Biology subjects

Shaw, J. M.

Publications and source records attributed to Shaw, J. M..

6 recordsLinked to original sources

bioRxiv2024

Electrostatic interactions define nacubactam potency against OXA-48-like β-lactamases

Carbapenemases, {beta}-lactamases hydrolysing carbapenem antibiotics, severely challenge treatment of multi-drug resistant bacterial infections. OXA-48 is among the most widely disseminated carbapenemases in Enterobacterales, consequently new treatment options for OXA-48 producers are urgently required. Development of diazabicyclooctane (DBO) inhibitors to overcome {beta}-lactamase-mediated resistance is one attractive option, due to their efficacy against a wide range of {beta}-lactamases. The DBO avibactam is already licensed for clinical use, with the related compound nacubactam currently in phase III trials for carbapenem-resistant infections. Here we investigate the activities of avibactam and nacubactam towards OXA-48 and two variants, OXA-163 and OXA-405, that contain deletions in the {beta}5 - {beta}6 loop adjacent to the active site and show modified activity towards different {beta}-lactam classes. Compared to avibactam, nacubactam is c. 80-fold less potent towards OXA-48, but this difference is reduced in OXA-163 and OXA-405. Crystal structures of the respective avibactam and nacubactam complexes, and molecular dynamics simulations based upon these, reveal residue Arg214 on the OXA-48 {beta}5 - {beta}6 active-site loop to be electrostatically repelled by nacubactam, but not avibactam, binding. This increases flexibility of the OXA-48 {beta}5 - {beta}6 loop, as well as neighbouring active site loops, in simulations of the OXA-48:nacubactam, compared to the avibactam, complex. Such effects are not observed in simulations of the respective complexes of OXA-163 and OXA-405, which lack Arg214. These data indicate that interactions with Arg214 can determine DBO potency towards OXA-48-like enzymes, and suggest that sequence variation in this {beta}-lactamase family affects reactivity towards inhibitors as well as {beta}-lactam substrates.

biochemistry↗

bioRxiv2023

Harvesting and amplifying gene cassettes confers cross-resistance to critically important antibiotics

Amikacin and piperacillin/tazobactam are frequent antibiotic choices to treat bloodstream infection, which is commonly fatal and most often caused by bacteria from the family Enterobacterales. Here we show that two gene cassettes located side-by-side in and ancestral integron similar to In37 have been "harvested" by insertion sequence IS26 as a transposon that is already globally disseminated among the Enterobacterales. This transposon encodes the enzymes AAC(6)-Ib-cr and OXA-1, reported, respectively, as amikacin and piperacillin/tazobactam resistance mechanisms. However, by studying bloodstream infection isolates from 769 patients from, three hospitals serving a population of 1.5 million people in South West England, we show that increased enzyme production due to mutation in an IS26/In37-derived hybrid promoter or, more commonly, transposon copy number amplification is required to simultaneously remove these two key therapeutic options; in many cases leaving only the last-resort antibiotic, meropenem. These findings may help improve the accuracy of predicting piperacillin/tazobactam treatment failure, allowing stratification of patients to receive meropenem or piperacillin/tazobactam, which may improve outcome and slow the emergence of meropenem resistance.

microbiology↗

bioRxiv2020

Amino Acids Promote Mitochondrial-Derived Compartment Formation in Mammalian Cells

We recently identified a new cellular structure in yeast, called the Mitochondrial-Derived Compartment (MDC), that forms on mitochondria in response to amino acid excess. While emerging evidence supports an important function for MDCs in protecting cells from metabolic stress, whether this system exists beyond yeast remains unclear. Here, we show that MDCs are conserved in mammals, and like their yeast counterparts, are responsive to the intracellular amino acid content. Specifically, we find that inhibition of protein translation stimulates formation of dynamic, micron-sized compartments that associate with the mitochondrial network. These compartments are enriched for the carrier receptor Tomm70A and other select mitochondrial outer and inner membrane cargo, associate with the ER membrane, and require the conserved GTPase Miro1 for formation. Mammalian MDCs are responsive to changes in amino acid levels during translation inhibition, and are not activated by other common cellular stressors. Thus, MDCs represent an evolutionarily conserved nutrient-responsive mitochondrial remodeling system.

