Hydrogen sulfide (H2S) is a gas neurotransmitter that is synthesized in various mammalian tissues including vascular tissues and regulates vascular tone. The aim of this study is to investigate whether the endogenous L-cysteine/H2S pathway is impaired due to aging and endothelial denudation in mouse isolated thoracic aorta. For this purpose, young (3-4 months) and old (23-25 months) mice were used in the experiments. The effects of aging and endothelium on endogenous and exogenous H2S-induced vasorelaxation were investigated by cumulative L-cysteine-(1 microM-10 mM) and NaHS-(1 microM-3 mM) induced vasorelaxations, respectively. The L-cysteine-induced relaxations were reduced in old mice aorta compared to the young mice. Also, vasorelaxant responses to L-cysteine (1 microM-10 mM) were reduced on aorta rings with denuded-endothelium of young and old mice. However, the relaxation responses to NaHS were not altered by age or endothelium denudation. The loss of staining of CSE in the endothelial layer was observed in old thoracic aorta. Ach-induced (1-30 microM) relaxation almost abolished in endothelium-denuded rings from both mice group. Also, relaxation Ach reduced in intact endothelium tissue of old mice aorta. In conclusion, the vasorelaxant responses to L-cysteine but not NaHS decreased and the protein expression of CSE reduced in old thoracic aorta rings consistent with a decrease in H2S concentration with aging and endothelium damage, suggesting that aging may be lead to decrease in enzyme expression and H2S signaling system due to endothelium damage in mouse thoracic aorta. Key words Aging, Hydrogen sulfide, L-cysteine, Endothelium, Thoracic aorta.
- MeSH
- aorta thoracica * účinky léků metabolismus fyziologie MeSH
- cévní endotel * metabolismus účinky léků MeSH
- cystein metabolismus farmakologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- signální transdukce MeSH
- stárnutí * metabolismus MeSH
- sulfan * metabolismus MeSH
- vazodilatace * účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4+ T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity.
- MeSH
- cystatiny * farmakologie MeSH
- cystein metabolismus MeSH
- endopeptidasy metabolismus MeSH
- kathepsiny metabolismus MeSH
- klíště * chemie MeSH
- obratlovci MeSH
- proteasy metabolismus MeSH
- slinné cystatiny chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Under certain circumstances, any of the three termination codons can be read through by a near-cognate tRNA; i.e., a tRNA whose two out of three anticodon nucleotides base pair with those of the stop codon. Unless programed to synthetize C-terminally extended protein variants with expanded physiological roles, readthrough represents an undesirable translational error. On the other side of a coin, a significant number of human genetic diseases is associated with the introduction of nonsense mutations (premature termination codons [PTCs]) into coding sequences, where stopping is not desirable. Here, the tRNA's ability to induce readthrough opens up the intriguing possibility of mitigating the deleterious effects of PTCs on human health. In yeast, the UGA and UAR stop codons were described to be read through by four readthrough-inducing rti-tRNAs-tRNATrp and tRNACys, and tRNATyr and tRNAGln, respectively. The readthrough-inducing potential of tRNATrp and tRNATyr was also observed in human cell lines. Here, we investigated the readthrough-inducing potential of human tRNACys in the HEK293T cell line. The tRNACys family consists of two isoacceptors, one with ACA and the other with GCA anticodons. We selected nine representative tRNACys isodecoders (differing in primary sequence and expression level) and tested them using dual luciferase reporter assays. We found that at least two tRNACys can significantly elevate UGA readthrough when overexpressed. This indicates a mechanistically conserved nature of rti-tRNAs between yeast and human, supporting the idea that they could be used in the PTC-associated RNA therapies.
- MeSH
- antikodon MeSH
- cystein * genetika metabolismus MeSH
- HEK293 buňky MeSH
- lidé MeSH
- nesmyslný kodon genetika MeSH
- proteosyntéza MeSH
- RNA transferová Cys metabolismus MeSH
- RNA transferová Trp metabolismus MeSH
- RNA transferová Tyr MeSH
- RNA transferová genetika metabolismus MeSH
- Saccharomyces cerevisiae * genetika MeSH
- terminační kodon genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Stable isotope labeling by amino acids in cell culture (SILAC) and iodoacetyl tandem mass tag (iodoTMT) are well-implemented mass spectrometry-based approaches for quantification of proteins and for site-mapping of cysteine modification. We describe here a combination of SILAC and iodoTMT to assess ongoing changes in the global proteome and cysteine modification levels using liquid chromatography separation coupled with high-resolution mass spectrometry (LC-MS/MS).
Hepatitis B virus uses e antigen (HBe), which is dispensable for virus infectivity, to modulate host immune responses and achieve viral persistence in human hepatocytes. The HBe precursor (p25) is directed to the endoplasmic reticulum (ER), where cleavage of the signal peptide (sp) gives rise to the first processing product, p22. P22 can be retro-translocated back to the cytosol or enter the secretory pathway and undergo a second cleavage event, resulting in secreted p17 (HBe). Here, we report that translocation of p25 to the ER is promoted by translocon-associated protein complex. We have found that p25 is not completely translocated into the ER; a fraction of p25 is phosphorylated and remains in the cytoplasm and nucleus. Within the p25 sp sequence, we have identified three cysteine residues that control the efficiency of sp cleavage and contribute to proper subcellular distribution of the precore pool.
