-associatedNf-kappaBactivator,NP_665731 [Rattusnorvegicus] RCSD1protein,AAH98426[Homosapiens] SEQIDNO:49 HWKTH Noidenticalsequencematch SEQIDNO:50 DSKGA dentinsialophosphoproteinpreproprotein,NP_055023 [Homosapiens] Monoglyceridelipase,AAH00551[Homosapiens] EGFdomain-containingprotein,AAP35084[Homosapiens] Proteinphosphatase1,regulatory(inhibitor)subunit1A,AAH22470 [Homosapiens] RNAbindingmotifprotein19,NP_057280[Homosapiens] MEGF8,BAA32469[Homosapiens] zincfingerprotein608,NP_065798XP_114432[Homosapiens] valyl-tRNA-synthetaseG7a/Bat6,AAL14460[Musmusculus] SEQIDNO:51 SDKYH Deoxycytidylatedeaminase,P32321(dCMPdeaminase) SEQIDNO:52 NHKII zincfingerprotein588,NP_057304[Homosapiens] zincfingerprotein15-like1(KOX8),NP_067092[Homosapiens] UDP-Gal:betaGlcNAcbeta1,4-galactosyltransferase6variant, BAD92431[Homosapiens] SMAP-7,BAB20272[Homosapiens] SEQIDNO:53 WWKHG Noidenticalsequencematches SEQIDNO:54 PIKEQ procollagen,typeXII,alpha1,NP_031756[Musmusculus] SEQIDNO:55 RPKQF RNAguanylyltransferase,AA888903[Musmusculus] KIAA0992protein,BAA76836[Homosapiens] N-methylpurine-DNAglycosylaseisoform,aNP_002425 [Homosapiens] SEQIDNO:56 KPKQF RP11-334P12.2,CAH71251[Homosapiens] celladhesionkinasebeta,AAC05330[Homosapiens] RNAbindingmotifprotein7,AAH34381[Homosapiens] PTK2Bproteintyrosinekinase2betaisoforma,NP_775266 [Homosapiens] Neuronalamiloride-sensitivecationchannel1,isoform2,AAH75043 [Homosapiens] RNAhelicase,AAD19826[Homosapiens] focaladhesionkinase,AAB47217[Homosapiens] SEQIDNO:57 DVKMH Noidenticalsequencematches SEQIDNO:58 IYKND immunoglobulinsuperfamilyreceptortranslocationassociated 2(IRTA2),CAH71429[Homosapiens] ROS1,AAA60277[Homosapiens] Gprotein-coupledreceptor119,AAP72132[Musmusculus] transmembraneproteinkinase3),AAA36580[Homosapiens] Fcreceptor-likeprotein5,AAK93971[Homosapiens] v-rosUR2sarcomavirusoncogenehomolog1(avian),CAI42375 [Homosapiens] SEQIDNO:59 TPKNA zincfingerprotein440like,NP_001012771XP_371138 [Homosapiens] adenosinedeaminase,RNA-specific,82,NP_443209 [Musmusculus] SEQIDNO:60 PGKLY Phosphatidylethanolaminebindingprotein,AAH08169 [Musmusculus] SEQIDNO:61 RWKIT integrinbeta4,NP_037312[Rattusnorvegicus] SEQIDNO:62 KWKIT desmoglein2,NP_031909[Musmusculus] SEQIDNO:63 WRKIT chromosome6openreadingframe103,CAI16490[Homosapiens] SEQIDNO:64 WKKIT Noidenticalsequencematch SEQIDNO:65 WPKIT EPB41L5protein,AAH32822[Homosapiens] erythrocytemembraneproteinband4.1like4Bisoform1, NP_060894[Homosapiens] EHM2,BAA96079[Homosapiens] KIAA1548protein,BAB13374[Homosapiens] beta-crystallin,AAA52107[Homosapiens] SEQIDNO:66 PWKIT REPS1protein,AAH21211[Homosapiens] RALBP1associatedEpsdomaincontaining1,CAI42879 [Homosapiens] sodiumpotassiumchloridecotransporter2,NP_000329 [Homosapiens] SEQIDNO:67 RPKSI alpha(1,3)-fucosyltransferase;ELFT,AAB20349[Homosapiens] nuclearfactorI,AAB52369[Homosapiens] fucosyltransferase4,NP_002024[Homosapiens] ELAM-1ligandfucosyltransferase,AAA63172[Homosapiens] calmodulinregulatedspectrin-associatedprotein1-like1,NP_982284 XP_036589[Homosapiens] CAMSAP1L1protein,AAH11385[Homosapiens] SEQIDNO:68 KPKSI TransientreceptorpotentialcationchannelsubfamilyMmember7 (Longtransientreceptorpotentialchannel7)(LTrpC7)(Channel- kinase1),Q96QT4[Homosapiens] Synapse-associatedprotein102,AAH93864[Homosapiens] C21orf7formC,AAF81753[Homosapiens] c2lorf7formB,AAF81752[Homosapiens] TAK1-likeprotein,AA016519[Homosapiens] MAP4K3,AAN75849[Homosapiens] channel-kinase1,AAK19738[Homosapiens] transcriptionfactorGATA-6,NP_999493[Susscrota] SEQIDNO:69 