cell biology↗

bioRxiv2020

Mitochondrial-Derived Compartments Facilitate Cellular Adaptation to Amino Acid Stress

Amino acids are essential building blocks of life. However, increasing evidence suggests that elevated amino acids cause cellular toxicity associated with numerous metabolic disorders. How cells cope with elevated amino acids remains poorly understood. Here, we show that a previously identified cellular structure, the mitochondrial-derived compartment (MDC), is a dynamic, lumen-containing organelle that functions to protect cells from amino acid stress. In response to amino acid elevation, MDCs are generated from mitochondria, where they selectively sequester and remove Tom70, a surface receptor required for import of nutrient carriers of the SLC25 family. MDC formation is regulated by levels of mitochondrial carriers, and its activation by amino acids occurs simultaneously with removal of plasma membrane-localized transporters via the multi-vesicular body (MVB) pathway. Combined loss of MDC and MVB formation renders cells sensitive to elevated amino acids, suggesting these pathways operate as a coordinated network to protect cells from amino acid toxicity.

cell biology↗

bioRxiv2020

ER-Mitochondria Contacts Promote Mitochondrial-Derived Compartment Biogenesis

Mitochondria are dynamic organelles with essential roles in signaling and metabolism. We recently identified a new cellular structure called the mitochondrial-derived compartment (MDC) that is generated from mitochondria in response to amino acid elevation. MDCs protect cells from amino acid toxicity, but how cells form MDCs is unclear. Here, we show that MDCs are micron-sized, lumen-containing organelles that form at sites of contact between the ER and mitochondria. Upon formation, MDCs stably persist at ER-mitochondria contacts for extended periods of time. MDC formation requires the ER-mitochondria encounter structure (ERMES) and GTP hydrolysis by the conserved GTPase Gem1. Unexpectedly, MDC formation is not linked to the role of ERMES/Gem1 in the maintenance of mitochondrial phospholipid homeostasis. Our results identify an important role for ER-mitochondria contacts in the biogenesis of MDCs. Abbreviations used in this paper: ERMES, ER-mitochondria encounter structure; IMM, inner mitochondrial membrane; MDC, mitochondrial-derived compartment; OMM, outer mitochondrial membrane. SummaryEnglish et al. use super-resolution imaging to show that mitochondrial-derived compartments are lumen-containing organelles that form at sites of contact between the ER and mitochondria. Mitochondrial-derived compartment biogenesis requires a noncanonical function of the ERMES complex and the conserved GTPase Gem1.

cell biology↗

bioRxiv2017

Structural Basis of Mitochondrial Receptor Binding and GTP Driven Conformational Constriction by Dynamin-Related Protein 1

Mitochondrial inheritance, genome maintenance, and metabolic adaptation all depend on organelle fission by Dynamin-Related Protein 1 (DRP1) and its mitochondrial receptors. DRP1 receptors include the paralogs Mitochondrial Dynamics 49 and 51 (MID49/MID51) and Mitochondrial Fission Factor (MFF), but the mechanisms by which these proteins recruit DRP1 and regulate its activities are unknown. Here we present a cryoEM structure of human, full-length DRP1 bound to MID49 and an analysis of structure- and disease-based mutations. We report that GTP binding allosterically induces a remarkable elongation and rotation of the G-domain, Bundle-Signaling Element (BSE) and connecting hinge loops of DRP1. In this nucleotide-bound conformation, a distributed network of multivalent interactions promotes DRP1 copolymerization into a linear filament with MID49, MID51 or both. Subsequent GTP hydrolysis and exchange within the filament leads to receptor dissociation, shortening through disassembly, and concomitant curling of DRP1 oligomers into closed rings. The dimensions of the closed DRP1 rings are consistent with DRP1-constricted mitochondrial tubules observed in human cells. These structures are the first views of full-length, receptor- and nucleotide-bound dynamin-family GTPases and--in comparison with nucleotide-free crystal structures--teach us how these molecular machines perform mechanical work through nucleotide-driven allostery.

biophysics↗