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Biochim Biophys Acta Proteins Proteom ; 1870(3): 140754, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34995802

RESUMO

Protein tyrosine phosphatases (PTPs) are key virulence factors in pathogenic bacteria, consequently, they have become important targets for new approaches against these pathogens, especially in the fight against antibiotic resistance. Among these targets of interest YopH (Yersinia outer protein H) from virulent species of Yersinia is an example. PTPs can be reversibly inhibited by nitric oxide (NO) since the oxidative modification of cysteine residues may influence the protein structure and catalytic activity. We therefore investigated the effects of NO on the structure and enzymatic activity of Yersinia enterocolitica YopH in vitro. Through phosphatase activity assays, we observe that in the presence of NO YopH activity was inhibited by 50%, and that this oxidative modification is partially reversible in the presence of DTT. Furthermore, YopH S-nitrosylation was clearly confirmed by a biotin switch assay, high resolution mass spectrometry (MS) and X-ray crystallography approaches. The crystal structure confirmed the S-nitrosylation of the catalytic cysteine residue, Cys403, while the MS data provide evidence that Cys221 and Cys234 might also be modified by NO. Interestingly, circular dichroism spectroscopy shows that the S-nitrosylation affects secondary structure of wild type YopH, though to a lesser extent on the catalytic cysteine to serine YopH mutant. The data obtained demonstrate that S-nitrosylation inhibits the catalytic activity of YopH, with effects beyond the catalytic cysteine. These findings are helpful for designing effective YopH inhibitors and potential therapeutic strategies to fight this pathogen or others that use similar mechanisms to interfere in the signal transduction pathways of their hosts.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Cisteína/química , Óxido Nítrico/química , Proteínas Tirosina Fosfatases/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Biotina/metabolismo , Catálise , Cristalografia por Raios X/métodos , Cisteína/metabolismo , Humanos , Espectrometria de Massas/métodos , Estrutura Molecular , Óxido Nítrico/metabolismo , Oxirredução , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais , Yersinia enterocolitica/metabolismo
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