PRKSI poly(ADP-ribose)polymerasefamily,member6isoform1, NP_064598[Homosapiens] C19orf2protein,AAH14933[Homosapiens] RPB5-mediatingprotein,isoformb,AAH67259[Homosapiens] NNX3,AAD08679[Homosapiens] SEQIDNO:70 PKKSI zincfingerprotein318,CAH71374[Homosapiens] Alanyl-tRNAsynthetase,AAH11451[Homosapiens] histoneH1,AAN06703[Homosapiens] nucleoporin,BAB18537[Homosapiens] Innermembraneprotein,mitochondrial,AAH02412[Homosapiens] ZNF318protein,AAH30687[Homosapiens]
Example 2. Use of a Combinatorial Library to Identify Histone-Specific Protein Binding
(95) General
(96) An OBOC histone H4N-terminal tail combinatorial library was constructed to identify the binding preferences of the antibody toward all known possible histone modification states. The H4 histone tail library was comprised of the sequence corresponding to the first 21 amino acids of human histone H4 attached to a linker composed of 2 -alanines (B) and a methionine (M). The library included 800 unique peptide sequences, representing all known modification states for the first 21 amino acids of histone H4 in addition to all possible methylation states at lysines and arginines that are known to be methylated. Using an -phos (S1) H4 antibody as a primary antibody, the library was screened to determine histone H4N-terminal sequences to which the primary antibody specifically bound.
(97) Amino acid derivatives and resins were purchased from Peptides International (Louisville, Ky.), Novabiochem (San Diego, Calif.), or from Bachem (Bubendorf, Switzerland). Other chemical reagents were obtained from Sigma-Aldrich, Invitrogen (Carlsbad, Calif.), or Jackson ImmunoResearch Laboratories (West Grove, Pa.). The -phos (S1) H4 antibody was a gift from the laboratory of C. David Allis (Rockefeller University, New York, N.Y.). Peptides were synthesized on a Symphony synthesizer from Protein Technologies (Tucson, Ariz.). Filter columns for on-bead assays were obtained from Alltech (Deerfield, Ill.).
(98) Analytical gradient HPLC was performed on a Shimadzu series 2010C HPLC with a Vydac C18 column (10 m, 4.6250 mm). All runs employed linear gradients of 0.05% aqueous TFA and 0.02% TFA in acetonitrile. Microextraction tips for desalting peptides were purchased from Varian, Inc. (Palo Alto, Calif.). MALDI-TOF MS was performed on a Bruker REFLEX II and MALDI TOF-TOF MS was executed on an Applied Biosystems 4800. A Zeiss Axioplan 2 microscope (Jena, Germany) with a DAPI dye bandpass filter (390-410 nm) and an AxioCam MRm was used for fluorescence microscopy.
(99) On-Bead Assay with Peptide Standards
(100) Five-mg quantities of TentaGel Macrobead NH.sub.2 resin (280-320 m, 0.27 mmol/g loading, 65,550 beads/g) bearing either a phosphorylated or unphosphorylated histone H4 sequence (or a mixture) were added to 1.5 mL filter columns, washed thoroughly with DCM, MeOH, doubly distilled water (ddH.sub.2O) and PBST buffer (25 mM NaPi, pH 7.4, 150 mM NaCl, and 0.1% Tween 20). The resin was swelled for 1 hour with gentle rocking prior to drainage and one hour of blocking with 3% (w/v) bovine serum albumin (BSA) in PBST. After draining the blocking solution to the resin bed, 100 L of a 100:1 dilution of -phos (S1) H4 antibody in PBST containing 3% BSA was added and the resin was allowed to rock gently for one hour.
