Disulfide Reductase Research Articles

Initiation of ERAD by the bifunctional complex of Mnl1 mannosidase and protein disulfide isomerase.

Misfolded glycoproteins in the endoplasmic reticulum (ER) lumen are translocated into the cytosol and degraded by the proteasome, a conserved process called ER-associated protein degradation (ERAD). In S. cerevisiae , the glycan of these proteins is trimmed by the luminal mannosidase Mnl1 (Htm1) to generate a signal that triggers degradation. Curiously, Mnl1 is permanently associated with protein disulfide isomerase (Pdi1). Here, we have used cryo- electron microscopy, biochemical, and in vivo experiments to clarify how this complex initiates ERAD. The Mnl1-Pdi1 complex first de-mannosylates misfolded, globular proteins that are recognized through a C-terminal domain (CTD) of Mnl1; Pdi1 causes the CTD to ignore completely unfolded polypeptides. The disulfides of these globular proteins are then reduced by the Pdi1 component of the complex, generating unfolded polypeptides that can be translocated across the membrane. Mnl1 blocks the canonical oxidative function of Pdi1, but allows it to function as the elusive disulfide reductase in ERAD.

Thioredoxin-interacting protein (TXNIP) is a substrate of the NEDD4-like E3 ubiquitin-protein ligase WWP1 in cellular redox state regulation of acute myeloid leukemia cells.

The HECT-type E3 ubiquitin WWP1 (also known as NEDD4-like E3 ubiquitin-protein ligase WWP1) acts as an oncogenic factor in acute myeloid leukemia (AML) cells. WWP1 overexpression in AML confers a proliferative advantage to leukemic blasts (abnormal immature white bloodcells) and counteracts apoptotic cell death and differentiation. In aneffort to elucidate the molecular basis of WWP1 oncogenic activities, we identified WWP1 as a previously unknown negative regulator of thioredoxin-interacting protein (TXNIP)-mediated reactive oxygen species (ROS) production in AML cells. TXNIP inhibits the disulfide reductase enzymatic activity of thioredoxin (Trx), impairing its antioxidant function and, ultimately, leading to the disruption of cellular redox homeostasis. In addition, TXNIP restricts cell growth and survival by blocking glucose uptake and metabolism. Here, we found that WWP1 directly interacts with TXNIP, thus promoting its ubiquitin-dependent proteasomal proteolysis. As a result, accumulation of TXNIP in response to WWP1 inactivation inAML blasts reduces Trx activity and increases ROS production, henceinducing cellular oxidative stress. Increased ROS generation in WWP1-depleted cells culminates in DNA strand breaks and subsequent apoptosis. Coherently with TXNIP stabilization following WWP1 inactivation, we also observed an impairment of both glucose up-take and consumption. Hence, a contribution to the increased cell death observed in WWP1-depleted cells also possibly arises from the attenuation of glucose up-take and glycolytic flux resulting from TXNIP accumulation. Future studies are needed to establish whether TXNIP-dependent deregulation of redox homeostasis in WWP1-overexpressing blasts may affect the response of leukemic cells to chemotherapeutic drugs.

Analysis of Punicalin and Punicalagin Interaction with PDIA3 and PDIA1.

PDIA3 is a pleiotropic protein primarily located in the endoplasmic reticulum where it is involved in protein folding, catalyzing the formation, breakage, and rearrangement of disulfide bonds. PDIA3 is implicated in numerous pathologies such as cancer, inflammation, and neurodegeneration. Although punicalagin has been proven to be a highly promising PDIA3 inhibitor and can be used as target protein in glioblastoma, it does not have sufficient selectivity for PDIA3 and is a quite-large molecule. With the aim of finding punicalagin derivatives with a simplified structure, we selected punicalin, which lacks the hexahydroxy-diphenic acid moiety. Previous docking studies suggest that this part of the molecule is not involved in the binding with PDIA3. In this study we compared the ability of punicalin to bind and inhibit PDIA3 and PDIA1. Tryptophan fluorescence quenching and disulfide reductase activity (using both glutathione and insulin as substrates) were evaluated, demonstrating the ability of punicalin to bind and inhibit PDIA3 even to a lesser extent compared to punicalagin. On the other hand, punicalin showed a very low inhibition activity towards PDIA1, demonstrating a higher selectivity for PDIA3. Protein thermal shift assay evidenced that both proteins can be destabilized by punicalin as well as punicalagin, with PDIA3 much more sensitive. Additionally, punicalin showed a higher change in the thermal stability of PDIA3, with a shift up to 8 °C. This result could explain the presence of PDIA3 aggregates, evidenced by immunofluorescence analysis, that accumulate within treated cells and that are more evident in the presence of punicalin. The results here obtained show punicalin is able to bind both proteins but with a higher selectivity for PDIA3, suggesting the possibility of developing new molecules with a simplified structure that are still able to selectively bind and inhibit PDIA3.

