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Protein cysteine S-nitrosylation provides reducing power by enhancing lactate dehydrogenase activity in Trichomonas vaginalis under iron deficiency.

ABSTRACT: BACKGROUND:Iron plays essential roles in the pathogenesis and proliferation of Trichomonas vaginalis, the causative agent of the most prevalent non-viral human sexually transmitted infection. We previously demonstrated that under iron deficiency, the endogenous nitric oxide (NO) is accumulated and capable of regulating the survival of T. vaginalis. Herein, we aim to explore the influence of NO on the activity of the pyruvate-reducing enzyme lactate dehydrogenase in T. vaginalis (TvLDH). METHODS:Levels of lactate and pyruvate were detected for determining glycolysis activity in T. vaginalis under iron deficiency. Quantitative PCR was performed to determine the expression of TvLDH. S-nitrosylated (SNO) proteomics was conducted to identify the NO-modified proteins. The activities of glyceraldehyde-3-phosphate dehydrogenase (TvGAPDH) and TvLDH were measured after sodium nitrate treatment. The effects of protein nitrosylation on the production of cellular reducing power were examined by measuring the amount of nicotinamide adenine dinucleotide (NAD) and the ratio of the NAD redox pair (NAD+/NADH). RESULTS:We found that although the glycolytic pathway was activated in cells under iron depletion, the level of pyruvate was decreased due to the increased level of TvLDH. By analyzing the SNO proteome of T. vaginalis upon iron deficiency, we found that TvLDH is one of the glycolytic enzymes modified by SNO. The production of pyruvate was significantly reduced after nitrate treatment, indicating that protein nitrosylation accelerated the consumption of pyruvate by increasing TvLDH activity. Nitrate treatment also induced NAD oxidation, suggesting that protein nitrosylation was the key posttranslational modification controlling cellular redox status. CONCLUSIONS:We demonstrated that NO-mediated protein nitrosylation plays pivotal roles in the regulation of glycolysis, pyruvate metabolism, and the activity of TvLDH. The recycling of oxidized NAD catalyzed by TvLDH provided the reducing power that allowed T. vaginalis to adapt to the iron-deficient environment.

SUBMITTER: Cheng WH

PROVIDER: S-EPMC7501694 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Protein cysteine S-nitrosylation provides reducing power by enhancing lactate dehydrogenase activity in Trichomonas vaginalis under iron deficiency.

Cheng Wei-Hung WH Huang Kuo-Yang KY Ong Seow-Chin SC Ku Fu-Man FM Huang Po-Jung PJ Lee Chi-Ching CC Yeh Yuan-Ming YM Lin Rose R Chiu Cheng-Hsun CH Tang Petrus P

Parasites & vectors 20200918 1

<h4>Background</h4>Iron plays essential roles in the pathogenesis and proliferation of Trichomonas vaginalis, the causative agent of the most prevalent non-viral human sexually transmitted infection. We previously demonstrated that under iron deficiency, the endogenous nitric oxide (NO) is accumulated and capable of regulating the survival of T. vaginalis. Herein, we aim to explore the influence of NO on the activity of the pyruvate-reducing enzyme lactate dehydrogenase in T. vaginalis (TvLDH).< ...[more]

PMID: 32948226

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Project description:The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases.

Dataset Information

Protein cysteine S-nitrosylation provides reducing power by enhancing lactate dehydrogenase activity in Trichomonas vaginalis under iron deficiency.

Publications

Protein cysteine S-nitrosylation provides reducing power by enhancing lactate dehydrogenase activity in Trichomonas vaginalis under iron deficiency.

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