(101) After draining to the resin bed, the resin was washed 3100 L PBST and 100 L of 50 nM biotinylated goat-anti-rabbit antibody in PBST containing 3% BSA was added. One hour of gentle rocking was followed by draining the solution to the resin bed and washing 3100 L PBST.
(102) The resin was incubated with 100 L of 25 nM solution of Q-dot 605 streptavidin conjugate in PBST and gently rocked for 2 hours. Following drainage to the resin bed, the resin was washed 10200 L PBST. The resin was then resuspended in PBST and viewed under a fluorescence microscope.
(103) Library Construction
(104) The combinatorial histone H4 peptide library was constructed on TentaGel Macrobead NH.sub.2 resin (280-320 m, 0.27 mmol/g loading, 65,550 beads/g) using the split-pool approach (Lam et al., 1991, Nature 354: 82-84; Furka et al., 1991, Int. J. Pept. Protein Res. 37: 487-493) for sites of variability. Sites of variability include positions 20 (K, AcK, MeK, Me.sub.2K, Me.sub.3K), 16 (K,AcK), 12 (K,AcK), 8 (K,AcK), 5 (K,AcK), 3 (R, MeR, Me.sub.2R.sub.symmetric, Me.sub.2R.sub.asymmetric, citrulline) and 1 (S, pS). The synthesis was performed on a 50 mol scale with standard Fmoc/tBu chemistry (Bodanszky M., 1993, Principles of Peptide Synthesis, 2nd ed., Springer-Verlag, Germany). All amino acids (at least 4.7 equivalents/coupling) were double coupled for 2 hour time periods.
(105) Prior to the partially randomized histone H4 sequence, a 3 amino acid linker, BBM (where B=-alanine, M=methionine) was synthesized. After the final N-terminal deprotection, the N-termini of all the peptides were acetylated with acetic anhdyride. A 50 mg (13.5 mol) portion of the library was deprotected for 5 hours with Reagent K (TFA/EDT/thioanisole/water/phenol: 82.5%, 2.5%, 5%, 5%, 5%) prior to use, as described by King et al., 1990, Int. J. Pept. Protein Res. 36: 255-266. The remainder of the library was stored at 4 C. for later use.
(106) Evaluation of Integrity of the Peptide Library
(107) Twenty beads were randomly selected from the library and deposited into separate microcentrifuge tubes containing 20 L of cleavage cocktail (70% TFA, 30% ddH.sub.2O and 20% cyanogen bromide by weight; Hu et al., 1999, Biochemistry 38: 643-650). After incubation overnight in the dark, the samples were dried.
(108) Ten of the cleavage products were desalted and sequenced by MALDI TOF-TOF MS. The remaining 10 cleavage products were dissolved in 1004 quantities of ddH.sub.2O and analyzed by analytical RP-HPLC. Fractions corresponding to the primary peak at 214 nm were lyophilized and resuspended in 5 L of ddH.sub.2O. Each sample (1 L) was combined with saturated matrix solution (1 L) and dried on the target for MALDI-TOF MS analysis (positive ion mode).
(109) On-Bead Library Prescreen and Screen
(110) Prescreen was performed by adding 50 mg (13.5 mol) of the peptide library to a 4 mL filter column and washing it thoroughly with DCM, MeOH, ddH.sub.2O and PBST buffer. The resin was swelled for 1 hour with gentle rocking prior to drainage and one hour of blocking with 3% BSA in PBST. After draining the blocking solution to the resin bed, 1 mL of 50 nM biotinylated goat-anti-rabbit antibody in PBST containing 3% BSA was added. Following 1 hour of rocking, the solution was drained to the resin bed and washed 31 mL PBST. The resin was then rocked with 1 mL of a 25 nM solution of Q-dot 605 streptavidin conjugate in PBST for two hours. Following drainage to the resin bed, the resin was washed 102 mL PBST. At this point, the resin was resuspended in PBST and viewed under a fluorescent microscope and any fluorescent beads could be removed from the library.
