REVERSIBLE ACETYLATION

The Smith lab has two focus areas in the field of reversible acetylation: sirtuin deacetylases and bromodomain-containing proteins.

SIRTUINS

Sirtuins catalyze the NAD+-dependent deacylation of acyl-lysine residues, producing O-acyl-ADP-ribose and nicotinamide. Humans encode seven sirtuins (Sirt1-7) that are considered pro-survival proteins. Decreased sirtuin activity promotes inflammation and aging-related diseases. However, how sirtuin activity is inhibited during aging is largely unknown. We seek to define the physiological mechanisms regulating sirtuin activity post-translationally. It is known oxidative stress increases with age and inflammatory disease states; thus are interested in regulation of sirtuin activity by post-translational modification by cellular oxidants.

We have demonstrated that Sirt1 can be S-nitrosated by the small molecule nitrosothiol S-nitrosoglutathione (Kalous et al, 2016).  Sirt1 S-nitrosation correlated with Zn2+-release and loss of α-helical structure, suggesting the target of nitrosation is the Zn2+-tetrathiolate domain conserved among sirtuins. Sirt1 S-nitrosation was reversed upon exposure to thiol-based reducing-agents, resulting in restoration of Sirt1 activity. This restoration was dependent on the presence of Zn2+, consistent with nitrosation of the Zn2+-tetrathiolate as the source of Sirt1 inhibition.

More recently we have expanded our studies to examine the susceptibility of other sirtuins to S-nitrosation and other oxidative modifications (Kalous et al, 2020), along with the development of targeted activity-based probes for sirtuin family members (Goetz et al, 2020).

Finally, we have adopted cell-based systems to examine the role these modifications play under more physiological conditions.

SELECTED REFERENCES

Goetz CJ, Sprague DJ, Smith BC. "Development of activity-based probes for the protein deacylase Sirt1." Bioorganic Chemistry. 2020, 104:104232. PMID: 32911193.

Kalous KS, Wynia-Smith SL, Summers SB, Smith BC. "Human sirtuins are differentially sensitive to nitrosating agents and other cysteine oxidants." Journal of Biological Chemistry. 2020 [epub ahead of print] doi: . PMID 32371394

Kalous KS, Wynia-Smith SL, Olp MD, Smith BC. “Mechanism of Sirt1 NAD+-dependent Deacetylase Inhibition by Cysteine S-nitrosation”. Journal of Biological Chemistry. 2016, 291, 25398-410.

Smith BC, Settles B, Hallows WC, Craven MW, Denu JM. “SIRT3 substrate specificity determined by peptide arrays and machine learning”. ACS Chemical Biology. 2011, 6, 146–57.

Smith BC, Hallows WC, Denu JM. “A continuous microplate assay for sirtuins and nicotinamide-producing enzymes”. Analytical Biochemistry. 2009, 394, 101–9.

Smith BC and Denu JM. “Mechanism-based inhibition of Sir2 deacetylases by thioacetyl-lysine peptide”. Biochemistry. 2007, 46, 14478–86.

Smith BC and Denu JM. “Sir2 deacetylases exhibit nucleophilic participation of acetyl-lysine in NAD+ cleavage”. Journal of the American Chemical Society. 2007, 129, 5802–3.

 

BROMODOMAIN CONTAINING PROTEINS

Bromodomains are chromatin interaction domains that can control gene expression by directing assembly of transcriptional complexes on chromatin, doing so by binding acyl-lysine residues on histones and transcription factors. The importance of the normal function of these proteins is highlighted by their implication in several diseases, including cancer and type II diabetes.

Our most recent published work (Olp et al, 2020) identifies a novel ligandable site unique to the second bromodomain of BRD4, paving the way for further development of BRD4-specific inhibitors.

We have also published work reporting on the ability of BET family bromodomain proteins to bind several different acylation states of histones, including several that had never been tested for bromodomain binding. Additionally, effects of neighboring methylation, phosphorylation, and acylation were investigated.

Currently we have extended our studies of bromodomain containing proteins into cellular contexts with an eye towards determining their role in disease pathogenesis.

REFERENCES

Olp MD, Sprague DJ, Goetz CJ, Kathman SG, Wynia-Smith SL, Shishodia S, Summers SB, Xu Z, Statsyuk AV, Smith BC. “Covalent-Fragment Screening of BRD4 Identifies a Ligandable Site Orthogonal to the Acetyl-Lysine Binding Sites.” ACS Chemical Biology. 2020 March 23. PMID 32149490

Fahey JM, Stancill JS, Smith BC, Girotti AW. “Antagonistic effects of nitric oxide in a glioblastoma photodynamic therapy model: mitigation by BET bromodomain inhibitor JQ1”. Journal of Biological Chemistry. 2018, 293, 5345-59.

Egner JM, Jensen DR, Olp MD, Kennedy NW, Volkman BF, Peterson FC, Smith BC, Hill RB. “Development and Validation of 2D Difference Intensity Analysis for Chemical Library Screening by Protein-Detected NMR Spectroscopy”. ChemBioChem. 2017, 19, 448-58. Appeared as the Cover Article

Olp MD, Zhu N, Smith BC. “Metabolically Derived Lysine Acylations and Neighboring Modifications Tune the Binding of the BET Bromodomains to Histone H4”. Biochemistry. 2017, 56, 5485-5495.

 

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