Tumoral Malignancy Decreases Coupled with Higher ROS and Lipid Peroxidation in HCT116 Colon Cancer Cells upon Loss of PRDX6.

Peroxiredoxin 6 (PRDX6) is an atypical member of the peroxiredoxin family that presents not only peroxidase but also phospholipase A2 and lysophosphatidylcholine acyl transferase activities able to act on lipid hydroperoxides of cell membranes. It has been associated with the proliferation and invasive capacity of different tumoral cells including colorectal cancer cells, although the effect of its removal in these cells has not been yet studied. Here, using CRISPR/Cas9 technology, we constructed an HCT116 colorectal cancer cell line knockout for PRDX6 to study whether the mechanisms described for other cancer cells in terms of proliferation, migration, and invasiveness also apply in this tumoral cell line. HCT116 cells lacking PRDX6 showed increased ROS and lipid peroxidation, a decrease in the antioxidant response regulator NRF2, mitochondrial dysfunction, and increased sensitivity to ferroptosis. All these alterations lead to a decrease in proliferation, migration, and invasiveness in these cells. Furthermore, the reduced migratory and invasive capacity of HCT116 cancer cells is consistent with the observed cadherin switch and decrease in pro-invasive proteins such as MMPs. Therefore, the mechanism behind the effects of loss of PRDX6 in HCT116 cells could differ from that in HepG2 cells which is coherent with the fact that the correlation of PRDX6 expression with patient survival is different in hepatocellular carcinomas. Nonetheless, our results point to this protein as a good therapeutic target also for colorectal cancer.

Knockout of PRDX6 induces mitochondrial dysfunction and cell cycle arrest at G2/M in HepG2 hepatocarcinoma cells.

Peroxiredoxin 6 (PRDX6) has been associated with tumor progression and cancer metastasis. Its acting on phospholipid hydroperoxides and its phospholipase-A2 activity are unique among the peroxiredoxin family and add complexity to its action mechanisms. As a first step towards the study of PRDX6 involvement in cancer, we have constructed a human hepatocarcinoma HepG2PRDX6-/- cell line using the CRISPR/Cas9 technique and have characterized the cellular response to lack of PRDX6. Applying quantitative global and redox proteomics, flow cytometry, in vivo extracellular flow analysis, Western blot and electron microscopy, we have detected diminished respiratory capacity, downregulation of mitochondrial proteins and altered mitochondrial morphology. Autophagic vesicles were abundant while the unfolded protein response (UPR), HIF1A and NRF2 transcription factors were not activated, despite increased levels of p62/SQSTM1 and reactive oxygen species (ROS). Insulin receptor (INSR), 3-phosphoinositide-dependent protein kinase 1 (PDPK1), uptake of glucose and hexokinase-2 (HK2) decreased markedly while nucleotide biosynthesis, lipogenesis and synthesis of long chain polyunsaturated fatty acids (LC-PUFA) increased. 254 Cys-peptides belonging to 202 proteins underwent significant redox changes. PRDX6 knockout had an antiproliferative effect due to cell cycle arrest at G2/M transition, without signs of apoptosis. Loss of PLA2 may affect the levels of specific lipids altering lipid signaling pathways, while loss of peroxidase activity could induce redox changes at critical sensitive cysteine residues in key proteins. Oxidation of specific cysteines in Proliferating Cell Nuclear Antigen (PCNA) could interfere with entry into mitosis. The GSH/Glutaredoxin system was downregulated likely contributing to these redox changes. Altogether the data demonstrate that loss of PRDX6 slows down cell division and alters metabolism and mitochondrial function, so that cell survival depends on glycolysis to lactate for ATP production and on AMPK-independent autophagy to obtain building blocks for biosynthesis. PRDX6 is an important link in the chain of elements connecting redox homeostasis and proliferation.

Improved integrative analysis of the thiol redox proteome using filter-aided sample preparation.

