Biology subjects

Evans, C. N.

Publications and source records attributed to Evans, C. N..

4 recordsLinked to original sources

bioRxiv2025

Discovery of Chirally-dependent Protein O-2-Hydroxyglutarylation by D2HG and L2HG

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are common in multiple types of human cancer, leading to the accumulation of D-2-hydroxyglutarate (D2HG) and the promotion of tumorigenesis1. Here we discovered a novel O-2- hydroxyglutarylation by D2HG using chemical proteomics and further revealed distinct chiral preferences for D/L2HG modifications. Notably, we identified two kinases, MRCKA and SLK, modified by D2HG and L2HG respectively, and detected reduced phosphorylation of their substrates, suggesting an inhibitory effect of D/L 2HG modifications on the kinases activity.

biochemistry↗

bioRxiv2021

Protonation-Dependent Sequencing of 5-Formylcytidine in RNA

Chemical modification of cytidine in non-coding RNAs plays a key role in regulating translation and disease. However, the distribution and dynamics of many of these modifications remains unknown due to a lack of sensitive site-specific sequencing technologies. Here we report a protonation-dependent sequencing reaction for detection of 5-formylcytidine (5fC) and 5-carboxycytidine (5caC) in RNA. First, we evaluate how protonation combined with electron-withdrawing substituents alters the molecular orbital energies and reduction of modified cytidine nucleosides, highlighting 5fC and 5caC as reactive species. Next, we apply this reaction to detect these modifications in synthetic oligonucleotides as well as endogenous human tRNA. Finally, we demonstrate the utility of our method to characterize a patient-derived model of 5fC-deficiency, where it enables facile monitoring of both pathogenic loss and exogenous rescue of NSUN3-dependent 5fC within the wobble base of human mitochondrial tRNAMet. These studies showcase the ability of protonation to enhance the reactivity and sensitive detection of 5fC in RNA, and provide a molecular foundation for applying optimized sequencing reactions to better understand the role of oxidized RNA cytidine nucleobases in disease.

biochemistry↗

bioRxiv2021

Structure-guided bifunctional molecules hit a DEUBAD-lacking hRpn13 species upregulated in multiple myeloma

Proteasome substrate receptor hRpn13 is a promising anti-cancer target. By integrated in silico and biophysical screening, we identified a chemical scaffold that binds hRpn13 with non-covalent interactions that mimic the proteasome and a weak electrophile for Michael addition. hRpn13 Pru domain binds proteasomes and ubiquitin whereas its DEUBAD domain binds deubiquitinating enzyme UCHL5. NMR revealed lead compound XL5 to interdigitate into a hydrophobic pocket created by lateral movement of a Pru {beta}-hairpin with an exposed end for Proteolysis Targeting Chimeras (PROTACs). Implementing XL5-PROTACs as chemical probes identified a DEUBAD-lacking hRpn13 species (hRpn13Pru) present naturally with cell type-dependent abundance. XL5-PROTACs preferentially target hRpn13Pru, causing its ubiquitination. Gene-editing and rescue experiments established hRpn13 requirement for XL5-PROTAC-triggered apoptosis and increased p62 levels. These data establish hRpn13 as an anti-cancer target for multiple myeloma and introduce an hRpn13-targeting scaffold that can be optimized for preclinical trials against hRpn13Pru-producing cancer types.

molecular biology↗

bioRxiv2020

dbGuide: A database of functionally validated guide RNAs for genome editing in human and mouse cells

With the technologys accessibility and ease of use, CRISPR has been employed widely in many different organisms and experimental settings. As a result, thousands of publications have used CRISPR to make specific genetic perturbations, establishing in itself a resource of validated guide RNA sequences. While numerous computational tools to assist in the design and identification of candidate guide RNAs exist, these are still just at best predictions and generally, researchers inevitably will test multiple sequences for functional activity. Here, we present dbGuide (https://sgrnascorer.cancer.gov/dbguide), a database of functionally validated guide RNA sequences for CRISPR/Cas9-based knockout in human and mouse. Our database not only contains computationally determined candidate guide RNA sequences, but of even greater value, over 4000 sequences which have been functionally validated either through direct amplicon sequencing or manual curation of literature from over 1000 publications. Finally, our established framework will allow for continual addition of newly published and experimentally validated guide RNA sequences for CRISPR/Cas9-based knockout as well as incorporation of sequences from different gene editing systems, additional species, and other types of site-specific functionalities such as base editing, gene activation, repression, and epigenetic modification.

bioinformatics↗