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Dr. Smith welcomes graduate students interested in learning and applying a variety of quantitative and chemical approaches to the study of protein post-translational modifications. Potential postdoctoral research fellows are also encouraged to apply. The Smith lab uses an interdisciplinary approach that combines protein biochemistry, enzymology, organic and peptide synthesis, high-throughput screening, structural biology, cell biology, proteomics, metabolomics, and other techniques to understand the control of protein function through post-translational modification and exploit these new insights in the design of novel chemical probes or drug lead compounds.
Recent estimates indicate that the human genome only contains ~19,000 protein encoding genes, or slightly less than the nematode worm genome. Protein post-translational modification is largely responsible for the complexity arising from the small number of genes that humans possess. The Smith lab focuses on the post-translational modification of critical charged and nucleophilic residues by lysine acylation and cysteine S-nitrosation.
Post-translational modification of histones, transcription factors, and other nuclear proteins forms the basis of epigenetics and regulates transcription through unknown mechanisms. The combinatorial effect of these post-translational modifications underlies the “histone language” that is interpreted by three broad protein classes: “writers”, “readers”, and “erasers”. Lysine residues are particularly abundant targets of epigenetic regulation and are modified by an astounding array of modifications including methylation, ubiquitinylation, sumoylation, ADP-ribosylation, oxidation, and acylation. One focus of my laboratory will be on the mechanism and regulation of the sirtuin and bromodomain family of proteins that erase and read sites of epigenetic lysine acylation.
Bromodomain reading of the acyl-lysine histone language
Bromodomain inhibitors are being developed to treat leukemia, lymphoma, and inflammation. Although bromodomains were originally described as readers of acetyl-lysine, many other histone acyl-lysine modifications were recently discovered. The distinct functions of these unique acyl-lysines are unknown; these functions will be elucidated in this project. The lab also aims to develop chemical probes allowing discovery of bromodomain drug targets and to profile bromodomain inhibitors.
Physiological and pharmacological regulation of sirtuin histone deacylases
The pharmaceutical industry is intensely interested in developing sirtuin activators for the treatment of a variety of aging-associated diseases including cancer, type II diabetes, and neurodegenerative disorders. However, existing activators of the human sirtuin, Sirt1, act through unknown mechanisms. In this project area, the lab seeks to investigate the binding sites and activation mechanisms of Sirt1 activators using hydrogen-deuterium exchange mass spectrometry. Additionally, Sirt1 is inactivated by cysteine S-nitrosation, but the mechanism of inactivation is unknown. The mechanism of Sirt1 inactivation by S-nitrosation will be determined. The lab seeks to also investigate potential sites of post-translational modification in other sirtuin family proteins.
Nitric Oxide (NO) plays critical roles in mammalian physiology including the immune response where cytotoxic levels of NO are generated at sites of infection or inflammation and in signaling where local bursts of NO mediate intercellular communication in cellular processes such as neurotransmission and smooth muscle relaxation. “Classical” NO signaling involves the production of NO by nitric oxide synthase (NOS) and activation of soluble guanylate cyclase (sGC). Recently, an alternate, sGC-independent NO signaling pathway has emerged as a new frontier in NO biology. S-nitrosation, the post-translational modification of cysteine residues by NO to form nitrosothiols controls protein structure and activity. Dysregulation of protein S-nitrosation has been implicated in a broad range of human diseases including heart disease, stroke, Parkinson’s disease, asthma, and cystic fibrosis. The overall goal of this project will be to provide the molecular underpinnings for cysteine S-nitrosation in vivo.
Develop chemical tools and proteomic techniques for detection of protein S-nitrosation
Since existing techniques to detect cellular S-nitrosation are plagued by high background, low selectivity, and low sensitivity, new chemical tools and proteomic techniques will be developed to detect and study protein S-nitrosation. Several groups recently published intriguing new chemistry for the direct reaction of nitrosothiols with triphenylphosphine derivatives. However, the application of arylphosphines to protein S-nitrosation has been severely hampered by poor water solubility. We seek to design, synthesize, and characterize water-soluble probes for direct detection and enrichment of protein S-nitrosation sites.
Existing proteomic methods to detect NO-dependent cysteine modifications are designed to exclusively identify S-nitrosation. These approaches only detect a subset of possible NO-dependent cysteine modifications. We will develop a proteomics approach to allow identification and direct quantitation of all possible NO-dependent cysteine modifications.
Identify and characterize metabolic pathways targeted by cysteine S-nitrosation using proteomics and metabolomics
Recent evidence suggests that nearly every major metabolic enzyme is nitrosated on at least one cysteine. To determine which of these S-nitrosation sites control protein function, we performed metabolomic analysis of nitric oxide synthase knockout mice and correlated dysregulated metabolites with known S-nitrosation sites. We will use the chemical tools and proteomic techniques that we develop to characterize these S-nitrosation sites in vitro and in cell culture. Overall, these studies will contribute to understanding the molecular mechanisms through which cysteine S-nitrosation contributes to normative and disease states.