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Browsing by Author "Runnebohm, Avery M."
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Item Methionine Restriction Impairs Degradation of a Protein that Aberrantly Engages the Endoplasmic Reticulum Translocon(Caltech LIbrary, 2023-11-09) Runnebohm, Avery M.; Indovina, Christopher J.; Turk, Samantha M.; Bailey, Connor G.; Orchard, Cade J.; Wade, Lauren; Overton, Danielle L.; Snow, Brian J.; Rubenstein, Eric M.; Biochemistry and Molecular Biology, School of MedicineProteins that persistently engage endoplasmic reticulum (ER) translocons are degraded by multiple translocon quality control (TQC) mechanisms. In Saccharomyces cerevisiae , the model translocon-associated protein Deg1 -Sec62 is subject to ER-associated degradation (ERAD) by the Hrd1 ubiquitin ligase and, to a lesser extent, proteolysis mediated by the Ste24 protease. In a recent screen, we identified nine methionine-biosynthetic genes as candidate TQC regulators. Here, we found methionine restriction impairs Hrd1-independent Deg1 -Sec62 degradation. Beyond revealing methionine as a novel regulator of TQC, our results urge caution when working with laboratory yeast strains with auxotrophic mutations, often presumed not to influence cellular processes under investigation.Item Obtaining Functional Proteomics Insights From Thermal Proteome Profiling Through Optimized Melt Shift Calculation and Statistical Analysis With InflectSSP(American Society for Biochemistry and Molecular Biology, 2023) McCracken, Neil A.; Liu, Hao; Runnebohm, Avery M.; Wijeratne, H. R. Sagara; Wijeratne, Aruna B.; Staschke, Kirk A.; Mosley, Amber L.; Biochemistry and Molecular Biology, School of MedicineThermal proteome profiling (TPP) is an invaluable tool for functional proteomics studies that has been shown to discover changes associated with protein–ligand, protein–protein, and protein–RNA interaction dynamics along with changes in protein stability resulting from cellular signaling. The increasing number of reports employing this assay has not been met concomitantly with new approaches leading to advancements in the quality and sensitivity of the corresponding data analysis. The gap between data acquisition and data analysis tools is important to fill as TPP findings have reported subtle melt shift changes related to signaling events such as protein posttranslational modifications. In this study, we have improved the Inflect data analysis pipeline (now referred to as InflectSSP, available at https://CRAN.R-project.org/package=InflectSSP) to increase the sensitivity of detection for both large and subtle changes in the proteome as measured by TPP. Specifically, InflectSSP now has integrated statistical and bioinformatic functions to improve objective functional proteomics findings from the quantitative results obtained from TPP studies through increasing both the sensitivity and specificity of the data analysis pipeline. InflectSSP incorporates calculation of a “melt coefficient” into the pipeline with production of average melt curves for biological replicate studies to aid in identification of proteins with significant melts. To benchmark InflectSSP, we have reanalyzed two previously reported datasets to demonstrate the performance of our publicly available R-based program for TPP data analysis. We report new findings following temporal treatment of human cells with the small molecule thapsigargin that induces the unfolded protein response as a consequence of inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase 2A. InflectSSP analysis of our unfolded protein response study revealed highly reproducible and statistically significant target engagement over a time course of treatment while simultaneously providing new insights into the possible mechanisms of action of the small molecule thapsigargin.