RSS 1.0Faculty, Student Works & Conferences School of Pharmacy
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The role of a cysteine residue within an ERK1/2 substrate docking site on signaling and proliferation of melanoma cells containing BRAF mutationsThe role of extracellular signal-regulated kinase 1/2 (ERK1/2) in signaling pathways in cells is crucial for cell proliferation. Within specific types of cancers, a member of this pathway, BRAF, is mutated at the valine (V)600 position so that this pathway is continuously activated, leading to uncontrolled proliferation. A docking site in ERK1/2 is of interest for inhibitors to control activation of downstream proteins responsible for transcription.1 A compound has been developed to target a substrate docking site and was found to target a specific cysteine2. This residue has been mutated via CRISPR CAS9 in both ERK1 and ERK2 and the proposed studies investigate the effects the ERK1/2 cysteine mutations have on A375 cell melanoma cells regarding cell signaling and proliferation.
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Zinc Fingers are a General Target for Persulfidation by Hydrogen Sulfide (H2S)Persulfidation (or "sulfhydration") of protein thiols by the gasotransmitter hydrogen sulfide (H2S) has been recently established as an important signaling event associated with oxidative stress and cellular aging. While H2S, mostly bisulfide (HS-) anion at physiological pH, likely does not persulfidate protein thiols directly due to incompatible electric potential, it may act through small- molecule thiols (ex: glutathione) to add a sulfur to cysteine thiols (CysSH → CysSSH). This highly reactive persulfide species can alter protein function and/or scavenge intracellular radical species during oxidative stress by direct reaction with radicals. Zinc finger proteins (ZFs) are potential targets for this PTM as they contain Cys-rich zinc-binding domains and our laboratory has previously reported the direct reaction of the ZF protein TTP with H2S. This reaction requires O2 and involves in-situ persulfidation. To understand how general ZF persulfidation is, we applied a persulfide- specific proteomics approach and observed a trend between Cys content of ZF domains and frequency of persulfidation. A series of TTP variants were prepared and analyzed for H2S reactivity via cryo-electrospray ionization mass spectrometry, as well as UV-visible, circular dichroism, and fluorescence spectroscopies. We found that all peptide variants bound Zn(II) and were persulfidated by H2S to some extent, with higher Cys content contributing to greater persulfide labeling and ROS in the -CCCH and -CCCC peptides. Current work is focused on proteomic classification of persulfidated ZFs and elucidating the radical mechanism of this PTM using chemical tags, fluorescence, and spin-traps.
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Development of SILCS kinetics methodology for the determination of ligand dissociation pathways and free energy barriersThe fast and accurate assessment of unbinding kinetics of ligands from proteins remains challenging due to high computational requirements and the lack of the information about the molecular transition states due to limited conformational sampling. Therefore, in the present study we investigate the extension of the site-identification by ligand competitive saturation (SILCS) methodology towards estimation of ligand unbinding kinetics. The proposed SILCS-kinetics (SILCS-KIN) method is implemented to sample the free-energy landscape of drug dissociation pathways. SILCS-KIN methodology will be expected as a potential tool for the discovery and design of drug-like compounds with optimized ligand dissociation properties
