Downregulation of thioredoxin-1-dependent CD95 S-nitrosation by Sorafenib reduces liver cancer.

Hepatocellular carcinoma (HCC) represents 80% of the primary hepatic neoplasms. It is the sixth most frequent neoplasm, the fourth cause of cancer-related death, and 7% of registered malignancies. Sorafenib is the first line molecular targeted therapy for patients in advanced stage of HCC. The present study shows that Sorafenib exerts free radical scavenging properties associated with the downregulation of nuclear factor E2-related factor 2 (Nrf2)-regulated thioredoxin 1 (Trx1) expression in liver cancer cells. The experimental downregulation and/or overexpression strategies showed that Trx1 induced activation of nitric oxide synthase (NOS) type 3 (NOS3) and S-nitrosation (SNO) of CD95 receptor leading to an increase of caspase-8 activity and cell proliferation, as well as reduction of caspase-3 activity in liver cancer cells. In addition, Sorafenib transiently increased mRNA expression and activity of S-nitrosoglutathione reductase (GSNOR) in HepG2 cells. Different experimental models of hepatocarcinogenesis based on the subcutaneous implantation of HepG2 cells in nude mice, as well as the induction of HCC by diethylnitrosamine (DEN) confirmed the relevance of Trx1 downregulation during the proapoptotic and antiproliferative properties induced by Sorafenib. In conclusion, the induction of apoptosis and antiproliferative properties by Sorafenib were related to Trx1 downregulation that appeared to play a relevant role on SNO of NOS3 and CD95 in HepG2 cells. The transient increase of GSNOR might also participate in the deactivation of CD95-dependent proliferative signaling in liver cancer cells.

Saccharomyces cerevisiae Cytosolic Thioredoxins Control Glycolysis, Lipid Metabolism, and Protein Biosynthesis under Wine-Making Conditions.

Thioredoxins are small proteins that regulate the cellular redox state, prevent oxidative damage, and play an active role in cell repair. Oxidative stress has proven to be of much relevance in biotechnological processes when the metabolism of Saccharomyces cerevisiae is mainly respiratory. During wine yeast starter production, active dry yeast cytosolic thioredoxin Trx2p is a key player in protecting metabolic enzymes from being oxidized by carbonylation. Less is known about the role of redox control during grape juice fermentation. A mutant strain that lacked both cytosolic thioredoxins, Trx1p and Trx2p, was tested for grape juice fermentation. Its growth and sugar consumption were greatly impaired, which indicates the system's relevance under fermentative conditions. A proteomic analysis indicated that deletion of the genes TRX1 and TRX2 caused a reduction in the ribosomal proteins and factors involved in translation elongation in addition to enzymes for glycolysis and amino acid biosynthesis. A metabolomic analysis of the trx1Δ trx2Δ mutant showed an increase in most proteogenic amino acids, phospholipids, and sphingolipids and higher fatty acid desaturase Ole1p content. Low glycolytic activity was behind the reduced growth and fermentative capacity of the thioredoxin deletion strain. All three hexokinases were downregulated in the mutant strain, but total hexokinase activity remained, probably due to posttranslational regulation. Pyruvate kinase Cdc19p presented an early level of aggregation in the trx1Δ trx2Δ mutant, which may contribute to a diminished hexose metabolism and trigger regulatory mechanisms that could influence the level of glycolytic enzymes.IMPORTANCE Oxidative stress is a common hazardous condition that cells have to face in their lifetime. Oxidative damage may diminish cell vitality and viability by reducing metabolism and eventually leading to aging and ultimate death. Wine yeast Saccharomyces cerevisiae also faces oxidative attack during its biotechnological uses. One of the main yeast antioxidant systems involves two small proteins called thioredoxins. When these two proteins are removed, wine yeast shows diminished growth, protein synthesis, and sugar metabolism under wine-making conditions, and amino acid and lipid metabolism are also affected. Altogether, our results indicate that proper redox regulation is a key factor for metabolic adaptations during grape juice fermentation.

Targeting hepatoma using nitric oxide donor strategies.