Design, development and characterization of a chimeric protein with disulfide reductase and protease domain showing keratinase activity

Keratin is one of the major components of solid waste, and the degradation products have extensive applications in various commercial industries. Due to the complexity of the structure of keratin, especially the disulfide bonds between keratin polypeptides, keratinolytic activity is efficient with a mixture of proteins with proteases, peptidases, and oxidoreductase activity. The present work aimed to create an engineered chimeric protein with a disulfide reductase domain and a protease domain connected with a flexible linker. The structure, stability, and substrate interaction were analyzed using the protein modeling tools and codon-optimized synthetic gene cloned, expressed, and purified using Ni2+-NTA chromatography. The keratinolytic activity of the protein was at its maximum at 70 °C. The suitable pH for the enzyme activity was pH 8. While Ni2+, Mg2+, and Na+ inhibited the keratinolytic activity, Cu2+, Ca2+, and Mn2+ enhanced it significantly. Biochemical characterization of the protease domain indicated significant keratinolytic activity at 70 °C at pH 10.0 but was less efficient than the chimeric protein. Experiments using feathers as the substrate showed a clear degradation pattern in the SEM analysis. The samples collected from the degradation experiments indicated the release of proteins (2-fold) and amino acids (8.4-fold) in a time-dependent manner. Thus, the protease with an added disulfide reductase domain showed excellent keratin degradation activity and has the potential to be utilized in the commercial industries.

Bacteria of Deep-Sea Sediments of the Black Sea Breaking Down Keratin

Down and feather raw materials generated at food processing plants are among the main environmental pollutants. Most enterprises use burial and burning methods to deal with this waste, which negatively affects the environmental situation. The use of enzymatic hydrolysis by decomposer microorganisms is a promising and safe method for processing keratin waste. Earlier, it was shown that bacteria isolated from the bottom sediments of the Black Sea are active producers of elastases, fibrinogenases, and fibrinases. Therefore, the aim of this work was to evaluate the ability of bacteria isolated from the deep-sea sediments of the Black Sea to exhibit other types of proteolytic activity, in particular, to decompose hard-to-reach protein keratin. Меthods. The objects of the study were 20 cultures of bacteria isolated from deep-sea sediments of the Black Sea represented by the genera Bacillus, Metabacillus, Priestia, and Robertmurraya. The cultures were grown under conditions of submerged cultivation at 28 °C, with a nutrient medium stirring rate of 232 rpm for 4 days. For growth, a basic nutrient medium containing 0.5 % defatted chicken feathers as sole sources of carbon and nitrogen was used. The keratinase activity was assessed by UV absorption at 280 nm of hydrolysis products of keratin-containing materials. Protein was determined by the Lowry method, and caseinolytic (total proteolytic) activity was determined by the Anson method. Disulfide reductase activity was measured spectrophotometrically at 412 nm by evaluating the yellow sulfide formed during the reduction of 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB). Results. It was shown that the cultures of Metabacillus idriensis 2 and Robertmurraya siralis 57, out of twenty studied, did not grow on a nutrient medium with chicken feathers as the only source of carbon and nitrogen. The remaining 18 cultures exhibited varying degrees of keratinase activity (from 3 to 32 U/mL). The highest level of activity is characteristic of the culture Priestia megaterium 035 (32 U/mL). A study of the ability to break down casein showed that the level of total proteolytic (caseinolytic) activity of most cultures ranged from 0.015 U/mL to 0.14 U/mL. The highest total proteolytic activity was demonstrated by Bacillus pumilus A (0.3 U/mL) and Priestia megaterium 55 (0.24 U/mL) cultures, which also demonstrated high keratinase activity. The highest level of disulfide reductase activity was observed in Bacillus pumilus A (63.3 µmol/min), Bacillus subtilis 248 (62.0 µmol/min), and Priestia megaterium 035 (61.3 µmol/min), and the lowest in Bacillus licheniformis 249. Thus, from the deep-sea sediments of the Black Sea, we have isolated a number of active producers of keratinases, representatives of the two genera Bacillus and Priestia, which, after studying their physicochemical and catalytic properties, may turn out to be promising for practical application, in particular in the development of new technologies for the utilization of down and feather poultry farm waste.

