A white paper on Phospholipid Hydroperoxide Glutathione Peroxidase (GPx4) forty years later.

The purification of a protein inhibiting lipid peroxidation led to the discovery of the selenoperoxidase GPx4 forty years ago. Thus, the evidence of the enzymatic activity was reached after identifying the biological effect and unambiguously defined the relationship between the biological function and the enzymatic activity. In the syllogism where GPx4 inhibits lipid peroxidation and its inhibition is lethal, cell death is operated by lipid peroxidation. Based on this rationale, this form of cell death emerged as regulated iron-enforced oxygen toxicity and was named ferroptosis in 2012. In the last decades, we learned that reduction of lipid hydroperoxides is indispensable and, in cooperation with prooxidant systems, controls the critical steady state of lipid peroxidation. This concept defined the GPx4 reaction as both the target for possible anti-cancer therapy and if insufficient, as cause of degenerative diseases. We know the reaction mechanism, but the details of the interaction at the membrane cytosol interface are still poorly defined. We know the gene structure, but the knowledge about expression control is still limited. The same holds true for post-transcriptional modifications. Reverse genetics indicate that GPx4 has a role in inflammation, immunity, and differentiation, but the observations emerging from these studies need a more specifically addressed biochemical evidence. Finally, the role of GPx4 in spermatogenesis disclosed an area unconnected to lipid peroxidation. In its mitochondrial and nuclear form, the peroxidase catalyzes the oxidation of protein thiols in two specific aspects of sperm maturation: stabilization of the mid-piece and chromatin compaction. Thus, although available evidence converges to the notion that GPx4 activity is vital due to the inhibition of lipid peroxidation, it is reasonable to foresee other unknown aspects of the GPx4 reaction to be disclosed.

Suppression of a SARS-CoV-2 outbreak in the Italian municipality of Vo'.

On 21 February 2020, a resident of the municipality of Vo', a small town near Padua (Italy), died of pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection1. This was the first coronavirus disease 19 (COVID-19)-related death detected in Italy since the detection of SARS-CoV-2 in the Chinese city of Wuhan, Hubei province2. In response, the regional authorities imposed the lockdown of the whole municipality for 14 days3. Here we collected information on the demography, clinical presentation, hospitalization, contact network and the presence of SARS-CoV-2 infection in nasopharyngeal swabs for 85.9% and 71.5% of the population of Vo' at two consecutive time points. From the first survey, which was conducted around the time the town lockdown started, we found a prevalence of infection of 2.6% (95% confidence interval (CI): 2.1-3.3%). From the second survey, which was conducted at the end of the lockdown, we found a prevalence of 1.2% (95% CI: 0.8-1.8%). Notably, 42.5% (95% CI: 31.5-54.6%) of the confirmed SARS-CoV-2 infections detected across the two surveys were asymptomatic (that is, did not have symptoms at the time of swab testing and did not develop symptoms afterwards). The mean serial interval was 7.2 days (95% CI: 5.9-9.6). We found no statistically significant difference in the viral load of symptomatic versus asymptomatic infections (P = 0.62 and 0.74 for E and RdRp genes, respectively, exact Wilcoxon-Mann-Whitney test). This study sheds light on the frequency of asymptomatic SARS-CoV-2 infection, their infectivity (as measured by the viral load) and provides insights into its transmission dynamics and the efficacy of the implemented control measures.

Copper/Zinc Superoxide Dismutase from the Crocodile Icefish Chionodraco hamatus: Antioxidant Defense at Constant Sub-Zero Temperature.

In the present study, we describe the purification and molecular characterization of Cu,Zn superoxide dismutase (SOD) from Chionodraco hamatus, an Antarctic teleost widely distributed in many areas of the Ross Sea that plays a pivotal role in the Antarctic food chain. The primary sequence was obtained using biochemical and molecular biology approaches and compared with Cu,Zn SODs from other organisms. Multiple sequence alignment using the amino acid sequence revealed that Cu,Zn SOD showed considerable sequence similarity with its orthologues from various vertebrate species, but also some specific substitutions directly linked to cold adaptation. Phylogenetic analyses presented the monophyletic status of Antartic Teleostei among the Perciformes, confirming the erratic differentiation of these proteins and concurring with the theory of the "unclock-like" behavior of Cu,Zn SOD evolution. Expression of C. hamatus Cu,Zn SOD at both the mRNA and protein levels were analyzed in various tissues, highlighting the regulation of gene expression related to environmental stress conditions and also animal physiology. The data presented are the first on the antioxidant enzymes of a fish belonging to the Channichthyidae family and represent an important starting point in understanding the antioxidant systems of these organisms that are subject to constant risk of oxidative stress.

