First Structural Insights into alpha-L-Arabinofuranosidases from the Two GH62 Glycoside Hydrolase Subfamilies*

Background: -l-Arabinofuranosidases hydrolyze arabinofuranosyl side chains from xylans. Results: The first crystal structures of two fungal -l-arabinofuranosidases representing two distinct subfamilies from the glycoside hydrolase GH62 family are presented. The examination of these unveils specificity determinants. Conclusion: The structures of complexes with arabinose and cellotriose provide preliminary insight into substrate recognition and catalysis. Significance: This work provides the first structural description members of the GH62 family. -l-Arabinofuranosidases are glycoside hydrolases that specifically hydrolyze non-reducing residues from arabinose-containing polysaccharides. In the case of arabinoxylans, which are the main components of hemicellulose, they are part of microbial xylanolytic systems and are necessary for complete breakdown of arabinoxylans. Glycoside hydrolase family 62 (GH62) is currently a small family of -l-arabinofuranosidases that contains only bacterial and fungal members. Little is known about the GH62 mechanism of action, because only a few members have been biochemically characterized and no three-dimensional structure is available. Here, we present the first crystal structures of two fungal GH62 -l-arabinofuranosidases from the basidiomycete Ustilago maydis (UmAbf62A) and ascomycete Podospora anserina (PaAbf62A). Both enzymes are able to efficiently remove the -l-arabinosyl substituents from arabinoxylan. The overall three-dimensional structure of UmAbf62A and PaAbf62A reveals a five-bladed -propeller fold that confirms their predicted classification into clan GH-F together with GH43 -l-arabinofuranosidases. Crystallographic structures of the complexes with arabinose and cellotriose reveal the important role of subsites +1 and +2 for sugar binding. Intriguingly, we observed that PaAbf62A was inhibited by cello-oligosaccharides and displayed binding affinity to cellulose although no activity was observed on a range of cellulosic substrates. Bioinformatic analyses showed that UmAbf62A and PaAbf62A belong to two distinct subfamilies within the GH62 family. The results presented here provide a framework to better investigate the structure-function relationships within the GH62 family

Affinity Chromatography: A Valuable Strategy to Isolate Substrates of Methionine Sulfoxide Reductases?

Reactive oxygen species fulfill key roles in development and signaling, but lead at high concentration to damage in macromolecules. In proteins, methionine (Met) is particularly prone to oxidative modification and can be oxidized into Met sulfoxide (MetO). MetO reduction is catalyzed by specialized enzymes, termed methionine sulfoxide reductases (MSRs), involved in senescence and protection against diseases and environmental constraints. The precise physiological functions of MSRs remain often elusive because of very poor knowledge of their substrates. In this study, affinity chromatography was used to isolate partners of Arabidopsis thaliana plastidial methionine sulfoxide reductase B1 (MSRB1). Twenty-four proteins involved in photosynthesis, translation, and protection against oxidative stress, as well as in metabolism of sugars and amino acids, were identified. Statistical analysis shows that the abundance of MSRB1 partners in chromatography affinity samples is proportional to Met content. All proteins, for which structural modeling was feasible, display surface-exposed Met and are thus potentially susceptible to oxidation. Biochemical analyses demonstrated that H2O2 treatment actually converts several MSRB1-interacting proteins into MSRB substrates. In consequence, we propose that affinity chromatography constitutes an efficient tool to isolate physiological targets of MSRs. Antioxid. Redox Signal. 16, 79-84

Bisubstrate-Type Chemical Probes Identify GRP94 as a Potential Target of Cytosine-Containing Adenosine Analogs

International audienceWe synthesized affinity-based chemical probes of cytosine-adenosine bisubstrate analogs and identified several potential targets by proteomic analysis. The validation of the proteomic analysis identified the chemical probe as a specific inhibitor of glucose-regulated protein 94 (GRP94), a potential drug target for several types of cancers. Therefore, as a result of the use of bisubstrate-type chemical probes and a chemical-biology methodology, this work opens the way to the development of a new family of GRP94 inhibitors that could potentially be of therapeutic interest

Chronic estradiol treatment reduces platelet responses and protects mice from thromboembolism through the hematopoietic estrogen receptor α.

