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1.
Proc Natl Acad Sci U S A ; 118(3)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33441483

RESUMO

Flaviviruses, including dengue and Zika, are widespread human pathogens; however, no broadly active therapeutics exist to fight infection. Recently, remodeling of endoplasmic reticulum (ER) proteostasis by pharmacologic regulators, such as compound 147, was shown to correct pathologic ER imbalances associated with protein misfolding diseases. Here, we establish an additional activity of compound 147 as an effective host-centered antiviral agent against flaviviruses. Compound 147 reduces infection by attenuating the infectivity of secreted virions without causing toxicity in host cells. Compound 147 is a preferential activator of the ATF6 pathway of the ER unfolded protein response, which requires targeting of cysteine residues primarily on protein disulfide isomerases (PDIs). We find that the antiviral activity of 147 is independent of ATF6 induction but does require modification of reactive thiols on protein targets. Targeting PDIs and additional non-PDI targets using RNAi and other small-molecule inhibitors was unable to recapitulate the antiviral effects, suggesting a unique polypharmacology may mediate the activity. Importantly, 147 can impair infection of multiple strains of dengue and Zika virus, indicating that it is suitable as a broad-spectrum antiviral agent.

2.
J Bacteriol ; 200(9)2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29440253

RESUMO

The actinobacterium Corynebacterium matruchotii has been implicated in nucleation of oral microbial consortia leading to biofilm formation. Due to the lack of genetic tools, little is known about basic cellular processes, including protein secretion and folding, in this organism. We report here a survey of the C. matruchotii genome, which encodes a large number of exported proteins containing paired cysteine residues, and identified an oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA (MdbACd). Crystallization studies uncovered that the 1.2-Å resolution structure of C. matruchotii MdbA (MdbACm) possesses two conserved features found in actinobacterial MdbA enzymes, a thioredoxin-like fold and an extended α-helical domain. By reconstituting the disulfide bond-forming machine in vitro, we demonstrated that MdbACm catalyzes disulfide bond formation within the actinobacterial pilin FimA. A new gene deletion method supported that mdbA is essential in C. matruchotii Remarkably, heterologous expression of MdbACm in the C. diphtheriae ΔmdbA mutant rescued its known defects in cell growth and morphology, toxin production, and pilus assembly, and this thiol-disulfide oxidoreductase activity required the catalytic motif CXXC. Altogether, the results suggest that MdbACm is a major thiol-disulfide oxidoreductase, which likely mediates posttranslocational protein folding in C. matruchotii by a mechanism that is conserved in ActinobacteriaIMPORTANCE The actinobacterium Corynebacterium matruchotii has been implicated in the development of oral biofilms or dental plaque; however, little is known about the basic cellular processes in this organism. We report here a high-resolution structure of a C. matruchotii oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA. By biochemical analysis, we demonstrated that C. matruchotii MdbA catalyzes disulfide bond formation in vitro Furthermore, a new gene deletion method revealed that deletion of mdbA is lethal in C. matruchotii Remarkably, C. matruchotii MdbA can replace C. diphtheriae MdbA to maintain normal cell growth and morphology, toxin production, and pilus assembly. Overall, our studies support the hypothesis that C. matruchotii utilizes MdbA as a major oxidoreductase to catalyze oxidative protein folding.


Assuntos
Proteínas de Bactérias/química , Corynebacterium/enzimologia , Corynebacterium/genética , Proteína Dissulfeto Redutase (Glutationa)/química , Proteínas de Bactérias/genética , Biofilmes , Catálise , Corynebacterium diphtheriae/enzimologia , Corynebacterium diphtheriae/genética , Dissulfetos/química , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Deleção de Genes , Genoma Bacteriano , Modelos Moleculares , Oxirredução , Oxirredutases/química , Oxirredutases/genética , Proteína Dissulfeto Redutase (Glutationa)/genética
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