Inactivation of the glutathione peroxidase GPx4 by the ferroptosis-inducing molecule RSL3 requires the adaptor protein 14-3-3ε.

Ras-selective lethal small molecule 3 (RSL3), a drug candidate prototype for cancer chemotherapy, triggers ferroptosis by inactivating the glutathione peroxidase glutathione peroxidase 4 (GPx4). Here, we report the purification of the protein indispensable for GPx4 inactivation by RSL3. Mass spectrometric analysis identified 14-3-3 isoforms as candidates, and recombinant human 14-3-3ε confirms the identification. The function of 14-3-3ε is redox-regulated. Moreover, overexpression or silencing of the gene coding for 14-3-3ε consistently controls the inactivation of GPx4 by RSL3. The interaction of GPx4 with a redox-regulated adaptor protein operating in cell signaling further contributes to frame it within redox-regulated pathways of cell survival and death and opens new therapeutic perspectives.

Loss of cardiac Wnt/ß-catenin signalling in desmoplakin-deficient AC8 zebrafish models is rescuable by genetic and pharmacological intervention.

Aims: Arrhythmogenic cardiomyopathy (AC) is an inherited heart disease characterized by life-threatening ventricular arrhythmias and fibro-fatty replacement of the myocardium. More than 60% of AC patients show pathogenic mutations in genes encoding for desmosomal proteins. By focusing our attention on the AC8 form, linked to the junctional protein desmoplakin (DSP), we present here a zebrafish model of DSP deficiency, exploited to identify early changes of cell signalling in the cardiac region. Methods and results: To obtain an embryonic model of Dsp deficiency, we first confirmed the orthologous correspondence of zebrafish Dsp genes (dspa and dspb) to the human DSP counterpart. Then, we verified their cardiac expression, at embryonic and adult stages, and subsequently we targeted them by antisense morpholino strategy, confirming specific and disruptive effects on desmosomes, like those identified in AC patients. Finally, we exploited our Dsp-deficient models for an in vivo cell signalling screen, using pathway-specific reporter transgenes. Out of nine considered, three pathways (Wnt/ß-catenin, TGFß/Smad3, and Hippo/YAP-TAZ) were significantly altered, with Wnt as the most dramatically affected. Interestingly, under persistent Dsp deficiency, Wnt signalling is rescuable both by a genetic and a pharmacological approach. Conclusion: Our data point to Wnt/ß-catenin as the final common pathway underlying different desmosomal AC forms and support the zebrafish as a suitable model for detecting early signalling pathways involved in the pathogenesis of DSP-associated diseases, possibly responsive to pharmacological or genetic rescue.

Redox status in a model of cancer stem cells.

Reversible oxidation of Cys residues is a crucial element of redox homeostasis and signaling. According to a popular concept in oxidative stress signaling, the oxidation of targets of signals can only take place following an overwhelming of the cellular antioxidant capacity. This concept, however, ignores the activation of feedback mechanisms possibly leading to a paradoxical effect. In a model of cancer stem cells (CSC), stably overexpressing the TAZ oncogene, we observed that the increased formation of oxidants is associated with a globally more reduced state of proteins. Redox proteomics revealed that several proteins, capable of undergoing reversible redox transitions, are indeed more reduced while just few are more oxidized. Among the proteins more oxidized, G6PDH emerges as both more expressed and activated by oxidation. This accounts for the observed more reduced state of the NADPH/NADP+ couple. The dynamic redox flux generating this apparently paradoxical effect is rationalized in a computational system biology model highlighting the crucial role of G6PDH activity on the rate of redox transitions eventually leading to the reduction of reversible redox switches.

Characterization of Intra-Type Variants of Oncogenic Human Papillomaviruses by Next-Generation Deep Sequencing of the E6/E7 Region.

