Publications by authors named "Katherine A Sitko"
Article Synopsis
- - The Cytochrome P450 family of enzymes metabolizes around 80% of small molecule drugs, but genetic variants can significantly affect how these drugs are processed, leading to risks of incorrect dosing and severe side effects.
- - Using a technique called VAMP-seq, researchers measured the protein abundance of over 7,600 single amino acid variants in CYP2C19, revealing key structural features essential for enzyme function and showcasing how variants at specific positions can impact protein levels.
- - The study also compared variants in CYP2C19 and CYP2C9, showing that while most amino acid exchanges had little effect, certain changes in substrate recognition sites diminished abundance in CYP2C19, indicating evolutionary trade-offs between stability and
Article Synopsis
- - Kinase inhibitors are important treatments for cancer, but their effectiveness is often undermined by resistance that can develop in cancer cells.
- - Researchers studied around 3,500 Src tyrosine kinase mutants and found resistance mutations spread throughout Src's catalytic domain, impacting both inhibitor binding and the enzyme's activity.
- - A specific cluster of residues in Src was identified as key in promoting drug resistance by enhancing the kinase's activity and reducing the binding affinity for the inhibitors, providing insights into both resistance pathways and kinase regulation.
Hsp90 is a molecular chaperone involved in the refolding and activation of numerous protein substrates referred to as clients. While the molecular determinants of Hsp90 client specificity are poorly understood and limited to a handful of client proteins, strong clients are thought to be destabilized and conformationally extended. Here, we measured the phosphotransferase activity of 3929 variants of the tyrosine kinase Src in both the presence and absence of an Hsp90 inhibitor.
View Article and Find Full Text PDFCYP2C9 encodes a cytochrome P450 enzyme responsible for metabolizing up to 15% of small molecule drugs, and CYP2C9 variants can alter the safety and efficacy of these therapeutics. In particular, the anti-coagulant warfarin is prescribed to over 15 million people annually and polymorphisms in CYP2C9 can affect individual drug response and lead to an increased risk of hemorrhage. We developed click-seq, a pooled yeast-based activity assay, to test thousands of variants.
View Article and Find Full Text PDFVitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin. Despite VKOR's pivotal role in coagulation, its structure and active site remain poorly understood.
View Article and Find Full Text PDFArticle Synopsis
- - The study investigates the regulation of Src kinase using saturation mutagenesis and chemical genetics, uncovering both known and new mechanisms that control its function and location in cells.
- - A previously unknown interaction between Src's SH4 and catalytic domains was found, which affects its activity, cellular position, and even cell shape changes independently of its kinase action.
- - The findings suggest that the SH4 domain acts as an internal regulator for Src kinases and may be a conserved feature in other related kinases, indicating that this research method could help uncover regulatory mechanisms in various kinases.
CYP4Z1 is an "orphan" cytochrome P450 (P450) enzyme that has provoked interest because of its hypothesized role in breast cancer through formation of the signaling molecule 20-hydroxyeicosatetraenoic acid (20-HETE). We expressed human CYP4Z1 in and evaluated its catalytic capabilities toward arachidonic and lauric acids (AA and LA). Specific and sensitive mass spectrometry assays enabled discrimination of the regioselectivity of hydroxylation of these two fatty acids.
View Article and Find Full Text PDFCellular control of protein activities by modulation of their abundance or compartmentalization is not easily measured on a large scale. We developed and applied a method to globally interrogate these processes that is widely useful for systems-level analyses of dynamic cellular responses in many cell types. The approach involves subcellular fractionation followed by comprehensive proteomic analysis of the fractions, which is enabled by a data-independent acquisition mass spectrometry approach that samples every available mass to charge channel systematically to maximize sensitivity.
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