(111) After prescreening the library for nonspecific interactions with the secondary antibody or the quantum dots, a screen was performed. The only difference from the prescreen was a one hour incubation with 1 mL of a 100:1 dilution of -phos (S1) H4 in PBST with 3% BSA after the swell step and washing 31 mL PBST prior to addition of the secondary antibody. When viewed under the microscope, a number of brightly fluorescent, moderately fluorescent and dark beads were manually selected.
(112) Peptide Sequencing with MALDI TOF-TOF MS
(113) Beads that were selected under the microscope were incubated with 200 L of 8 M guanidinium hydrochloride prior to washing 3500 L ddH.sub.2O and drying. Peptides were cyanogen bromide cleaved from each bead and desalted before sequencing with MALDI TOF-TOF MS.
(114) On-Bead Western Analysis
(115)
(116)
(117) After demonstrating detection of serine phosphorylation of a histone H4 sequence immobilized on a bead (
(118) The library synthesis was followed by rigorous evaluation of the synthetic product. RP-HPLC analysis of the cleavage products from 10 individual beads revealed peptides of 90-95% purity within the correct mass range. In addition, the cleavage products from 10 additional randomly selected beads were successfully sequenced with MALDI TOF-TOF MS.
(119) The library was first prescreened with only the secondary antibody and quantum dots. The fact that none of the beads exhibited fluorescence due to quantum dots suggested the absence of non-specific interactions between the immobilized peptides with either the secondary antibody or the quantum dots. Therefore, when the primary antibody was included in a screening experiment, the fluorescence observed was due to a specific interaction with the primary antibody (
(120)
(121) Data from the screen indicate the binding preferences of the -phos (S1) H4 antibody for certain sequences (Table 5). Twenty beads were manually selected from a screen of a histone H4 tail library. Sequences were elucidated by MALDI TOF-TOF MS. All sequences obtained from fluorescent beads were phosphorylated while the moderately fluorescent beads displayed peptides that were typically phosphorylated (and generally highly-modified). Eighty percent of the dark beads harbored peptides that were unphosphorylated. Legend: pS=phosphorylated serine, AcK=acetylated lysine, MeK, Me.sub.2K, Me.sub.3K=the correspondingly methylated states of lysine, MeR and Me.sub.2R=the correspondingly methylated states of arginine where (a) and (s) refer to symmetric and asymmetric respectively.
(122) TABLE-US-00005 TABLE 5 Binding preferences of the -phos (S1) H4 antibody Bead Position Position Position Fluorescence Position 1 Position 3 Position 5 Position 8 12 16 20 Fluorescent pS Mc.sub.2R (a) AcK K K AcK MeK Fluorescent pS Me.sub.2R (a) K/AcK K/AcK K K MeK Fluorescent pS Me.sub.2R (a) K AcK MeK K AcK Fluorescent pS MeR AcK AcK K K AcK/Me.sub.3K Fluorescent pS R K AcK Me.sub.2K K MeK Fluorescent pS MeR K AcK K K AcK/Me.sub.3K Fluorescent pS R K K AcK/Me.sub.3K K MeK Fluorescent Poor quality (3 beads) Moderately pS MeR AcK AcK Ack/Me.sub.3K AcK Me.sub.2K Fluorescent Moderately pS Me.sub.2R (s) AcK AcK MeK AcK Me.sub.2K Fluorescent Moderately S Me.sub.2R (s) AcK AcK Me2K K K Fluorescent Moderately Poor quality (but appear to be phosphorylated Fluorescent and heavily modified - 2 beads) Dark S Me.sub.2R (s) K K MeK K K Dark S R K K Me.sub.2K K AcK/Me.sub.3K Dark pS Me.sub.2R (s) K AcK AcK/Me.sub.3K AcK MeK Dark S MeR K K AcK K MeK Dark S MeR K K Me.sub.2K K Me.sub.3K
(123) It is to be understood that this invention is not limited to the particular devices, methodology, protocols, subjects, or reagents described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the claims. Other suitable modifications and adaptations of a variety of conditions and parameters normally encountered in clinical prevention and therapy, obvious to those skilled in the art, are within the scope of this invention. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.