Different human papillomavirus (HPV) types are characterized by differences in tissue tropism and ability to promote cell proliferation and transformation. In addition, clinical and experimental studies have shown that some genetic variants/lineages of high-risk HPV (HR-HPV) types are characterized by increased oncogenic activity and probability to induce cancer. In this study, we designed and validated a new method based on multiplex PCR-deep sequencing of the E6/E7 region of HR-HPV types to characterize HPV intra-type variants in clinical specimens. Validation experiments demonstrated that this method allowed reliable identification of the different lineages of oncogenic HPV types. Advantages of this method over other published methods were represented by its ability to detect variants of all HR-HPV types in a single reaction, to detect variants of HR-HPV types in clinical specimens with multiple infections, and, being based on sequencing of the full E6/E7 region, to detect amino acid changes in these oncogenes potentially associated with increased transforming activity.

Selenocysteine oxidation in glutathione peroxidase catalysis: an MS-supported quantum mechanics study.

Glutathione peroxidases (GPxs) are enzymes working with either selenium or sulfur catalysis. They adopted diverse functions ranging from detoxification of H(2)O(2) to redox signaling and differentiation. The relative stability of the selenoenzymes, however, remained enigmatic in view of the postulated involvement of a highly unstable selenenic acid form during catalysis. Nevertheless, density functional theory calculations obtained with a representative active site model verify the mechanistic concept of GPx catalysis and underscore its efficiency. However, they also allow that the selenenic acid, in the absence of the reducing substrate, reacts with a nitrogen in the active site. MS/MS analysis of oxidized rat GPx4 complies with the predicted structure, an 8-membered ring, in which selenium is bound as selenenylamide to the protein backbone. The intermediate can be re-integrated into the canonical GPx cycle by glutathione, whereas, under denaturing conditions, its selenium moiety undergoes ß-cleavage with formation of a dehydro-alanine residue. The selenenylamide bypass prevents destruction of the redox center due to over-oxidation of the selenium or its elimination and likely allows fine-tuning of GPx activity or alternate substrate reactions for regulatory purposes.

Understanding mammalian glutathione peroxidase 7 in the light of its homologs.

The glutathione peroxidase homologs (GPxs) efficiently reduce hydroperoxides using electrons from glutathione (GSH), thioredoxin (Trx), or protein disulfide isomerase (PDI). Trx is preferentially used by the GPxs of the majority of bacteria, invertebrates, plants, and fungi. GSH or PDI, instead, is preferentially used by vertebrate GPxs that operate by Sec or Cys catalysis, respectively. Mammalian GPx7 and GPx8 are unique homologs that contain a peroxidatic Cys (CP). Being reduced by PDI and located within the endoplasmic reticulum (ER), these enzymes have been involved in oxidative protein folding. Kinetic analysis indicates that oxidation of PDI by recombinant GPx7 occurs at a much faster rate than that of GSH. Nonetheless, activity measurement suggests that, at physiological concentrations, a competition between these two substrates takes place, with the rate of PDI oxidation by GPx7 controlled by the concentration of GSH, whereas the GSSG produced in the competing reaction contributes to the ER redox buffer. A mechanism has been proposed for GPx7 involving two Cys residues, in which an intramolecular disulfide of the CP is formed with an alleged resolving Cys (CR) located in the strongly conserved FPCNQ motif (C86 in humans), a noncanonical position in GPxs. Kinetic measurements and comparison with the other thiol peroxidases containing a functional CR suggest that a resolving function of C86 in the catalytic cycle is very unlikely. We propose that GPx7 is catalytically active as a 1-Cys-GPx, in which CP both reduces H2O2 and oxidizes PDI, and that the CP-C86 disulfide has instead the role of stabilizing the oxidized peroxidase in the absence of the reducing substrate.

Quantitative label-free redox proteomics of reversible cysteine oxidation in red blood cell membranes.

Reversible oxidation of cysteine residues is a relevant posttranslational modification of proteins. However, the low activation energy and transitory nature of the redox switch and the intrinsic complexity of the analysis render quite challenging the aim of a rigorous high-throughput screening of the redox status of redox-sensitive cysteine residues. We describe here a quantitative workflow for redox proteomics, where the ratio between the oxidized forms of proteins in the control vs treated samples is determined by a robust label-free approach. We critically present the convenience of the procedure by specifically addressing the following aspects: (i) the accurate ratio, calculated from the whole set of identified peptides rather than just isotope-tagged fragments; (ii) the application of a robust analytical pipeline to frame the most consistent data averaged over the biological variability; (iii) the relevance of using stringent criteria of analysis, even at the cost of losing potentially interesting but statistically uncertain data. The pipeline has been assessed on red blood cell membrane challenged with diamide as a model of a mild oxidative condition. The cluster of identified proteins encompassed components of the cytoskeleton more oxidized. Indirectly, our analysis confirmed the previous observation that oxidized hemoglobin binds to membranes while oxidized peroxiredoxin 2 loses affinity.

