Shirran, S. L. | bioRxiv Science

bioRxiv2022

Identification of plasma proteins associated with oesophageal cancer chemotherapeutic treatment outcomes using SWATH-MS

Oesophageal adenocarcinoma (OAC) is an aggressive cancer with a five-year survival of <15%. Current chemotherapeutic strategies only benefit a minority (20-30%) of patients and there are no methods available to differentiate between responders and non-responders. We performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on albumin/IgG-depleted and non-depleted plasma samples from 23 patients with locally advanced OAC prior to treatment. Individuals were grouped based on tumour regression (TRG) score (TRG1/2/3 vs TRG4/5) after chemotherapy, and differentially abundant proteins were compared. Protein depletion of highly abundant proteins led to the identification of around twice as many proteins. SWATH-MS revealed significant quantitative differences in the abundance of several proteins between the two groups. These included complement c1q subunit proteins, C1QA, C1QB and C1QC, which were of higher abundance in the low TRG group. Of those that were found to be of higher abundance in the high TRG group, GSTP1 was found to exhibit the lowest p-value and highest classification accuracy and Cohens kappa value. Concentrations of these proteins were further examined using ELISA-based assays. This study provides quantitative information relating to differences in the plasma proteome that underpin response to chemotherapeutic treatment in oesophageal cancers.

cancer biology↗

bioRxiv2021

Structure, dynamics, and inhibition of Staphylococcus aureus m1A22-tRNA methyltransferase

The enzyme m1A22-tRNA methyltransferase (TrmK) catalyses the transfer of a methyl group from SAM to the N1 of adenine 22 in tRNAs. TrmK is essential for Staphylococcus aureus survival during infection, but has no homologue in mammals, making it a promising target for antibiotic development. Here we describe the structural and functional characterisation of S. aureus TrmK. Crystal structures are reported for S. aureus TrmK apoenzyme and in complexes with SAM and SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favourable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated by a large favourable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. Activity assays for S. aureus TrmK-catalysed methylation of WT and position 22 mutants of tRNALeu demonstrate that the enzyme requires an adenine at position 22 of the tRNA. Intriguingly, a small RNA hairpin of 18 nucleotides is methylated by TrmK depending on the position of the adenine. In-silico screening of compounds suggested plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92, in the vicinity of the SAM-binding site, as the sole residue modified. These results these results identify a cryptic binding pocket of S. aureus TrmK and lay the foundation for future structure-based drug discovery.

biochemistry↗