Changes in the oxidation state of protein Cys residues are involved in cell signalling and play a key role in a variety of pathophysiological states. We had previously developed GELSILOX, an in-gel method that enables the large-scale, parallel analysis of dynamic alterations to the redox state of Cys sites and protein abundance changes. Here we present FASILOX, a further development of the GELSILOX approach featuring: i) significantly increased peptide recovery, ii) enhanced sensitivity for the detection of Cys oxidative alterations, and iii) streamlined workflow that results in shortened assay duration. In mitochondria isolated from the adipose tissue of obese, diabetic patients, FASILOX revealed a sexually dimorphic trait of Cys oxidation involving mainly mitochondrial oxidative phosphorylation complexes. These results provide the first evidence for a decreased efficiency in the antioxidant response of men as compared to women.

Thioredoxin Downregulation Enhances Sorafenib Effects in Hepatocarcinoma Cells.

Sorafenib is the first-line recommended therapy for patients with advanced hepatocarcinoma (HCC) in de-differentiation stage (presenting epithelial-mesenchymal transition, EMT). We studied the role of the thioredoxin system (Trx1/TrxR1) in the sensitivity or resistance of HCC cells to the treatment with Sorafenib. As a model, we used a set of three established HCC cell lines with different degrees of de-differentiation as occurs in metastasis. By quantitative proteomics, we found that the expression levels of Trx1 and TrxR1 followed the same trend as canonical EMT markers in these cell lines. Treatment with Sorafenib induced thiol redox reductive changes in critical elements of oncogenic pathways in all three cell lines but induced drastic proteome reprograming only in HCC cell lines of intermediate stage. Trx1 downregulation counteracted the thiol reductive effect of Sorafenib on Signal Transducer and Activator of Transcription 3 (STAT3) but not on Mitogen-Activated Protein Kinase (MAPK) or Protein Kinase B (Akt) and transformed advanced HCC cells into Sorafenib-sensitive cells. Ten targets of the combined Sorafenib-siRNATrx1 treatment were identified that showed a gradually changing expression trend in parallel to changes in the expression of canonical EMT markers, likely as a result of the activation of Hippo signaling. These findings support the idea that a combination of Sorafenib with thioredoxin inhibitors should be taken into account in the design of therapies against advanced HCC.

Glutathione Is the Resolving Thiol for Thioredoxin Peroxidase Activity of 1-Cys Peroxiredoxin Without Being Consumed During the Catalytic Cycle.

AIMS: A three-step catalytic cycle is common to all peroxiredoxins (Prxs), despite structural and kinetic differences. The second step in 1-Cys type Prxs is a matter of debate since they lack an additional cysteine to play the resolving role, as happens with the 2-Cys Prxs. The aim of this study was to elucidate the role of glutathione (GSH) in the thioredoxin-dependent peroxidase activity of Saccharomyces cerevisiae mitochondrial Prx1p, a 1-Cys type Prx. RESULTS: The peroxidatic Cys91 residue of two Prx1p peptides can be linked by a disulfide, which can be reduced by thioredoxin and by GSH (Km=6.1 µM). GSH forms a mixed disulfide with the peroxidatic cysteine spontaneously in vitro and in vivo. Mitochondrial Trx3p deglutathionylates Prx1p without formation of GSSG so that GSH is not consumed in the process. The structural unit of native Prx1p is a dimer whose subunits are not covalently linked, but a hexameric assembly of three disulfide-bound dimers can also be formed. INNOVATION: GSH is presented as a protective cofactor of Prx1p, which is not consumed during the peroxidase reaction, but provides a robust mechanism as the resolving cysteine and efficiently prevents Prx1p overoxidation. GSH exerts these roles at concentrations well below those commonly considered necessary for its antioxidant and redox buffering functions. CONCLUSION: A 1-Cys peroxide scavenging mechanism operates in yeast mitochondria involving an autonomous glutathione molecule and the thioredoxin system, which could have universal validity. Prx1p is fairly well protected from overoxidation, questioning its role in a floodgate mechanism for H2O2 signaling.