- MeSH
- cystein metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- hepatitida B - antigeny e * metabolismus MeSH
- hepatitida B * metabolismus MeSH
- lidé MeSH
- membránové glykoproteiny MeSH
- proteiny - lokalizační signály genetika MeSH
- proteiny vázající vápník MeSH
- receptory cytoplazmatické a nukleární MeSH
- receptory peptidů MeSH
- virus hepatitidy B metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Decreasing the dietary intake of methionine exerts robust anti-adiposity effects in rodents but modest effects in humans. Since cysteine can be synthesized from methionine, animal diets are formulated by decreasing methionine and eliminating cysteine. Such diets exert both methionine restriction (MR) and cysteine restriction (CR), that is, sulfur amino acid restriction (SAAR). Contrarily, SAAR diets formulated for human consumption included cysteine, and thus might have exerted only MR. Epidemiological studies positively correlate body adiposity with plasma cysteine but not methionine, suggesting that CR, but not MR, is responsible for the anti-adiposity effects of SAAR. Whether this is true, and, if so, the underlying mechanisms are unknown. Using methionine- and cysteine-titrated diets, we demonstrate that the anti-adiposity effects of SAAR are due to CR. Data indicate that CR increases serinogenesis (serine biosynthesis from non-glucose substrates) by diverting substrates from glyceroneogenesis, which is essential for fatty acid reesterification and triglyceride synthesis. Molecular data suggest that CR depletes hepatic glutathione and induces Nrf2 and its downstream targets Phgdh (the serine biosynthetic enzyme) and Pepck-M. In mice, the magnitude of SAAR-induced changes in molecular markers depended on dietary fat concentration (60% fat >10% fat), sex (males > females), and age-at-onset (young > adult). Our findings are translationally relevant as we found negative and positive correlations of plasma serine and cysteine, respectively, with triglycerides and metabolic syndrome criteria in a cross-sectional epidemiological study. Controlled feeding of low-SAA, high-polyunsaturated fatty acid diets increased plasma serine in humans. Serinogenesis might be a target for treating hypertriglyceridemia.
- MeSH
- aminokyseliny sírové * metabolismus MeSH
- cystein * metabolismus MeSH
- lidé MeSH
- metabolismus lipidů MeSH
- methionin metabolismus MeSH
- myši MeSH
- obezita metabolismus MeSH
- průřezové studie MeSH
- serin metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- cystein metabolismus MeSH
- cysteinové proteasy * metabolismus MeSH
- fagozomy metabolismus MeSH
- lidé MeSH
- lyzozomy metabolismus MeSH
- proteasy metabolismus MeSH
- proteomika MeSH
- Trichomonas vaginalis * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Metabolic rewiring is a characteristic hallmark of cancer cells. This phenomenon sustains uncontrolled proliferation and resistance to apoptosis by increasing nutrients and energy supply. However, reprogramming comes together with vulnerabilities that can be used against tumor and can be applied in targeted therapy. In the last years, the genetic background of tumors has been identified thoroughly and new therapies targeting those mutations tested. Nevertheless, we propose that targeting the phenotype of cancer cells could be another way of treatment aiming to avoid drug resistance and non-responsiveness of cancer patients. Amino acid metabolism is part of the altered processes in cancer cells. Amino acids are building blocks and also sensors of signaling pathways regulating main biological processes. In this comprehensive review, we described four amino acids (asparagine, arginine, methionine, and cysteine) which have been actively investigated as potential targets for anti-tumor therapy. Asparagine depletion is successfully used for decades in the treatment of acute lymphoblastic leukemia and there is a strong implication to apply it to other types of tumors. Arginine auxotrophic tumors are great candidates for arginine-starvation therapy. Higher requirement for essential amino acids such as methionine and cysteine point out promising targetable weaknesses of cancer cells.
- MeSH
- aminokyseliny metabolismus MeSH
- arginin metabolismus terapeutické užití MeSH
- asparagin * metabolismus terapeutické užití MeSH
- cystein metabolismus MeSH
- lidé MeSH
- methionin MeSH
- nádory * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Persulfidation contributes to a group of redox post-translational modifications (PTMs), which arise exclusively on the sulfhydryl group of cysteine as a result of hydrogen sulfide (H2S) action. Redox-active molecules, including H2S, contribute to sperm development; therefore, redox PTMs represent an extremely important signalling pathway in sperm life. In this path, persulfidation prevents protein damage caused by irreversible cysteine hyperoxidation and thus maintains this signalling pathway. In our study, we detected both H2S and its production by all H2S-releasing enzymes (cystathionine γ-lyase (CTH), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MPST)) in male reproduction, including spermatozoa. We provided evidence that sperm H2S leads to persulfidation of proteins, such as glyceraldehyde-3-phosphate dehydrogenase, tubulin, and anchor protein A-kinase. Overall, this study suggests that persulfidation, as a part of the redox signalling pathway, is tightly regulated by enzymatic H2S production and is required for sperm viability.
- MeSH
- cystathionin-gama-lyasa metabolismus MeSH
- cystein metabolismus MeSH
- lidé MeSH
- rozmnožování MeSH
- sperma metabolismus MeSH
- sulfan * metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
