Biology subjects

Sitko, K.

Publications and source records attributed to Sitko, K..

3 recordsLinked to original sources

bioRxiv2023

Deep mutational scanning of CYP2C19 reveals a substrate specificity-abundance tradeoff

Cytochrome P450s (CYPs) are a family of enzymes responsible for metabolizing nearly 80% of small molecule drugs. Variants in CYPs can substantially alter drug metabolism, which may result in improper dosing and severe adverse drug reactions. CYPs have low sequence conservation, making it difficult to anticipate whether variant effects measured in one CYP may extend to others based on sequence alone. Even closely related CYPs, like CYP2C9 and its closest homolog CYP2C19, have distinct phenotypic properties despite sharing 92% amino acid sequence identity. Thus, we used Variant Abundance by Massively Parallel sequencing (VAMP-seq) to measure the steady-state protein abundance, a proxy for protein stability, of 7,660 missense variants in CYP2C19 expressed in cultured human cells. Our results confirmed positions and structural features critical for CYP function and revealed how variants at positions conserved across all eukaryotic CYPs influence abundance. We jointly analyzed 4,670 variants whose abundance was measured in both CYP2C19 and CYP2C9, finding that the homologs have different variant abundances in substrate recognition sites within the hydrophobic core, and that substitutions in some regions reduced abundance in CYP2C19 but not CYP2C9. We also measured the abundance of all single and some multiple WT amino acid exchanges between CYP2C19 and CYP2C9. While most exchanges had no effect, substitutions in substrate recognition site 4 (SRS4) reduced abundance in CYP2C19. When nearby amino acids were exchanged in double and triple mutants, we found distinct interactions between the sites in CYP2C19 and CYP2C9, revealing a region that is partially responsible for the difference in thermodynamic stability between the two homologs. Since these positions are also important for determining substrate specificity, there may be an evolutionary tradeoff between stability and altered enzymatic function. Finally, we used our data to analyze 368 previously unannotated human variants, finding that 43% had decreased abundance. Thus, by comparing variant effects between two closely related and important human genes, we have uncovered regions underlying their functional differences and paved the way for a more complete understanding of one of the most versatile families of enzymes.

genetics↗

bioRxiv2021

Profiling of the drug resistance of thousands of Src tyrosine kinase mutants uncovers a regulatory network that couples autoinhibition to the dynamics of the catalytic domain

Kinase inhibitors are effective cancer therapies but resistance often limits clinical efficacy. Despite the cataloguing of numerous resistance mutations, our understanding of kinase inhibitor resistance is still incomplete. Here, we comprehensively profiled the resistance of [~]3500 Src tyrosine kinase mutants to four different ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Srcs catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Srcs phosphotransferase activity were prone to the development of resistance. Unexpectedly, we found that a resistance-prone cluster of residues located on the top face of the N-terminal lobe of Srcs catalytic domain contributes to autoinhibition by reducing catalytic domain dynamics, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how drug resistance profiling can be used to define potential resistance pathways and uncover new mechanisms of kinase regulation.

biochemistry↗

bioRxiv2019

Elucidating the molecular determinants of Aβ aggregation with deep mutational scanning

Despite the importance of A{beta} aggregation in Alzheimers disease etiology, our understanding of the sequence determinants of aggregation is sparse and largely derived from in vitro studies. For example, in vitro proline and alanine scanning mutagenesis of A{beta}40 proposed core regions important for aggregation. However, we lack even this limited mutagenesis data for the more disease-relevant A{beta}42. Thus, to better understand the molecular determinants of A{beta}42 aggregation in a cell-based system, we combined a yeast DHFR aggregation assay with deep mutational scanning. We measured the effect of 791 of the 798 possible single amino acid substitutions on the aggregation propensity of A{beta}42. We found that ~75% of substitutions, largely to hydrophobic residues, maintained or increased aggregation. We identified 11 positions at which substitutions, particularly to hydrophilic and charged amino acids, disrupted A{beta} aggregation. These critical positions were similar but not identical to critical positions identified in previous A{beta} mutagenesis studies. Finally, we analyzed our large-scale mutagenesis data in the context of different A{beta} aggregate structural models, finding that the mutagenesis data agreed best with models derived from fibrils seeded using brain-derived A{beta} aggregates.

molecular biology↗