Genome-wide analysis of Smad and Schnurri transcription factors in C. elegans demonstrates widespread interaction and a function in collagen secretion.

Smads and their transcription factor partners mediate the transcriptional responses of target cells to secreted ligands of the Transforming Growth Factor-β (TGF-β) family, including those of the conserved bone morphogenetic protein (BMP) family, yet only a small number of direct target genes have been well characterized. In C. elegans, the BMP2/4 ortholog DBL-1 regulates multiple biological functions, including body size, via a canonical receptor-Smad signaling cascade. Here, we identify functional binding sites for SMA-3/Smad and its transcriptional partner SMA-9/Schnurri based on ChIP-seq peaks (identified by modEncode) and expression differences of nearby genes identified from RNA-seq analysis of corresponding mutants. We found that SMA-3 and SMA-9 have both overlapping and unique target genes. At a genome-wide scale, SMA-3/Smad acts as a transcriptional activator, whereas SMA-9/Schnurri direct targets include both activated and repressed genes. Mutations in sma-9 partially suppress the small body size phenotype of sma-3, suggesting some level of antagonism between these factors and challenging the prevailing model for Schnurri function. A functional analysis of target genes revealed a novel role in body size for genes involved in one-carbon metabolism and in the endoplasmic reticulum (ER) secretory pathway, including the disulfide reductase dpy-11. Our findings indicate that Smads and SMA-9/Schnurri have previously unappreciated complex genetic and genomic regulatory interactions that in turn regulate the secretion of extracellular components like collagen into the cuticle to mediate body size regulation.

A synergistic catalytic system of keratinases and disulfide reductases for the bioconversion of feather wastes to feed alternative protein

Effective hydrolysis of feather waste by keratinase is an important way to develop high-quality alternative proteins for feed. It has been shown that the reduction of disulfide bonds is the key rate-limiting step in the hydrolysis of feathers by keratinases. However, the reductive activity of keratinase itself is low. In order to increase the reductive activity of the catalytic system, we screened endogenous disulfide reductase from Bacillus subtilis and constructed a synergistic catalytic system, KerZ1/yjbI-S2-P1, consisting of keratinase and disulfide reductase by co-expression optimization. The reductive activity and feather hydrolytic activity of the synergistic system were 5.67-fold and 5-fold higher than the original system. Feathers were hydrolyzed to a higher extent compared with the original system. The total amino acid and soluble protein concentrations in the products increased by 99% and 59%, respectively. This study provides a more environmentally friendly and efficient reduction capability for keratinase to hydrolysis waste feathers, and also demonstrates a new way of alternative protein feedstock mining.

Bacillus velezensis strain NA16 shows high poultry feather-degrading efficiency, protease and amino acid production

Isolated Bacillus velezensis strain NA16, which produces proteases, amino acids and the transcription levels of different keratinolytic enzymes and disulfide reductase genes in whole gene sequencing, was evaluated during feather degradation. The result shows under optimum fermentation conditions, chicken feather fermentation showed total amino acid concentration of 7599 mg/L, degradation efficiency of 99.3% at 72 h, and protease activity of 1058 U/mL and keratinase activity of 288 U/mL at 48 h. Goose feather fermentation showed total amino acid concentration of 4918 mg/L (96 h), and degradation efficiency was 98.9% at 120 h. Chicken feather fermentation broth at 72 h showed high levels of 17 amino acids, particularly phenylalanine (1050 ± 1.90 mg/L), valine (960 ± 1.04 mg/L), and glutamic (950 ± 3.00 mg/L). Scanning electron microscopy and Fourier transform infrared analysis revealed the essential role of peptide bond cleavage in structural changes and degradation of feathers. Protein purification and zymographic analyses revealed a key role in feather degradation of the 39-kDa protein encoded by gene1031, identified as an S8 family serine peptidase. Whole genome sequencing of NA16 revealed 26 metalloproteinase genes and 22 serine protease genes. Among the proteins, S8 family serine peptidase (gene1031, gene1428) and S9 family peptidase (gene3132) were shown by transcription analysis to play major roles in chicken feather degradation. These findings revealed the transcription levels of different families of keratinolytic enzymes in the degradation of feather keratin by microorganisms, and suggested potential applications of NA16 in feather waste management and amino acid production.