AIMS: The study evaluated the role of increased intracellular nitric oxide (NO) concentration using NO donors or stably NO synthase-3 (NOS-3) overexpression during CD95-dependent cell death in hepatoma cells. The expression of cell death receptors and caspase activation, RhoA kinase activity, NOS-3 expression/activity, oxidative/nitrosative stress, and p53 expression were analyzed. The antitumoral activity of NO was also evaluated in the subcutaneous implantation of NOS-3-overexpressing hepatoma cells, as well NO donor injection into wild-type hepatoma-derived tumors implanted in xenograft mouse models. RESULTS: NO donor increased CD95 expression and activation of caspase-8 and 3 in HepG2, Huh7, and Hep3B cells. NOS-3 overexpression increased oxidative/nitrosative stress, p53 and CD95 expression, cellular Fas-associated death domain (FADD)-like IL-1beta converting enzyme (FLICE) inhibitory protein long (cFLIP(L)) and its short isoform (cFLIP(S)) shift, and cell death in HepG2 (4TO-NOS) cells. The inhibition of RhoA kinase and p53 knockdown using RNA interference reduced cell death in 4TO-NOS cells. The supplementation with hydrogen peroxide (H(2)O(2)) increased NOS-3 activity and cell death in 4TO-NOS cells. NOS-3 overexpression or NO donor injection into hepatoma-derived tumors reduced the size and increased p53 and cell death receptor expression in nude mice. INNOVATION AND CONCLUSIONS: The increase of intracellular NO concentration promoted oxidative and nitrosative stress, Rho kinase activity, p53 and CD95 expression, and cell death in cultured hepatoma cells. NOS-3-overexpressed HepG2 cells or intratumoral NO donor administration reduced tumor cell growth and increased the expression of p53 and cell death receptors in tumors developed in a xenograft mouse model.

Nitrogen starvation induces extensive changes in the redox proteome of Prochlorococcus sp. strain SS120.

Very low nitrogen concentration is a critical limitation in the oligotrophic oceans inhabited by the cyanobacterium Prochlorococccus, one of the main primary producers on Earth. It is well known that nitrogen starvation affects redox homeostasis in cells. We have studied the effect of nitrogen starvation on the thiol redox proteome in the Prochlorococcus sp. SS120 strain, by using shotgun proteomic techniques to map the cysteine modified in each case and to quantify the ratio of reversibly oxidized/reduced species. We identified a number of proteins showing modified cysteines only under either control or N-starvation, including isocitrate dehydrogenase and ribulose phosphate 3-epimerase. We detected other key enzymes, such as glutamine synthetase, transporters and transaminases, showing that nitrogen-related pathways were deeply affected by nitrogen starvation. Reversibly oxidized cysteines were also detected in proteins of other important metabolic pathways, such as photosynthesis, phosphorus metabolism, ATP synthesis and nucleic acids metabolism. Our results demonstrate a wide effect of nitrogen limitation on the redox status of the Prochlorococcus proteome, suggesting that besides previously reported transcriptional changes, this cyanobacterium responds with post-translational redox changes to the lack of nitrogen in its environment.

Overcoming function annotation errors in the Gram-positive pathogen Streptococcus suis by a proteomics-driven approach.

BACKGROUND: Annotation of protein-coding genes is a key step in sequencing projects. Protein functions are mainly assigned on the basis of the amino acid sequence alone by searching of homologous proteins. However, fully automated annotation processes often lead to wrong prediction of protein functions, and therefore time-intensive manual curation is often essential. Here we describe a fast and reliable way to correct function annotation in sequencing projects, focusing on surface proteomes. We use a proteomics approach, previously proven to be very powerful for identifying new vaccine candidates against Gram-positive pathogens. It consists of shaving the surface of intact cells with two proteases, the specific cleavage-site trypsin and the unspecific proteinase K, followed by LC/MS/MS analysis of the resulting peptides. The identified proteins are contrasted by computational analysis and their sequences are inspected to correct possible errors in function prediction. RESULTS: When applied to the zoonotic pathogen Streptococcus suis, of which two strains have been recently sequenced and annotated, we identified a set of surface proteins without cytoplasmic contamination: all the proteins identified had exporting or retention signals towards the outside and/or the cell surface, and viability of protease-treated cells was not affected. The combination of both experimental evidences and computational methods allowed us to determine that two of these proteins are putative extracellular new adhesins that had been previously attributed a wrong cytoplasmic function. One of them is a putative component of the pilus of this bacterium. CONCLUSION: We illustrate the complementary nature of laboratory-based and computational methods to examine in concert the localization of a set of proteins in the cell, and demonstrate the utility of this proteomics-based strategy to experimentally correct function annotation errors in sequencing projects. This approach also contributes to provide strong experimental evidences that can be used to annotate those proteins for which a Gene Ontology (GO) term has not been assigned so far. Function annotation correction would then improve the identification of surface-associated proteins in bacterial pathogens, thus accelerating the discovery of new vaccines in infectious disease research.