Thiol redox sensitivity of two key enzymes of heme biosynthesis and pentose phosphate pathways: uroporphyrinogen decarboxylase and transketolase.

Uroporphyrinogen decarboxylase (Hem12p) and transketolase (Tkl1p) are key mediators of two critical processes within the cell, heme biosynthesis, and the nonoxidative part of the pentose phosphate pathway (PPP). The redox properties of both Hem12p and Tkl1p from Saccharomyces cerevisiae were investigated using proteomic techniques (SRM and label-free quantification) and biochemical assays in cell extracts and in vitro with recombinant proteins. The in vivo analysis revealed an increase in oxidized Cys-peptides in the absence of Grx2p, and also after treatment with H2O2 in the case of Tkl1p, without corresponding changes in total protein, demonstrating a true redox response. Out of three detectable Cys residues in Hem12p, only the conserved residue Cys52 could be modified by glutathione and efficiently deglutathionylated by Grx2p, suggesting a possible redox control mechanism for heme biosynthesis. On the other hand, Tkl1p activity was sensitive to thiol redox modification and although Cys622 could be glutathionylated to a limited extent, it was not a natural substrate of Grx2p. The human orthologues of both enzymes have been involved in certain cancers and possess Cys residues equivalent to those identified as redox sensitive in yeast. The possible implication for redox regulation in the context of tumour progression is put forward.

Application of iTRAQ Reagents to Relatively Quantify the Reversible Redox State of Cysteine Residues.

Cysteines are one of the most rarely used amino acids, but when conserved in proteins they often play critical roles in structure, function, or regulation. Reversible cysteine modifications allow for potential redox regulation of proteins. Traditional measurement of the relative absolute quantity of a protein between two samples is not always necessarily proportional to the activity of the protein. We propose application of iTRAQ reagents in combination with a previous thiol selection method to relatively quantify the redox state of cysteines both within and between samples in a single analysis. Our method allows for the identification of the proteins, identification of redox-sensitive cysteines within proteins, and quantification of the redox status of individual cysteine-containing peptides. As a proof of principle, we applied this technique to yeast alcohol dehydrogenase-1 exposed in vitro to H(2)O(2) and also in vivo to the complex proteome of the Gram-negative bacterium Bacillus subtilis.

Biosynthetic and iron metabolism is regulated by thiol proteome changes dependent on glutaredoxin-2 and mitochondrial peroxiredoxin-1 in Saccharomyces cerevisiae.

Redoxins are involved in maintenance of thiol redox homeostasis, but their exact sites of action are only partly known. We have applied a combined redox proteomics and transcriptomics experimental strategy to discover specific functions of two interacting redoxins: dually localized glutaredoxin 2 (Grx2p) and mitochondrial peroxiredoxin 1 (Prx1p). We have identified 139 proteins showing differential postranslational thiol redox modifications when the cells do not express Grx2p, Prx1p, or both and have mapped the precise cysteines involved in each case. Some of these modifications constitute functional switches that affect metabolic and signaling pathways as the primary effect, leading to gene transcription remodeling as the secondary adaptive effect as demonstrated by a parallel high throughput gene expression analysis. The results suggest that in the absence of Grx2p, the metabolic flow toward nucleotide and aromatic amino acid biosynthesis is slowed down by redox modification of the key enzymes Rpe1p (D-ribulose-5-phosphate 3-epimerase), Tkl1p (transketolase) and Aro4p (3-deoxy-D-arabino-heptulosonate-7-phosphate synthase). The glycolytic mainstream is then diverted toward carbohydrate storage by induction of trehalose and glycogen biosynthesis genes. Porphyrin biosynthesis may also be compromised by inactivation of the redox-sensitive cytosolic enzymes Hem12p (uroporphyrinogen decarboxylase) and Sam1p (S-adenosyl methionine synthetase) and a battery of respiratory genes sensitive to low heme levels are induced. Genes of the Aft1p-dependent iron regulon were induced specifically in the absence of Prx1p despite optimal mitochondrial Fe-S biogenesis, suggesting dysfunction of the mitochondria to the cytosol signaling pathway. Strikingly, requirement of Grx2p for these events places dithiolic Grx2 in the framework of iron metabolism.