Genome-based taxonomic classification of the genus Sulfitobacter along with the proposal of a new genus Parasulfitobacter gen. nov. and exploring the gene clusters associated with sulfur oxidation

BackgroundThe genus Sulfitobacter, a member of the family Roseobacteraceae, is widely distributed in the ocean and is believed to play crucial roles in the global sulfur cycle. However, gene clusters associated with sulfur oxidation in genomes of the type strains of this genus have been poorly studied. Furthermore, taxonomic errors have been identified in this genus, potentially leading to significant confusion in ecological and evolutionary interpretations in subsequent studies of the genus Sulfitobacter. This study aims to investigate the taxonomic status of this genus and explore the metabolism associated with sulfur oxidation.ResultsThis study suggests that Sulfitobacter algicola does not belong to Sulfitobacter and should be reclassified into a novel genus, for which we propose the name Parasulfitobacter gen. nov., with Parasulfitobacter algicola comb. nov. as the type species. Additionally, enzymes involved in the sulfur oxidation process, such as the sulfur oxidization (Sox) system, the disulfide reductase protein family, and the sulfite dehydrogenase (SoeABC), were identified in almost all Sulfitobacter species. This finding implies that the majority of Sulfitobacter species can oxidize reduced sulfur compounds. Differences in the modular organization of sox gene clusters among Sulfitobacter species were identified, along with the presence of five genes with unknown function located in some of the sox gene clusters. Lastly, this study revealed the presence of the demethylation pathway and the cleavage pathway used by many Sulfitobacter species to degrade dimethylsulfoniopropionate (DMSP). These pathways enable these bacteria to utilize DMSP as important source of sulfur and carbon or as a defence strategy.ConclusionsOur findings contribute to interpreting the mechanism by which Sulfitobacter species participate in the global sulfur cycle. The taxonomic rearrangement of S. algicola into the novel genus Parasulfitobacter will prevent confusion in ecological and evolutionary interpretations in future studies of the genus Sulfitobacter.

Hypothiocyanous acid reductase is critical for host colonization and infection by Streptococcus pneumoniae

The major human pathogen Streptococcus pneumoniae encounters the immune-derived oxidant hypothiocyanous acid (HOSCN) at sites of colonization and infection. We recently identified the pneumococcal hypothiocyanous acid reductase (Har), a member of the flavoprotein disulfide reductase enzyme family, and showed that it contributes to the HOSCN tolerance of S. pneumoniae in vitro. Here, we demonstrate in mouse models of pneumococcal infection that Har is critical for colonization and invasion. In a colonization model, bacterial load was attenuated dramatically in the nasopharynx when har was deleted in S. pneumoniae. The Δhar strain was also less virulent compared to wild type in an invasion model as reflected by a significant reduction in bacteria in the lungs and no dissemination to the blood and brain. Kinetic measurements with recombinant Har demonstrated that this enzyme reduced HOSCN with near diffusion limited catalytic efficiency, using either NADH (kcat/KM = 1.2 x 108 M−1s−1) or NADPH (kcat/KM = 2.5 x 107 M−1s−1) as electron donors. We determined the X-ray crystal structure of Har in complex with the FAD cofactor to 1.50 Å resolution, highlighting the active site architecture characteristic for this class of enzymes. Collectively, our results demonstrate that pneumococcal Har is a highly efficient HOSCN reductase, enabling survival against oxidative host immune defenses. In addition, we provide structural insights that may aid the design of Har inhibitors.

