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Graphical abstract, Highlights • Corneal stromal dystrophies are a group of hereditary disorders caused by mutations in the TGFBI gene and affect the corneal stroma and epithelium. • The disease is characterized by the accumulation of insoluble deposits of the mutant TGFBIp leading to poor visual acuity in patients. • Mutations are hypothesized to disrupt the protein folding and stability, leading oligomerization of the mutant protein. • Current treatment relies on surgical intervention, either tissue removal or substitution, both of which are associated with disease recurrence. • The lead compounds reported here prevent/delay the atypical proteolysis of the mutant protein and the generation of amyloidogenic fragments., Corneal dystrophies are a group of genetically inherited disorders with mutations in the TGFBI gene affecting the Bowman’s membrane and the corneal stroma. The mutant TGFBIp is highly aggregation-prone and is deposited in the cornea. Depending on the type of mutation the protein deposits may vary (amyloid, amorphous powdery aggregate or a mixed form of both), making the cornea opaque and thereby decreases visual acuity. The aggregation of the mutant protein is found to be specific with a unique aggregation mechanism distinct to the cornea. The proteolytic processing of the mutant protein is reported to be different compared to the WT protein. The proteolytic processing of mutant protein gives rise to highly amyloidogenic peptide fragments. The current treatment option, available for patients, is tissue replacement surgery that is associated with high recurrence rates. The clinical need for a simple treatment option for corneal dystrophy patients has become highly essential either to prevent the protein aggregation or to dissolve the preformed aggregates. Here, we report the screening of 2500 compounds from the Maybridge RO3 fragment library using weak affinity chromatography (WAC). The primary hits from WAC were validated by 15N-HSQC NMR assays and specific regions of binding were identified. The recombinant mutant proteins (4th FAS-1 domain of R555W and H572R) were subjected to limited proteolysis by trypsin together with the lead compounds identified by NMR assays. The lead compounds (MO07617, RJF00203 and, BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the R555W mutant and compounds (RJF00203 and BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the H572R mutant. Thus the lead compounds reported here upon further validation and/or modification might be proposed as a potential treatment option to prevent/delay aggregation by inhibiting the formation of amyloidogenic peptides in TGFBI-corneal dystrophy.

Highlights • XyrB is involved in conversion of d-xylose and l-arabinose in A. niger. • The xyrB expression is induced both by d-xylose and l-arabinose. • XyrB expression is controlled by xlnR and araR regulators., d-xylose reductase is a member of the aldo-keto reductase family, and is involved in d-xylose and l-arabinose conversion through the Pentose Catabolic Pathway (PCP) in fungi. In this study, we biochemically characterized a newly identified second d-xylose reductase (XyrB) from Aspergillus niger. This NADPH-dependent reductase is able to efficiently convert d-xylose and l-arabinose, and it has the highest affinity for these sugars of all currently known fungal pentose reductases. A combination of biochemical data, transcriptomics and phylogenetic analysis further illustrated the role of XyrB in the PCP. Enzymes: D-xylose reductase (EC 1.1.1.307), L-arabinose reductase (EC 1.1.1.21).

