Biology subjects

Oltion, K.

Publications and source records attributed to Oltion, K..

3 recordsLinked to original sources

bioRxiv2022

An E3 ligase network engages GCN1 to promote elongation factor-1α degradation on stalled ribosomes

How cells monitor the status of translating ribosomes is a major question in gene regulation. Elongating ribosomes frequently stall during mRNA translation, resulting in context- dependent activation of quality control pathways. However, surveillance mechanisms that specifically respond to stalled ribosomes with an elongation factor occupying the GTPase center have not been identified. By employing ternatin-4, an allosteric elongation factor-1 (eEF1A) inhibitor, we unveil an E3 ligase network that triggers ubiquitination and degradation of eEF1A on stalled ribosomes. A CRISPRi screen revealed two E3 ligases of unknown function, RNF14 and RNF25, which are both essential for ternatin-induced eEF1A degradation. Based on quantitative proteomics analysis, we find that RNF14 and RNF25 promote ubiquitination of eEF1A and a discrete set of ribosomal proteins. By forming a complex with RNF14, the ribosome collision sensor GCN1 plays an essential role in eEF1A degradation. Our findings illuminate a translation elongation checkpoint that monitors the ribosomal GTPase center.

cell biology↗

bioRxiv2020

Total synthesis and biological characterization of SR-A3, a ternatin-related eEF1A inhibitor with enhanced cellular residence time

Ternatin-family cyclic peptides inhibit protein synthesis by targeting the eukaryotic elongation factor-1 (eEF1A). A potentially related cytotoxic natural product ("A3") was isolated from Aspergillus, but only 4 of its 11 stereocenters could be assigned. Here, we synthesized SR-A3 and SS-A3 - two out of 128 possible A3 epimers - and discovered that synthetic SR-A3 is indistinguishable from naturally derived A3. Relative to SS-A3, SR-A3 exhibits enhanced residence time and rebinding kinetics, as revealed by single-molecule fluorescence imaging of elongation reactions catalyzed by eEF1A in vitro. Increased residence time - stereospecifically conferred by the unique {beta}-hydroxyl in SR-A3 - was also observed in cells. Consistent with its prolonged duration of action, thrice-weekly dosing with SR-A3 led to dramatically increased survival in an aggressive Myc-driven mouse lymphoma model. Our results demonstrate the potential of SR-A3 as a cancer therapeutic and exemplify an evolutionary mechanism for enhancing cyclic peptide binding kinetics via stereospecific side-chain hydroxylation.

pharmacology and toxicology↗

bioRxiv2017

Extending Chemical Perturbations Of The Ubiquitin Fitness Landscape In A Classroom Setting

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary time scales. Building on our previous work (Mavor et al 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. We found sensitization of Lys63 in eight new conditions. In total, our experiments have uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the Ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.\n\nBuilds onMavor D, Barlow KA, Thompson S, Barad BA, Bonny AR, Cario CL, Gaskins G, Liu Z, Deming L, Axen SD, Caceres E, Chen W, Cuesta A, Gate R, Green EM, Hulce KR, Ji W, Kenner LR, Mensa B, Morinishi LS, Moss SM, Mravic M, Muir RK, Niekamp S, Nnadi CI, Palovcak E, Poss EM, Ross TD, Salcedo E, See S, Subramaniam M, Wong AW, Li J, Thorn KS, Conchuir SO, Roscoe BP, Chow ED, DeRisi JL, Kortemme T, Bolon DN, Fraser JS. Determination of Ubiquitin Fitness Landscapes Under Different Chemical Stresses in a Classroom Setting. eLife. 2016.\n\nImpact StatementWe organized a project-based course that used deep mutational scanning in multiple chemical conditions to resolve the inconsistencies between tolerance to mutations in laboratory conditions and sequence conservation over evolutionary timescales.

bioinformatics↗