Functional diversity of YbbN/CnoX proteins: Insights from a comparative analysis of three thioredoxin-like oxidoreductases from Pseudomonas aeruginosa, Xylella fastidiosa and Escherichia coli

YbbN/CnoX are proteins that display a Thioredoxin (Trx) domain linked to a tetratricopeptide domain. YbbN from Escherichia coli (EcYbbN) displays a co-chaperone (holdase) activity that is induced by HOCl. Here, we compared EcYbbN with YbbN proteins from Xylella fastidiosa (XfYbbN) and from Pseudomonas aeruginosa (PaYbbN). EcYbbN presents a redox active Cys residue at Trx domain (Cys63), 24 residues away from SQHC motif (SQHC[N24]C) that can form mixed disulfides with target proteins. In contrast, XfYbbN and PaYbbN present two Cys residues in the CXXC (CAPC) motif, while only PaYbbN shows the Cys residue equivalent to Cys63 of EcYbbN. Our phylogenetic analysis revealed that most of the YbbN proteins are in the bacteria domain of life and that their members can be divided into four groups according to the conserved Cys residues. EcYbbN (SQHC[N24]C), XfYbbN (CAPC[N24]V) and PaYbbN (CAPC[N24]C) are representatives of three sub-families. In contrast to EcYbbN, both XfYbbN and PaYbbN: (1) reduced an artificial disulfide (DTNB) and (2) supported the peroxidase activity of Peroxiredoxin Q from X. fastidiosa, suggesting that these proteins might function similarly to the canonical Trx enzymes. Indeed, XfYbbN was reduced by XfTrx reductase with a high catalytic efficiency (kcat/Km = 1.27 x 107 M−1 s−1), similar to the canonical XfTrx (XfTsnC). Furthermore, EcYbbN and XfYbbN, but not PaYbbN displayed HOCl-induced holdase activity. Remarkably, EcYbbN gained disulfide reductase activity while lost the HOCl-activated chaperone function, when the SQHC was replaced by CQHC. In contrast, the XfYbbN CAPA mutant lost the disulfide reductase activity, while kept its HOCl-induced chaperone function. In all cases, the induction of the holdase activity was accompanied by YbbN oligomerization. Finally, we showed that deletion of ybbN gene did not render in P. aeruginosa more sensitive stressful treatments. Therefore, YbbN/CnoX proteins display distinct properties, depending on the presence of the three conserved Cys residues.

ERdj5-mediated regulation of proliferation and differentiation in colon organoids under inflammatory stress

Background: Endoplasmic reticulum (ER) stress is resolved by ERdj5, which functions as a disulfide reductase. In a previous study, we confirmed that ERdj5 was not indispensable for the formation of intestinal epithelial cells in mice. However, studies evaluating the association of ERdj5 and growth factors are limited. In the present study, we observed changes in the proliferation of colon organoids cultured from intestinal crypts obtained from wild-type (WT) and ERdj5 knockout (KO) mice.Methods: We cultured colon organoids from WT and ERdj5 KO mice, and we then assessed differentiation- and proliferation-related markers after exposure to various stimuli, such as irradiation and the Toll-like receptor (TLR) 2 ligand Pam3CSK4.Results: Over 15 days of organoid culture, ERdj5 KO colon organoids demonstrated a similar increase in leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) expression as WT colon organoids. However, in response to Pam3CSK4-induced stress, the ERdj5 KO organoids exhibited decreased Lgr5 expression and increased C/EBP homologous protein (CHOP) levels. Moreover, after irradiation-induced stress, ERdj5 downregulated epithelial-related genes, including Lgr5, Villin 1, and Prominin 1.Conclusion: Inflammation through irradiation or TLR2-induced mild or severe ER stress in both groups, whereas ERdj5 deficiency eventually led to the downregulation of Lgr5. In summary, these findings suggest that colon organoids lack the ability to mount an effective defense against various inflammatory stimuli when there is a deficiency in the ERdj5-related ER stress resolution pathway.

The crystal structure of mycothiol disulfide reductase (Mtr) provides mechanistic insight into the specific low-molecular-weight thiol reductase activity of Actinobacteria.

Low-molecular-weight (LMW) thiols are involved in many processes in all organisms, playing a protective role against reactive species, heavy metals, toxins and antibiotics. Actinobacteria, such as Mycobacterium tuberculosis, use the LMW thiol mycothiol (MSH) to buffer the intracellular redox environment. The NADPH-dependent FAD-containing oxidoreductase mycothiol disulfide reductase (Mtr) is known to reduce oxidized mycothiol disulfide (MSSM) to MSH, which is crucial to maintain the cellular redox balance. In this work, the first crystal structures of Mtr are presented, expanding the structural knowledge and understanding of LMW thiol reductases. The structural analyses and docking calculations provide insight into the nature of Mtrs, with regard to thebinding and reduction of the MSSM substrate, in the context of related oxidoreductases. The putative binding site for MSSM suggests a similar binding to that described for the homologous glutathione reductase and its respective substrate glutathione disulfide, but with distinct structural differences shaped to fit the bulkier MSSM substrate, assigning Mtrs as uniquely functioning reductases. As MSH has been acknowledged as an attractive antitubercular target, the structural findings presented in this work may contribute towards future antituberculosis drug development.