Staphylococcus aureus produces bacillithiol (BSH) as major low molecular weight (LMW) thiol which functions in thiol-protection and redox-regulation by protein S-bacillithiolation under hypochlorite stress. The aldehyde dehydrogenase AldA was identified as S-bacillithiolated at its active site Cys279 under NaOCl stress in S. aureus. Here, we have studied the expression, function, redox regulation and structural changes of AldA of S. aureus. Transcription of aldA was previously shown to be regulated by the alternative sigma factor SigmaB. Northern blot analysis revealed SigmaB-independent induction of aldA transcription under formaldehyde, methylglyoxal, diamide and NaOCl stress. Deletion of aldA resulted in a NaOCl-sensitive phenotype in survival assays, suggesting an important role of AldA in the NaOCl stress defense. Purified AldA showed broad substrate specificity for oxidation of several aldehydes, including formaldehyde, methylglyoxal, acetaldehyde and glycol aldehyde. Thus, AldA could be involved in detoxification of aldehyde substrates that are elevated under NaOCl stress. Kinetic activity assays revealed that AldA is irreversibly inhibited under H2O2 treatment in vitro due to overoxidation of Cys279 in the absence of BSH. Pre-treatment of AldA with BSH prior to H2O2 exposure resulted in reversible AldA inactivation due to S-bacillithiolation as revealed by activity assays and BSH-specific Western blot analysis. Using molecular docking and molecular dynamic simulation, we further show that BSH occupies two different positions in the AldA active site depending on the AldA activation state. In conclusion, we show here that AldA is an important target for S-bacillithiolation in S. aureus that is up-regulated under NaOCl stress and functions in protection under hypochlorite stress., Graphical abstract fx1, Highlights • AldA is S-bacillithiolated at Cys279 under NaOCl stress in Staphylococcus aureus. • AldA is strongly induced under NaOCl and aldehyde stress and protects S. aureus under hypochlorite stress. • S-bacillithiolation functions in redox-regulation of AldA activity. • The position of BSH in the AldA active site depends on the Cys279 activation state.

Mycothiol (MSH) functions as major low molecular weight (LMW) thiol in the industrially important Corynebacterium glutamicum. In this study, we genomically integrated an Mrx1-roGFP2 biosensor in C. glutamicum to measure dynamic changes of the MSH redox potential (EMSH) during the growth and under oxidative stress. C. glutamicum maintains a highly reducing intrabacterial EMSH throughout the growth curve with basal EMSH levels of ~− 296 mV. Consistent with its H2O2 resistant phenotype, C. glutamicum responds only weakly to 40 mM H2O2, but is rapidly oxidized by low doses of NaOCl. We further monitored basal EMSH changes and the H2O2 response in various mutants which are compromised in redox-signaling of ROS (OxyR, SigH) and in the antioxidant defense (MSH, Mtr, KatA, Mpx, Tpx). While the probe was constitutively oxidized in the mshC and mtr mutants, a smaller oxidative shift in basal EMSH was observed in the sigH mutant. The catalase KatA was confirmed as major H2O2 detoxification enzyme required for fast biosensor re-equilibration upon return to non-stress conditions. In contrast, the peroxiredoxins Mpx and Tpx had only little impact on EMSH and H2O2 detoxification. Further live imaging experiments using confocal laser scanning microscopy revealed the stable biosensor expression and fluorescence at the single cell level. In conclusion, the stably expressed Mrx1-roGFP2 biosensor was successfully applied to monitor dynamic EMSH changes in C. glutamicum during the growth, under oxidative stress and in different mutants revealing the impact of Mtr and SigH for the basal level EMSH and the role of OxyR and KatA for efficient H2O2 detoxification under oxidative stress., Highlights • Construction of stably integrated Mrx1-roGFP2 in C. glutamicum. • Highly reducing basal EMSH of ~− 296 mV along the growth curve. • Mtr and SigH contribute to basal EMSH in C. glutamicum. • KatA and OxyR required for EMSH regeneration under oxidative stress. • Live imaging revealed stable biosensor expression and fluorescence.

Abundant, low prices and a highly reduced nature make glycerol to be an ideal feedstock for the production of reduced biochemicals and biofuels. Escherichia coli has been paid much attention as the platform of microbial cell factories due to its high growth rate (giving higher metabolite production rate) and the capability of utilizing a wide range of carbon sources. However, one of the drawbacks of using E. coli as a platform is its mixed metabolite formation under anaerobic conditions. In the present study, it was shown that ethanol could be exclusively produced from glycerol by the wild type E. coli, while d-lactic acid could be exclusively produced from glucose by pflA.cra mutant, where the glucose uptake rate could be increased by this mutant as compared to the wild type strain. It was also shown that the growth rate is significantly reduced in pflA.cra mutant for the case of using glycerol as a carbon source due to redox imbalance. The metabolic regulation mechanisms behind the fermentation characteristic were clarified to some extent.