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7.

BACKGROUND: Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (CP) it lacks the resolving Cys (CR), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs. METHODS: Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme-substrate and protein-protein interaction were analyzed by molecular docking and surface plasmon resonance analysis. RESULTS: Oxidation of the CP is fast (k+1>10(3)M(-1)s(-1)), however the rate of reduction by GSH is slow (k'+2=12.6M(-1)s(-1)) even though molecular docking indicates a strong GSH-GPx7 interaction. Instead, the oxidized CP can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k+1>10(3)M(-1)s(-1)), but not by Trx. By surface plasmon resonance analysis, a KD=5.2µM was calculated for PDI-GPx7 complex. Participation of an alternative non-canonical CR in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo. CONCLUSIONS: GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates. GENERAL SIGNIFICANCE: In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.

Applications of next-generation sequencing technologies to diagnostic virology.

Novel DNA sequencing techniques, referred to as "next-generation" sequencing (NGS), provide high speed and throughput that can produce an enormous volume of sequences with many possible applications in research and diagnostic settings. In this article, we provide an overview of the many applications of NGS in diagnostic virology. NGS techniques have been used for high-throughput whole viral genome sequencing, such as sequencing of new influenza viruses, for detection of viral genome variability and evolution within the host, such as investigation of human immunodeficiency virus and human hepatitis C virus quasispecies, and monitoring of low-abundance antiviral drug-resistance mutations. NGS techniques have been applied to metagenomics-based strategies for the detection of unexpected disease-associated viruses and for the discovery of novel human viruses, including cancer-related viruses. Finally, the human virome in healthy and disease conditions has been described by NGS-based metagenomics.

Human papillomavirus genotyping by 454 next generation sequencing technology.

BACKGROUND: An accurate tool for human papillomavirus (HPV) typing is important both for management of patients with HPV infection and for surveillance studies. OBJECTIVES: Design and evaluation of an HPV typing method based on 454 next generation sequencing (NGS) technology. STUDY DESIGN: Development of an HPV typing method based on 454 NGS of HPV L1 amplicons generated with MY09/11-based primers. Evaluation of the NGS method in control samples and in a panel of cervical cytological samples. Comparison of the NGS typing method with cycle sequencing and with the reverse hybridization-based INNO-LiPA HPV Genotyping Extra assay (LiPA). RESULTS: In control samples carrying mixtures of HPV16 and HPV18 DNA, the NGS method could reliably detect genotype sequences occurring at a frequency of 1% in multiple infections with a sensitivity of 100 genome equivalents/µL. In cervical cytology samples, comparison with cycle sequencing demonstrated accuracy of HPV typing by NGS. The NGS method had however lower sensitivity for some HPV types than LiPA, conceivably due to the poor sensitivity of the MY09/11-based primers. At variance, LiPA could not detect HPV types which were present in low proportion in multiple infections (<10% of HPV reads obtained by NGS). In addition, NGS allowed identifying the presence of different variants of the same HPV type in a specimen. CONCLUSIONS: NGS is a promising method for HPV typing because of its high sensitivity in multiple infection and its potential ability to detect a broad spectrum of HPV types, subtypes, and variants.

A comparison of thiol peroxidase mechanisms.

Thiol peroxidases comprise glutathione peroxidases (GPx) and peroxiredoxins (Prx). The enzymes of both families reduce hydroperoxides with thiols by enzyme-substitution mechanisms. H(2)O(2) and organic hydroperoxides are reduced by all thiol peroxidases, most efficiently by SecGPxs, whereas fast peroxynitrite reduction is more common in Prxs. Reduction of lipid hydroperoxides is the domain of monomeric GPx4-type enzymes and of some Prxs. The catalysis starts with oxidation of an active-site selenocysteine (U(P)) or cysteine (C(P)). Activation of Cys (Sec) for hydroperoxide reduction in the GPx family is achieved by a typical tetrad composed of Cys (Sec), Asn, Gln, and Trp, whereas a triad of Cys Thr (or Ser) and Arg is the signature of Prx. In many of the CysGPxs and Prxs, a second Cys (C(R)) is required. In these 2-CysGPxs and 2-CysPrxs, the C(P) oxidized to a sulfenic acid forms an intra- or intermolecular disulfide (typical 2-CysPrx) with C(R), before a stepwise regeneration of ground-state enzyme by redoxin-type proteins can proceed. In SecGPxs and sporadically in Prxs, GSH is used as the reductant. Diversity combined with structural variability predestines thiol peroxidases for redox regulation via ROOH sensing and direct or indirect transduction of oxidant signals to specific protein targets.