Glutaredoxin participates in the reduction of peroxides by the mitochondrial 1-CYS peroxiredoxin in Saccharomyces cerevisiae.

The mechanism for regeneration of the active-site "peroxidatic" cysteine in 1-Cys peroxiredoxins is a matter of debate. Saccharomyces cerevisiae Prx1 is a mitochondrial enzyme belonging to the 1-Cys Prx, whereas Grx2 is involved in antioxidant defense and localizes at the mitochondria, so we hypothesized that it could be a perfect candidate to resolve the sulfenate in Prx1 with GSH. In vitro experiments with purified Prx1p and Grx2p demonstrate that Grx2p, at concentrations <1 microM, coupled to GSH, is a very efficient thiolic intermediary for the reduction of the peroxidatic Cys in Prx1p. Prx1p forms oligomeric aggregates natively, but depolymerizes down to a dimeric state after treatment with GSH. The catalytic cycle involves glutathionylation of dimeric Prx1p and deglutathionylation by Grx2p. Dihydrolipoamide, a genuine mitochondrial dithiol, can efficiently substitute for GSH. The activity is highest at alkaline pH, consistent with the conditions of active respiring mitochondria, and the process is highly specific for 1-Cys Prx because Grx2p is totally inactive with human PRX1, a typical 2-Cys Prx, as opposed to the promiscuity of Trx. Our results suggest that although Trx is the reductant involved in the reduction of peroxides by 2-Cys-Prx, Grx might be the natural resolving partner of 1-Cys Prx through a monothiolic mechanism.

Selection of thiol- and disulfide-containing proteins of Escherichia coli on activated thiol-Sepharose.

Activated thiol-Sepharose (ATS) facilitates selection of thiol-containing proteins. In control- and menadione-treated Escherichia coli, batch selection performed under denaturing conditions revealed distinct two-dimensional electrophoresis (2DE) patterns. Using shotgun proteomics, 183 thiol-containing proteins were identified in control ATS-selected extracts and 126 were identified in menadione-treated E. coli, with 85 proteins being common to both. More than 90% of identified proteins contained one or more cysteines. Blocking with N-ethyl maleimide followed by reduction facilitated ATS-based selection of disulfide-containing proteins. In total, 62 proteins were unique to control cells and 164 were identified in menadione-treated E. coli cells, with 29 proteins being common to both. Proteins from menadione-treated cells were excised from 2DE gels, digested with trypsin, and identified by peptide mass fingerprinting. This revealed 19 unique proteins, 14 of which were identified by shotgun proteomics. Outer membrane proteins A, C, W, and X and 30S ribosomal protein S1 were found in 2DE but not by shotgun proteomics. Foldases, ribosomal proteins, aminoacyl transfer RNA (tRNA) synthetases, and metabolic and antioxidant enzymes were prominent among identified proteins, and many had previously been found to respond to, and be targets for, oxidative stress in E. coli. ATS provides a convenient and rapid way to select thiol-containing proteins.

Shotgun redox proteomics identifies specifically modified cysteines in key metabolic enzymes under oxidative stress in Saccharomyces cerevisiae.

