Biology subjects

Dyotima,

Publications and source records attributed to Dyotima,.

3 recordsLinked to original sources

bioRxiv2024

Warfarin analogs target disulfide bond-forming enzymes and suggest a residue important for quinone and coumarin binding

Disulfide bond formation has a central role in protein folding of both eukaryotes and prokaryotes. DsbB and VKOR enzymes catalyze the oxidation of the oxidoreductase partner and the formation of de novo disulfide bonds using quinone as cofactor. We have used E. coli and a family of warfarin analogs to study de novo disulfide bond formation. We found that human VKORc1 can function in E. coli by removing two positive residues, allowing the search for novel anticoagulants. One analog was capable of inhibiting both bacterial DsbB and VKOR, and a second one antagonized only the mammalian enzymes. We identified the two amino acid residues responsible for binding. One of these is also essential for quinone binding in both DsbB and VKOR. Our studies highlight a conserved role of this residue in de novo disulfide-generating enzymes and enable the design of novel anticoagulants or antibacterials using coumarin as a scaffold.

microbiology↗

bioRxiv2023

Development of a sensor for disulfide bond formation in diverse bacteria

In bacteria, disulfide bonds contribute to the folding and stability of proteins important for processes in the cellular envelope. In E. coli, disulfide bond formation is catalyzed by DsbA and DsbB enzymes. DsbA is a periplasmic protein that catalyzes disulfide bond formation in substrate proteins while DsbB is an inner membrane protein that transfers electrons from DsbA to quinones, thereby regenerating the DsbA active state. Actinobacteria including mycobacteria use an alternative enzyme named VKOR which performs the same function as DsbB. Disulfide bond formation enzymes, DsbA and DsbB/ VKOR represent novel drug targets because their inhibition could simultaneously affect the folding of several cell envelope proteins including virulence factors, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. We have previously developed a cell-based and target-based assay to identify molecules that inhibit the DsbB and VKOR in pathogenic bacteria, using Escherichia coli cells expressing a periplasmic {beta}-Galactosidase sensor ({beta}-Galdbs) which is only active when disulfide bond formation is inhibited. Here we report the construction of plasmids that allow fine-tuning of the expression of the {beta}-Galdbs sensor and can be mobilized into other gram-negative organisms. As an example, when harbored in P. aeruginosa UCBPP-PA14, {beta}-Galdbs behaves similarly as in E. coli and the biosensor responds to the inhibition of the two DsbB proteins. Thus, these {beta}-Galdbs reporter plasmids provide a basis for identifying novel inhibitors of DsbA and DsbB/VKOR against multi-drug resistant, gram-negative pathogens and to further study oxidative protein folding in diverse gram-negative bacteria. ImportanceDisulfide bonds contribute to the folding and stability of proteins in the bacterial cell envelope. Disulfide bond-forming enzymes represent new drug targets against multidrug-resistant bacteria since inactivation of this process would simultaneously affect several proteins in the cell envelope, including virulence factors, toxins, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. Identifying the enzymes involved in disulfide bond formation in gram-negative pathogens as well as their inhibitors can contribute to the much-needed antibacterial innovation. In this work, we developed sensors of disulfide bond formation for gram-negative bacteria. These tools will enable the study of disulfide bond formation and the identification of inhibitors for this crucial process in diverse gram-negative pathogens.

microbiology↗

bioRxiv2023

Divergent Pairwise Epistasis in the Context of Unstable Membrane Protein Variants

Many membrane proteins are prone to misfolding, which compromises their functional expression at the plasma membrane. This is particularly true for the mammalian gonadotropin-releasing hormone receptor GPCRs (GnRHR). We recently demonstrated that evolutionary GnRHR modifications appear to have coincided with adaptive changes in cotranslational folding efficiency. Though protein stability is known to shape evolution, it is unclear how cotranslational folding constraints modulate the synergistic, epistatic interactions between mutations. We therefore compared the pairwise interactions formed by mutations that disrupt the membrane topology (V276T) or tertiary structure (W107A) of GnRHR. Using deep mutational scanning, we evaluated how the plasma membrane expression of these variants is modified by hundreds of secondary mutations. An analysis of 251 mutants in three genetic backgrounds reveals that V276T and W107A form distinct epistatic interactions that depend on both the severity and the mechanism of destabilization. V276T forms predominantly negative epistatic interactions with destabilizing mutations in soluble loops. In contrast, W107A forms positive interactions with mutations in both loops and transmembrane domains that reflect the diminishing impacts of the destabilizing mutations in variants that are already unstable. These findings reveal how epistasis is remodeled by conformational defects in membrane proteins and in unstable proteins more generally. Impact StatementMany of the synergistic interactions between mutations within a gene are modified in the context of unstable proteins in a manner that depends on how the variants promote misfolding in the cell.

biochemistry↗