Human cytomegalovirus productively infects adrenocortical cells and induces an early cortisol response.

Following our recent findings on the presence of human cytomegalovirus (HCMV) in the normal human adrenal cortex and in adrenocortical tumors, especially in cortisol-secreting tumors, aim of the present study was to investigate the direct effects of HCMV infection on human adrenocortical cells. To this aim, both clinical isolates and laboratory strains of HCMV were used to assess the early effects of infection on human adrenocortical cell morphology, proliferation, gene expression, and steroidogenic function. Both clinical and laboratory HCMV strains could infect and replicate in primary human adrenocortical cell cultures and in adrenocortical carcinoma cell lines, leading to cytopathic changes. Most importantly, in the first hours post-infection (p.i.), adrenocortical cells showed a significant increase of cortisol and estrogen production, paralleled by up-regulation of steroidogenic acute regulatory protein and expression of steroidogenic enzymes involved in the last steps of adrenal steroidogenesis. This effect was probably due to HCMV immediate-early gene expression, since it was most evident in the early phases p.i. and UV-inactivated viral particles did not affect hormone production. Moreover, the effect on steroidogenesis was HCMV specific, since it was not observed after infection with herpes simplex virus. These data suggest that human adrenocortical cells are permissive to HCMV infection and acutely respond to infection with increased cortisol production. An acute glucocorticoid response is typically triggered by infections and is considered to be critical to host defense against pathogens, although, in the case of HCMV infection, it might also enhance viral replication and reactivation from latency.

Catalytic mechanisms and specificities of glutathione peroxidases: variations of a basic scheme.

Kinetics and molecular mechanisms of GPx-type enzymes are reviewed with emphasis on structural features relevant to efficiency and specificity. In Sec-GPxs the reaction takes place at a single redox centre with selenocysteine as redox-active residue (peroxidatic Sec, U(P)). In contrast, most of the non-vertebrate GPx have the U(P) replaced by a cysteine (peroxidatic Cys, C(P)) and work with a second redox centre that contains a resolving cysteine (C(R)). While the former type of enzymes is more or less specific for GSH, the latter are reduced by "redoxins". The common denominator of the GPx family is the first redox centre comprising the (seleno)cysteine, tryptophan, asparagine and glutamine. In this architectural context the rate of hydroperoxide reduction by U(P) or C(P), respectively, is enhanced by several orders of magnitude compared to that of free selenolate or thiolate. Mammalian GPx-1 dominates H(2)O(2) metabolism, whereas the domain of GPx-4 is the reduction of lipid hydroperoxides with important consequences such as counteracting 12/15-lipoxygenase-induced apoptosis and regulation of inflammatory responses. Beyond, the degenerate GSH specificity of GPx-4 allows selenylation and oxidation to disulfides of protein thiols. Heterodimer formation of yeast GPx with a transcription factor is discussed as paradigm of a redox sensing that might also be valid in vertebrates.

Evolutionary and structural insights into the multifaceted glutathione peroxidase (Gpx) superfamily.

Glutathione peroxidase (GPx) is a widespread protein superfamily found in many organisms throughout all kingdoms of life. Although it was initially thought to use only glutathione as reductant, recent evidence suggests that the majority of GPxs have specificity for thioredoxin. We present a thorough in silico analysis performed on 724 sequences and 12 structures aimed to clarify the evolutionary, structural, and sequence determinants of GPx specificity. Structural variability was found to be limited to only two regions, termed oligomerization loop and functional helix, which modulate both reduced substrate specificity and oligomerization state. We show that mammalian GPx-1, the canonic selenocysteine-based tetrameric glutathione peroxidase, is a recent "invention" during evolution. Contrary to common belief, cysteine-based thioredoxin-specific GPx, which we propose the TGPx, are both more common and more ancient. This raises interesting evolutionary considerations regarding oligomerization and the use of active-site selenocysteine residue. In addition, phylogenetic analysis has revealed the presence of a novel member belonging to the GPx superfamily in Mammalia and Amphibia, for which we propose the name GPx-8, following the present numeric order of the mammalian GPxs.

Characterization of a novel complex BRAF mutation in a follicular variant papillary thyroid carcinoma.