Post-translational redox modification of thiol groups can form the molecular basis of antioxidative protection and redox control. We have implemented a shotgun redox proteomic technique to identify the precise cysteines reversibly oxidised in key proteins. The method was applied to Saccharomyces cerevisiae subjected to peroxide treatment. Enrichment by covalent redox affinity chromatography allowed the isolation of a "redox subpeptidome" that was analysed by LC-MS/MS. Unique peptides containing specific reversibly oxidised cysteines were used to identify over 70 proteins in control and treated samples of which 27 were consistently present in all replicates. In most cases, the redox modification negatively affects their function and slows down their metabolic pathways. Integration of the data provides a snapshot consistent with a metabolic defensive strategy, regulating key enzymes by redox modification, redirecting energy toward ribulose-5-phosphate recycling for NADPH production and antioxidative defence.This generally applicable method has allowed us to discover new redox regulated proteins (DAHP and carbamoylphosphate synthases, Doa1p) and to precisely identify target cysteines in a number of known ones.

Structural aspects of the distinct biochemical properties of glutaredoxin 1 and glutaredoxin 2 from Saccharomyces cerevisiae.

Glutaredoxins (Grxs) are small (9-12 kDa) heat-stable proteins that are ubiquitously distributed. In Saccharomyces cerevisiae, seven Grx enzymes have been identified. Two of them (yGrx1 and yGrx2) are dithiolic, possessing a conserved Cys-Pro-Tyr-Cys motif. Here, we show that yGrx2 has a specific activity 15 times higher than that of yGrx1, although these two oxidoreductases share 64% identity and 85% similarity with respect to their amino acid sequences. Further characterization of the enzymatic activities through two-substrate kinetics analysis revealed that yGrx2 possesses a lower K(M) for glutathione and a higher turnover than yGrx1. To better comprehend these biochemical differences, the pK(a) of the N-terminal active-site cysteines (Cys27) of these two proteins and of the yGrx2-C30S mutant were determined. Since the pK(a) values of the yGrx1 and yGrx2 Cys27 residues are very similar, these parameters cannot account for the difference observed between their specific activities. Therefore, crystal structures of yGrx2 in the oxidized form and with a glutathionyl mixed disulfide were determined at resolutions of 2.05 and 1.91 A, respectively. Comparisons of yGrx2 structures with the recently determined structures of yGrx1 provided insights into their remarkable functional divergence. We hypothesize that the substitutions of Ser23 and Gln52 in yGrx1 by Ala23 and Glu52 in yGrx2 modify the capability of the active-site C-terminal cysteine to attack the mixed disulfide between the N-terminal active-site cysteine and the glutathione molecule. Mutagenesis studies supported this hypothesis. The observed structural and functional differences between yGrx1 and yGrx2 may reflect variations in substrate specificity.

Changes in the proteome of functional and regressing corpus luteum during pregnancy and lactation in the rat.

The corpus luteum (CL) is an exquisitely regulated transitory endocrine gland necessary for the onset and maintenance of pregnancy in mammals. Most of the data on the mechanisms of CL differentiation at the molecular level come from genomic studies, but direct protein data are scarce. Here we have undertaken a differential expression proteomic approach to identify, in an unbiased way, those proteins whose levels change significantly in the rat CL as it evolves from functionality during pregnancy to regression after parturition. Moreover, we have compared the regressing CL with the newly formed functional CL that coexist during lactation under the same endocrine environment. We have defined a "proteomic signature" of CL functionality, which is constituted by a set of 24 proteins with a few differences between pregnancy and lactation. Most of these markers are new and are involved in microtubule assembly, retinoic acid transport, and Raf kinase signaling cascade; 10 are enzymes that define a ketogenic metabolic landscape, demonstrating, for the first time, the prevalence of de novo cholesterol synthesis in luteal cells. The "proteomic signature of regression," on the other hand, is composed of nine proteins, one of which is 20alpha-hydroxysteroid dehydrogenase and two, ferritin and gamma-actin, are new. The discovery of unpredictable new actors in the differentiation process of CL reported here will contribute to new hypotheses that explain the complex female reproductive function at the protein level. It will also open new doors to research on each identified protein by relating them to cellular differentiation.