International audienceAlthough estrogens are known to have a deleterious effect on the venous thrombosis risk and a preventive action on the development of arterial atheroma, their effect on platelet function in vivo remains unclear. Here, we demonstrate that a chronic high physiologic level of estradiol (E2) in mice leads to a marked decrease in platelet responsiveness ex vivo and in vivo compared with ovariectomized controls. E2 treatment led to increased bleeding time and a resistance to thromboembolism. Hematopoietic chimera mice harboring a selective deletion of estrogen receptors (ERs) α or β were used to demonstrate that the effects of E2 were exclusively because of hematopoietic ERα. Within ERα the activation function-1 domain was not required for resistance to thromboembolism, as was previously shown for atheroprotection. This domain is mandatory for E2-mediated reproductive function and suggests that this role is controlled independently. Differential proteomics indicated that E2 treatment modulated the expression of platelet proteins including β1 tubulin and a few other proteins that may impact platelet production and activation. Overall, these data demonstrate a previously unrecognized role for E2 in regulating the platelet proteome and platelet function, and point to new potential antithrombotic and vasculoprotective therapeutic strategies

Looking for missing proteins in the proteome of human spermatozoa: an update

The Chromosome-Centric Human Proteome Project aims to identify proteins classed as « missing » in the neXtProt knowledgebase. In this article, we present an in-depth proteomics analysis of the human sperm proteome to identify testis-enriched missing proteins. Using a range of protein extraction procedures and LC-MS/MS analysis, we detected a total of 235 proteins (PE2-PE4) for which no previous evidence of protein expression was annotated. Through a combination of LC-MS/MS and LC-PRM analysis, data mining and immunohistochemistry, we were able to confirm the expression of 206 missing proteins (PE2-4) in line with current HPP guidelines (version 2.0). Parallel Reaction Monitoring (PRM) acquisition combined with synthetic heavy labeled peptides was used to target 36 « one-hit wonder » candidates selected on the basis of prior PSM assessment. Of this subset of candidates, 24 were validated with additional predicted and specifically targeted peptides. Evidence was found for a further 16 missing proteins using immunohistochemistry on human testis sections. The expression pattern for some of these proteins was specific to the testis, and they could potentially be valuable markers with applications in fertility assessment. Strong evidence was also found for the existence of 4 proteins labeled as "uncertain" (PE5); the status of these proteins should therefore be re-examined. Our results show how the use of a range of sample preparation techniques combined with MS-based analysis, expert knowledge and complementary antibody-based techniques can produce data of interest to the community. All MS/MS data are available via ProteomeXchange under identifier PXD003947. In addition to contributing to the Chromosome-Centric Human Proteome Project, we hope the availability of these data will stimulate the continued exploration of the sperm proteome

Optimization of adsorptive removal of α-toluic acid by CaO2 nanoparticles using response surface methodology

The present work addresses the optimization of process parameters for adsorptive removal of α-toluic acid by calcium peroxide (CaO2) nanoparticles using response surface methodology (RSM). CaO2 nanoparticles were synthesized by chemical precipitation method and confirmed by Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) analysis which shows the CaO2 nanoparticles size range of 5–15 nm. A series of batch adsorption experiments were performed using CaO2 nanoparticles to remove α-toluic acid from the aqueous solution. Further, an experimental based central composite design (CCD) was developed to study the interactive effect of CaO2 adsorbent dosage, initial concentration of α-toluic acid, and contact time on α-toluic acid removal efficiency (response) and optimization of the process. Analysis of variance (ANOVA) was performed to determine the significance of the individual and the interactive effects of variables on the response. The model predicted response showed a good agreement with the experimental response, and the coefficient of determination, (R2) was 0.92. Among the variables, the interactive effect of adsorbent dosage and the initial α-toluic acid concentration was found to have more influence on the response than the contact time. Numerical optimization of process by RSM showed the optimal adsorbent dosage, initial concentration of α-toluic acid, and contact time as 0.03 g, 7.06 g/L, and 34 min respectively. The predicted removal efficiency was 99.50%. The experiments performed under these conditions showed α-toluic acid removal efficiency up to 98.05%, which confirmed the adequacy of the model prediction

SARS-CoV-2 vaccination modelling for safe surgery to save lives: data from an international prospective cohort study

Background: Preoperative SARS-CoV-2 vaccination could support safer elective surgery. Vaccine numbers are limited so this study aimed to inform their prioritization by modelling. Methods: The primary outcome was the number needed to vaccinate (NNV) to prevent one COVID-19-related death in 1 year. NNVs were based on postoperative SARS-CoV-2 rates and mortality in an international cohort study (surgical patients), and community SARS-CoV-2 incidence and case fatality data (general population). NNV estimates were stratified by age (18-49, 50-69, 70 or more years) and type of surgery. Best- and worst-case scenarios were used to describe uncertainty. Results: NNVs were more favourable in surgical patients than the general population. The most favourable NNVs were in patients aged 70 years or more needing cancer surgery (351; best case 196, worst case 816) or non-cancer surgery (733; best case 407, worst case 1664). Both exceeded the NNV in the general population (1840; best case 1196, worst case 3066). NNVs for surgical patients remained favourable at a range of SARS-CoV-2 incidence rates in sensitivity analysis modelling. Globally, prioritizing preoperative vaccination of patients needing elective surgery ahead of the general population could prevent an additional 58 687 (best case 115 007, worst case 20 177) COVID-19-related deaths in 1 year. Conclusion: As global roll out of SARS-CoV-2 vaccination proceeds, patients needing elective surgery should be prioritized ahead of the general population