INTRODUCTION: Activating mutations of the BRAF oncogene are frequently detected in papillary thyroid carcinoma (PTC) and have been associated with a worse prognosis. The amino acid substitution V600E accounts for 90% of all oncogenic BRAF mutations and is typically detected in classic PTCs, whereas other less frequent BRAF mutations seem to be associated with other PTC histotypes. CASE: Screening for activating BRAF mutations in a series of 83 PTCs identified the most common V600E mutation in 39 cases (histologically, 38 classic PTCs and 1 sclerosing variant PTC) and a complex in-frame mutation involving amino acids V600-S605 in a stage III multicentric follicular variant PTC, occurring in a 50-year-old female patient, who was affected by hypothyroidism in autoimmune thyroiditis and had a family history of PTC and autoimmune thyroiditis. Since the identified BRAF mutation was novel in the literature, bioinformatic modeling was performed to predict its impact on BRAF activity. Although the mutation resulted in loss of a phosphorylation site in the activation loop of BRAF, it was predicted to increase BRAF kinase activity by mimicking an activating phosphorylation. CONCLUSIONS: This study, which reports a new BRAF mutation, highlights the usefulness of bioinformatic modeling in the prediction of functional effects of new mutations and indicates that mutation-specific screening tests might miss some rare BRAF mutations. These facts should be taken into consideration in the molecular diagnosis of thyroid cancer and in the design of therapeutic protocols based on inhibitors of the BRAF pathway.

Zebrafish spata2 is expressed at early developmental stages.

Spata2 (spermatogenesis-associated protein 2) was originally described as a novel gene involved in the spermatogenic process. In this study, we cloned a potential zebrafish spata2 orthologue. The consensus open reading frame (1650 bp) encodes a polypeptide of 550 amino acids which shares 37% identity with the human SPATA2. Bioinformatic analysis reveals a small pattern PW [KR] KE [YF][RK] which seems to be of particular interest in the light of its strong conservation between SPATA2 and the recently discovered TAMO protein of D. melanogaster. RT-PCR analysis in adult zebrafish tissues revealed that spata2 mRNA has a broad distribution. Whole-mount in situ hybridization demonstrated that spata2 transcripts are maternally derived and becomes strongly localized in the central nervous system at early developmental stages. From 5 dpf, spata2 expression becomes detectable in the gut and pronephric duct epithelium, suggesting a wide tissue function during vertebrate development.

The thioredoxin specificity of Drosophila GPx: a paradigm for a peroxiredoxin-like mechanism of many glutathione peroxidases.

Some members of the glutathione peroxidase (GPx) family have been reported to accept thioredoxin as reducing substrate. However, the selenocysteine-containing ones oxidise thioredoxin (Trx), if at all, at extremely slow rates. In contrast, the Cys homolog of Drosophila melanogaster exhibits a clear preference for Trx, the net forward rate constant, k'(+2), for reduction by Trx being 1.5x10(6) M(-1) s(-1), but only 5.4 M(-1) s(-1) for glutathione. Like other CysGPxs with thioredoxin peroxidase activity, Drosophila melanogaster (Dm)GPx oxidized by H(2)O(2) contained an intra-molecular disulfide bridge between the active-site cysteine (C45; C(P)) and C91. Site-directed mutagenesis of C91 in DmGPx abrogated Trx peroxidase activity, but increased the rate constant for glutathione by two orders of magnitude. In contrast, a replacement of C74 by Ser or Ala only marginally affected activity and specificity of DmGPx. Furthermore, LC-MS/MS analysis of oxidized DmGPx exposed to a reduced Trx C35S mutant yielded a dead-end intermediate containing a disulfide between Trx C32 and DmGPx C91. Thus, the catalytic mechanism of DmGPx, unlike that of selenocysteine (Sec)GPxs, involves formation of an internal disulfide that is pivotal to the interaction with Trx. Hereby C91, like the analogous second cysteine in 2-cysteine peroxiredoxins, adopts the role of a "resolving" cysteine (C(R)). Molecular modeling and homology considerations based on 450 GPxs suggest peculiar features to determine Trx specificity: (i) a non-aligned second Cys within the fourth helix that acts as C(R); (ii) deletions of the subunit interfaces typical of tetrameric GPxs leading to flexibility of the C(R)-containing loop. Based of these characteristics, most of the non-mammalian CysGPxs, in functional terms, are thioredoxin peroxidases.