Entwicklung und Nutzung chemischer Werkzeuge zur Untersuchung von Histon-Modifikationen und den zugehörigen Enzymen
Final Report Abstract
The scientific results and further perspectives can be summarized as follows: Identification and characterization of phosphorylation-dependent histone binding proteins and phosphatases: Results: We used phosphonomethylene alanine (Pma), a non-hydrolyzable analog of phospho-Ser, as a pSer substitute in proteomic investigations. - We established a new strategy for uncovering specific binding proteins of unmodified histone tails by proteomic screens with histone tail peptides synthesized from L- and D-amino acids. - We conducted a proteomic screen based on SILAC (stable isotope labeling by amino acids in cell culture) and identified a) H3pSer10-dependent binding proteins and b) binding proteins of unmodified histone H3, which are blocked by H3-Ser10 phosphorylation. - We did not detect histone phosphatases that bound to H3Pma10. - We identified several 14-3-3 isoforms that bound to H3pSer10, some of which have not been described in this context before. - We uncovered HAT1 and RBBP7 as specific binders of H3pSer10 which are blocked by H3 phosphorylation. Future perspectives: Importantly, the developed strategy of screening interactions profiles with peptide baits synthesized from L- and D-amino acids is generally applicable for uncovering specific binding proteins of unmodified histone tails. Based on this it is conceivable to extend this approach to the tails of H4, H2A and H2B. - The initial concept of using synthetic amino acids as traps for de-modifying enzymes was further pursuit for HDACs enabling a substrate-selectivity profiling. New methods for detecting HDAC and HAT activities: Results: - Three types of HDAC assays were developed: 1.) simple colorimetric assays that can be used in a high-throughput format and 2.) assays that report the catalytic activities of HDACs and HATs on several modification sites at once by NMR and 3.) miniaturized assays based on MALDI-TOF which enables kinetic measurements with very limited amounts of enzymes. - The colorimetric assay allows simple access to the required substrates, can be adapted to a high-throughput format and is not affected by a common assay artifact of conventional assays. - The NMR-bases assay allows non-destructive analysis at several acetylation sites in real time. The assay can be used for other types of PTMs as well. - The miniaturized assay is highly applicable for studying native HDACs immunoprecipitated from mammalian cells. - Applications of all of these assays enabled various insights into the function and substrate selectivity of HDACs. Future perspectives: - Investigating HDACs has become a central focus of our working group and sophisticated HDAC assays will be used in order to conduct this research. - We plan to use the above-described assays to validate the findings obtained by substrate selectivity profiling of HDACs. Establishing the transpeptidase sortase A as a tool for chromatin research: Results: - We have established a phage-display scheme for selecting sortase mutants that recognize altered sorting sequences. - We have evolved a sortase mutant (F40) that recognizes an amino acid sequence found in histone H3. - We could demonstrate that this F40 mutant is able to catalyze ‘traceless’ semisynthesis of histone H3. - We have further used the phage-displayed sortases in combination with “bar-coding” of phage genomes to encode synthetic peptides with defined modification patterns. - These modified and “bar-coded” phages have been used to analyze the phosphorylation-dependent interactions of SH2 domains and 14-3-3 proteins with substrate peptides. Future Perspectives: - The phage-display setup can be further used for evolving the substrate selectivity and catalytic properties of sortases. A grant application is currently in preparation. - The “bar-coded” phages displaying sortase will be used for determining the modification preferences of predicted histone binding proteins. - Using sortases for semisynthesis of histone H3 on the level of reconstituted nucleosomes served as that bases for a successful grant application aimed to analyze the epigenetic histone code. Substrate selectivity profiling of HDACs: Results: - We have established hydroxamate-based amino acids imbedded in peptides as probes for capturing HDACs. - These probes enable the profiling of the substrate selectivity of endogenous HDACs in cellular lysates. - The probes can be further used to co-isolate HDAC-binding proteins. Future Perspectives: - Our group will continue to use and establish new HDAC probes in order to understand the biology of these enzymes. - Future investigations will include the development of new HDAC-trapping amino acids and a brought range of peptides containing these HDAC-traps.
Publications
- Chemoselective Ligation and Modification Strategies for Peptides and Proteins. Angew. Chem. Int. Ed. 47 (2008) 10030-10074
Hackenberger C.P.R., Schwarzer D.
- TRIM24 links a noncanonical histone signature to breast cancer. Nature 468 (2010) 927-932
Tsai W.-W., Wang Z., Yiu T.T., Akdemir K.C., Xia W., Winter S., Tsai C.-Y., Shi X., Schwarzer D., Plunkett W., Aronow B., Gozani O., Fischle W., Hung M.-C., Patel D.J., Barton M.C.
- Directed evolution of sortase A mutants with altered substrate selectivity profiles. J. Am. Chem. Soc. 133 (2011) 17536-17539
Piotukh K., Geltinger B., Heinrich N., Gerth F., Beyermann M., Freund C., Schwarzer D.
- NMR profiling of histone deacetylase and acetyl-transferase activities in real time (2011), ACS Chem Biol. 6 (2011) 419-424
Dose A., Liokatis S., Theillet F.-X., Selenko P., Schwarzer D.
- Facile synthesis of colorimetric histone deacetylase substrates. Chem Commun. 48 (2012) 9525-9527
Dose A., Jost J.O., Spieß A.C., Henklein P., Beyermann M., Schwarzer D.
(See online at https://doi.org/10.1039/c2cc34422j) - Phosphorylation of histone H3 Ser10 establishes a hierarchy for subsequent intramolecular modification events. Nat. Struct. Mol. Biol. 19 (2012) 819-823
Liokatis S., Stützer A., Elsässer S.J., Theillet F.-X., Klingberg R., van Rossum B., Schwarzer D., Allis C.D., Fischle W., Selenko P.
(See online at https://doi.org/10.1038/nsmb.2310) - The Alzheimer's Disease Related Tau Protein as a New Target for Chemical Protein Engineering. Chemistry. 18 (2012) 2488-2492
Broncel M., Krause E., Schwarzer D., Hackenberger C.P.
(See online at https://doi.org/10.1002/chem.201103032) - Probing the acetylation code of histone H4. Proteomics. 13 (2013) 2989-2997
Lang D., Schümann M., Gelato K., Fischle W., Schwarzer D., Krause E.
(See online at https://doi.org/10.1002/pmic.201200568) - Probing the recognition of post-translational modifications by combining sortase-mediated ligation and phage-assisted selection. ACS Chem Biol. 8 (2013) 1692-1697
Teschke T., Geltinger B., Dose A., Freund C., Schwarzer D.
(See online at https://doi.org/10.1021/cb4001487) - Accessibility of Different Histone H3-Binding Domains of UHRF1 Is Allosterically Regulated by Phosphatidylinositol 5-Phosphate. Mol Cell. 54 (2014) 905-919
Gelato K.A., Tauber M., Ong M.S., Winter S., Hiragami-Hamada K., Sindlinger J., Lemak A., Bultsma Y., Houliston S., Schwarzer D., Divecha N., Arrowsmith C.H., Fischle W.
(See online at https://doi.org/10.1016/j.molcel.2014.04.004)
