Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade

Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system–induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence (HSQC) spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys66, or by the non-conserved residue Cys127. We noted that the overall structural changes during the disulfide cascade expose the Cys122–Cys66 disulfide to recycling through thioredoxin (Trx). In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the non-conserved Cys127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure–function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys122–Cys66 disulfide for Trx reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation

Hypocrates is a genetically encoded fluorescent biosensor for (pseudo)hypohalous acids and their derivatives

The lack of tools to monitor the dynamics of (pseudo)hypohalous acids in live cells and tissues hinders a better understanding of inflammatory processes. Here we present a fluorescent genetically encoded biosensor, Hypocrates, for the visualization of (pseudo)hypohalous acids and their derivatives. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from Escherichia coli. We show that Hypocrates is ratiometric, reversible, and responds to its analytes in the 106 M-1s-1 range. Solving the Hypocrates X-ray structure provided insights into its sensing mechanism, allowing determination of the spatial organization in this circularly permuted fluorescent protein-based redox probe. We exemplify its applicability by imaging hypohalous stress in bacteria phagocytosed by primary neutrophils. Finally, we demonstrate that Hypocrates can be utilized in combination with HyPerRed for the simultaneous visualization of (pseudo)hypohalous acids and hydrogen peroxide dynamics in a zebrafish tail fin injury model

Cross-cultural adaptation of the VISA-A questionnaire, an index of clinical severity for patients with Achilles tendinopathy, with reliability, validity and structure evaluations

BACKGROUND: Achilles tendinopathy is considered to be one of the most common overuse injuries in elite and recreational athletes and the recommended treatment varies. One factor that has been stressed in the literature is the lack of standardized outcome measures that can be used in all countries. One such standardized outcome measure is the Victorian Institute of Sports Assessment – Achilles (VISA-A) questionnaire, which is designed to evaluate the clinical severity for patients with Achilles tendinopathy. The purpose of this study was to cross-culturally adapt the VISA-A questionnaire to Swedish, and to perform reliability, validity and structure evaluations. METHODS: Cross-cultural adaptation was performed in several steps including translations, synthesis of translations, back translations, expert committee review and pre-testing. The final Swedish version, the VISA-A Swedish version (VISA-A-S) was tested for reliability on healthy individuals (n = 15), and patients (n = 22). Tests for internal consistency, validity and structure were performed on 51 patients. RESULTS: The VISA-A-S had good reliability for patients (r = 0.89, ICC = 0.89) and healthy individuals (r = 0.89–0.99, ICC = 0.88–0.99). The internal consistency was 0.77 (Cronbach's alpha). The mean [95% confidence interval] VISA-A-S score in the 51 patients (50 [44–56]) was significantly lower than in the healthy individuals (96 [94–99]). The VISA-A-S score correlated significantly (Spearman's r = -0.68) with another tendon grading system. Criterion validity was considered good when comparing the scores of the Swedish version with the English version in both healthy individuals and patients. The factor analysis gave the factors pain/symptoms and physical activity CONCLUSION: The VISA-A-S questionnaire is a reliable and valid instrument and comparable to the original version. It measures two factors: pain/symptoms and physical activity, and can be used in both research and the clinical setting

High-confidence glycosome proteome for procyclic form <em>Trypanosoma brucei</em> by epitope-tag organelle enrichment and SILAC proteomics

The glycosome of the pathogenic African trypanosome Trypanosoma brucei is a specialized peroxisome that contains most of the enzymes of glycolysis and several other metabolic and catabolic pathways. The contents and transporters of this membrane-bounded organelle are of considerable interest as potential drug targets. Here we use epitope tagging, magnetic bead enrichment, and SILAC quantitative proteomics to determine a high-confidence glycosome proteome for the procyclic life cycle stage of the parasite using isotope ratios to discriminate glycosomal from mitochondrial and other contaminating proteins. The data confirm the presence of several previously demonstrated and suggested pathways in the organelle and identify previously unanticipated activities, such as protein phosphatases. The implications of the findings are discussed

Evaluation of chloroform/methanol extraction to facilitate the study of membrane proteins of non-model plants

Membrane proteins are of great interest to plant physiologists because of their important function in many physiological processes. However, their study is hampered by their low abundance and poor solubility in aqueous buffers. Proteomics studies of non-model plants are generally restricted to gel-based methods. Unfortunately, all gel-based techniques for membrane proteomics lack resolving power. Therefore, a very stringent enrichment method is needed before protein separation. In this study, protein extraction in a mixture of chloroform and methanol in combination with gel electrophoresis is evaluated as a method to study membrane proteins in non-model plants. Benefits as well as disadvantages of the method are discussed. To demonstrate the pitfalls of working with non-model plants and to give a proof of principle, the method was first applied to whole leaves of the model plant Arabidopsis. Subsequently, a comparison with proteins extracted from leaves of the non-model plant, banana, was made. To estimate the tissue and organelle specificity of the method, it was also applied on banana meristems. Abundant membrane or lipid-associated proteins could be identified in both tissues, with the leaf extract yielding a higher number of membrane proteins

Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase Modulates Oscillations of Pancreatic Islet Metabolism

Pulses of insulin from pancreatic beta-cells help maintain blood glucose in a narrow range, although the source of these pulses is unclear. It has been proposed that a positive feedback circuit exists within the glycolytic pathway, the autocatalytic activation of phosphofructokinase-1 (PFK1), which endows pancreatic beta-cells with the ability to generate oscillations in metabolism. Flux through PFK1 is controlled by the bifunctional enzyme PFK2/FBPase2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) in two ways: via (1) production/degradation of fructose-2,6-bisphosphate (Fru2,6-BP), a potent allosteric activator of PFK1, as well as (2) direct activation of glucokinase due to a protein-protein interaction. In this study, we used a combination of live-cell imaging and mathematical modeling to examine the effects of inducibly-expressed PFK2/FBPase2 mutants on glucose-induced Ca2+ pulsatility in mouse islets. Irrespective of the ability to bind glucokinase, mutants of PFK2/FBPase2 that increased the kinase:phosphatase ratio reduced the period and amplitude of Ca2+ oscillations. Mutants which reduced the kinase:phosphatase ratio had the opposite effect. These results indicate that the main effect of the bifunctional enzyme on islet pulsatility is due to Fru2,6-BP alteration of the threshold for autocatalytic activation of PFK1 by Fru1,6-BP. Using computational models based on PFK1-generated islet oscillations, we then illustrated how moderate elevation of Fru-2,6-BP can increase the frequency of glycolytic oscillations while reducing their amplitude, with sufficiently high activation resulting in termination of slow oscillations. The concordance we observed between PFK2/FBPase2-induced modulation of islet oscillations and the models of PFK1-driven oscillations furthermore suggests that metabolic oscillations, like those found in yeast and skeletal muscle, are shaped early in glycolysis