Unresolved questions regarding cellular cysteine sources and their possible relationships to ferroptosis.

Cysteine is required for synthesis of glutathione (GSH), coenzyme A, other sulfur-containing metabolites, and most proteins. In most cells, cysteine comes from extracellular disulfide sources including cystine, glutathione-disulfide, and peptides. The thioredoxin reductase-1 (TrxR1)- or glutathione-disulfide reductase (GSR)-driven enzymatic systems can fuel cystine reduction via thioredoxins, glutaredoxins, or other thioredoxin-fold proteins.Free cystine enters cells thorough the cystine-glutamate antiporter, xCT, but systemically, plasma glutathione-disulfide might predominate as a cystine source. Erastin, inhibiting both xCT and voltage-dependent anion channels, induces ferroptotic cell death, so named because this type of cell death is antagonized by iron-chelators. Many cancer cells seem to be predisposed to ferroptosis, which has been proposed as a targetable cancer liability. Ferroptosis is associated with lipid peroxidation and loss of either glutathione peroxidase-4 (GPX4) or ferroptosis suppressor protein-1 (FSP1), which each prevent accumulation of lipid peroxides. It has been suggested that an xCT inhibition-induced cellular cysteine-deficiency lowers GSH levels, starving GPX4 for reducing power and allowing membrane lipid peroxides to accumulate, thereby causing ferroptosis. Aspects of ferroptosis are however not fully understood and need to be further scrutinized, for example that neither disruption of GSH synthesis, loss of GSH, nor disruption of glutathione disulfide reductase (GSR), triggers ferroptosis in animal models. Here we reevaluate the relationships between Erastin, xCT, GPX4, cellular cysteine and GSH, RSL3 or ML162, and ferroptosis. We conclude that, whereas both Cys and ferroptosis are potential liabilities in cancer, their relationship to each other remains insufficiently understood.

Proteolytic Activity of Bacillus Strains Isolated from Soil of Rice Agrocenosis

Microorganisms are the most common sources of commercial enzymes due to their physiological and biochemical properties, facile culture conditions, and ease of cell manipulation. Among microbial enzymes, proteases are ubiquitous in nature and have been found in all living forms encompassing the eukaryotes like plants, animals, fungi, and protists as well as the prokaryotic domains of bacteria and archaea. Proteases are the most important for the industry and constitute approximately 60% of the total industrial enzyme market. Among the bacteria, the genus Bacillus has a very prominent place in terms of the commercial production of proteases. Earlier from the water and bottom sediments of the Black Sea, we have isolated a number of producers of proteolytic enzymes from Bacillus species. The aim of this work was to investigate the ability of representatives of a number of soil bacilli species to synthesize enzymes that hydrolyze such protein substrates as elastin, fibrin, fibrinogen, and keratin. Methods. The objects of the study were 8 cultures (KS 1 — KS 8) isolated from the soil of the rice agrocenosis. Cultures were grown under conditions of deep cultivation at 28 °С, with a mixing speed of for the nutrient medium of 230 rpm for 4 days. Methods of determining proteolytic (caseinolytic, elastolytic, fibrinolytic, fibrinogenolytic, and keratinase) activity in the culture liquid supernatant were used. Disulfide reductase activity was measured spectrophotometrically at 412 nm by evaluating the yellow sulfide formed during the reduction of 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB). Results. The study of the spectrum of proteolytic activities of 8 freshly isolated strains showed that only KS 6 under experimental conditions did not show the ability to hydrolyze any of the studied substrates (casein, elastin, fibrin, fibrinogen, and keratin). Strains KS 1, KS 2, KS 7, and KS 8 showed higher levels of activity compared to other strains studied. The most interesting for further research are: І) strain KS 1, which showed the highest fibrinolytic activity, ІІ) strain KS 2 as the most effective producer with elastase and fibrinogenolytic activity, III) KS 7 and KS 8, which simultaneously showed the highest rates as keratinase (7 U/mL and 9 U/mL) and sulfate reductase (33 μmol/min and 31 μmol/min) activity, respectively. Conclusions. According to the catalytic properties, a number of representatives of Bacillus, isolated from the soil of the rice agrocenosis may be promising for further research as an enzyme producer with proteolytic activity.

