January 2011 Meeting Announcement, Delaware Valley Mass Spectrometry Discussion Group
PLEASE NOTE: We will meet in Mendel 154.
- Topic: "Complementary mass spectrometry-based proteomics provides novel insights into phosphorylation-dependent mechanisms of histone deacetylase regulation"
- Speaker: Todd Greco, Princeton
- Date: Monday, January 10, 2011. 6:30 PM
- Time: Social Hour: 6:30 PM.
Talk: 7:30 PM.
Please RSVP to Karen Wendling WendlingK@chc.edu by Thursday January 6th.
- Place: Department of Chemistry, Villanova University (Room 154, Mendel Hall)
Chromatin remodeling protein complexes provide the enzymatic and structural scaffolding that control cellular gene transcription, in part due to their recruitment of histone deacetylase (HDAC) enzymes. For a subset of HDAC enzymes, their influence on transcriptional regulation is regulated by a unique mechanism--a nucleo-cytoplasmic shuttling dependent on phosphorylation. Of interest to this particular study is histone deacetylase 5 (HDAC5), which plays critical roles in promoting cellular differentiation and growth during cardiac development and in response to hypertrophic stimuli. Previous studies have established that nucleo-cytoplasmic shuttling can be regulated by phosphorylation of two key serine residues, facilitating recruitment of 14-3-3 proteins. Given the importance of phosphorylation in regulating HDAC5 and the fact that no unbiased mapping of histone deacetylases had been performed, this study comprehensively investigated the phosphorylation status of immunoisolated HDAC5 by using complementary enzymatic digestion and peptide fragmentation techniques. Mass spectrometry analysis on a MALDI and ESI LTQ-Orbitrap XL and ESI LTQ-Orbitrap Velos-ETD afforded 17 in vivo phosphorylation sites; 14 of which had not been previously identified. Of note, HCD and ETD fragmentation on the Velos platform provided complementary high mass accuracy determination of fragment ions and near-complete backbone sequence ion coverage, which aided in phosphosite localization. Using HDAC5 serine-to-alanine phosphomutants the effect of individual phosphorylation sites on deacetylase activity and cellular localization were assessed. Stable cells lines expressing HDAC5 phosphomutants and live cell imaging allowed the identification of phosphoserines within the nuclear localization signal (NLS) as critical for efficient nuclear import of HDAC5. Interestingly, label-free proteomic analysis of immunoisolated HDAC5 NLS phosphomutants revealed decreases in nuclear corepressor and protein kinase D protein-protein interactions. These results provide the first evidence for phosphorylation events outside of the known 14-3-3 sites that result in downstream alterations in protein interactions. Overall, these studies identified Ser279 as a critical phosphorylation within the NLS, which provides a regulatory point in nucleo-cytoplasmic shuttling that may be conserved in HDAC4 and HDAC9.
- Bio: Todd Greco is currently a post-doctoral research associate in the lab of Ileana Cristea at Princeton University. He obtained his undergraduate degree from the University of Delaware (Honors B.S. in Biochemistry), where he completed his senior thesis on the substrate specificity of the pseudouridine synthase RluA in the lab of Eugene Mueller. He earned his graduate degree (Ph.D. in Neuroscience) in the lab of Harry Ischiropoulos at the University of Pennsylvania and Children’s Hospital of Philadelphia Research Institute, where he used mass spectrometry-based proteomics to profile nitric oxide-derived protein post-translational modifications as well as to develop quantitative proteomic methods for the characterization of astrocyte cell secretomes. Currently, Todd is utilizing these techniques in conjunction with cell biology tools to understand the molecular events underlying herpes simplex virus infection and how cellular chromatin remodeling protein complexes are influenced by viral infection. An important aspect of his research involves the development of proteomic methods to characterize viral-host cell protein complexes and identification of novel protein-protein interactions and protein post-translational modifications that are dynamically regulated during the viral life cycle. Todd is funded through the Human Frontier Science Program Grant, co-awarded to Drs. Ileana Cristea, Kay Grünewald (Oxford University), Frank Alber (University of Southern California), and Maya Topf (University of London).
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