Role of glutaredoxin 2 and cytosolic thioredoxins in cysteinyl-based redox modification of the 20S proteasome.

The yeast 20S proteasome is subject to sulfhydryl redox alterations, such as the oxidation of cysteine residues (Cys-SH) into cysteine sulfenic acid (Cys-SOH), followed by S-glutathionylation (Cys-S-SG). Proteasome S-glutathionylation promotes partial loss of chymotrypsin-like activity and post-acidic cleavage without alteration of the trypsin-like proteasomal activity. Here we show that the 20S proteasome purified from stationary-phase cells was natively S-glutathionylated. Moreover, recombinant glutaredoxin 2 removes glutathione from natively or in vitro S-glutathionylated 20S proteasome, allowing the recovery of chymotrypsin-like activity and post-acidic cleavage. Glutaredoxin 2 deglutathionylase activity was dependent on its entry into the core particle, as demonstrated by stimulating S-glutathionylated proteasome opening. Under these conditions, deglutathionylation of the 20S proteasome and glutaredoxin 2 degradation were increased when compared to non-stimulated samples. Glutaredoxin 2 fragmentation by the 20S proteasome was evaluated by SDS-PAGE and mass spectrometry, and S-glutathionylation was evaluated by either western blot analyses with anti-glutathione IgG or by spectrophotometry with the thiol reactant 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. It was also observed in vivo that glutaredoxin 2 was ubiquitinated in cellular extracts of yeast cells grown in glucose-containing medium. Other cytoplasmic oxido-reductases, namely thioredoxins 1 and 2, were also active in 20S proteasome deglutathionylation by a similar mechanism. These results indicate for the first time that 20S proteasome cysteinyl redox modification is a regulated mechanism coupled to enzymatic deglutathionylase activity.

Role of glutaredoxin 2 and cytosolic thioredoxins incysteinyl-based redox modification of the 20S proteasome

The yeast 20S proteasome is subject to sulfhydryl redox alterations, such as the oxidation of cysteine residues (Cys-SH) into cysteine sulfenic acid (Cys-SOH), followed by S-glutathionylation (Cys-S-SG). Proteasome S-glutathionylation promotes partial loss of chymotrypsin-like activity and post-acidic cleavage without alteration of the trypsin-like proteasomal activity. Here we show that the 20S proteasome purified from stationary-phase cells was natively S-glutathionylated. Moreover, recombinant glutaredoxin 2 removes glutathione from natively or in vitro S-glutathionylated 20S proteasome, allowing the recovery of chymotrypsin-like activity and post-acidic cleavage. Glutaredoxin 2 deglutathionylase activity was dependent on its entry into the core particle, as demonstrated by stimulating S-glutathionylated proteasome opening. Under these conditions, deglutathionylation of the 20S proteasome and glutaredoxin 2 degradation were increased when compared to non-stimulated samples. Glutaredoxin 2 fragmentation by the 20S proteasome was evaluated by SDS-PAGE and mass spectrometry, and S-glutathionylation was evaluated by either western blot analyses with anti-glutathione IgG or by spectrophotometry with the thiol reactant 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. It was also observed in vivo that glutaredoxin 2 was ubiquitinated in cellular extracts of yeast cells grown in glucose-containing medium. Other cytoplasmic oxido-reductases, namely thioredoxins 1 and 2, were also active in 20S proteasome deglutathionylation by a similar mechanism. These results indicate for the first time that 20S proteasome cysteinyl redox modification is a regulated mechanism coupled to enzymatic deglutathionylase activity.