Functional validation of Lobularia maritima thioredoxin-h2 protein for its ability to combat bacterial and fungal infections

Thioredoxins (Trxs) belong to a family of multifunctional redox proteins that is critical for maintaining and regulating the cellular redox environment during plant cell growth. Also, they are important for the development of plant's response to biotic and abiotic stress; however, the specific biological functions of h-type thioredoxins (Trxhs) in plants have not been fully elucidated. Herein, we investigated the role of LmTrxh2, a specific member of the Trxh family, in response to various biotic stress simulants, including mechanical wounding, exposure to jasmonic acid (JA), picolinic acid (PA), salicylic acid (SA), ethephon (ETP), and hydrogen peroxide (H2O2). We observed that LmTrxh2 transcripts were significantly upregulated upon exposure to these stress simulants. The characterization of enzymatic activity revealed that the recombinant LmTrxh2 protein functions as a disulfide reductase. While the role of Trx proteins in redox regulation is well known, their involvement in antimicrobial activity is still unexplored. Therefore, we assessed the antimicrobial effect of LmTrxh2 towards various microorganisms and observed a concentration-dependent inhibition of microbial growth. The minimum inhibitory and minimum bactericidal concentrations and the diameters of the inhibition zones were 40–1250 μg/mL, 40–1250 μg/mL, and 12.5–32.5 mm, respectively. In addition, we used previously developed LmTrxh2-transgenic tobacco lines and found that they showed enhanced resistance to fungal infections triggered by Fusarium graminearum and Aspergillus niger. This resistance was associated with an upregulation of known defense-related genes. Overall, our findings suggest that LmTrxh2 is responsive to multiple biotic stress simulants and plays a critical role in the basal resistance of plants to pathogen infections. These results highlight the potential of LmTrxh2 in the development of strategies to protect crops from various stress factors and emphasize its importance in the adaptation of plants to different stress conditions.

Structural and Functional Analyses of the Flavoprotein Disulfide Reductase FN0820 of Fusobacterium nucleatum.

Escherichia coli RclA and Staphylococcus aureus MerA are part of the Group I flavoprotein disulfide reductase (FDR) family and have been implicated in the contribution to bacterial pathogenesis by defending against the host immune response. Fusobacterium nucleatum is a pathogenic, anaerobic Gram-negative bacterial species commonly found in the human oral cavity and gastrointestinal tract. In this study, we discovered that the F. nucleatum protein FN0820, belonging to the Group I FDR family, exhibited a higher activity of a Cu2+-dependent NADH oxidase than E. coli RclA. Moreover, FN0820 decreased the dissolved oxygen level in the solution with higher NADH oxidase activity. We found that L-tryptophan and its analog 5-hydroxytryptophan inhibit the FN0820 activities of NADH oxidase and the concomitant reduction of oxygen. Our results have implications for developing new treatment strategies against pathogens that defend the host immune response with Group I FDRs.

Comprehensive insights into the mechanism of keratin degradation and exploitation of keratinase to enhance the bioaccessibility of soybean protein

Keratin is a recalcitrant protein and can be decomposed in nature. However, the mechanism of keratin degradation is still not well understood. In this study, Bacillus sp. 8A6 can completely degrade the feather in 20 h, which is an efficient keratin degrader reported so far. Comprehensive transcriptome analysis continuously tracks the metabolism of Bacillus sp. 8A6 throughout its growth in feather medium. It reveals for the first time how the strain can acquire nutrients and energy in an oligotrophic feather medium for proliferation in the early stage. Then, the degradation of the outer lipid layer of feather can expose the internal keratin structure for disulfide bonds reduction by sulfite from the newly identified sulfite metabolic pathway, disulfide reductases and iron uptake. The resulting weakened keratin has been further proposedly de-assembled by the S9 protease and hydrolyzed by synergistic effects of the endo, exo and oligo-proteases from S1, S8, M3, M14, M20, M24, M42, M84 and T3 families. Finally, bioaccessible peptides and amino acids are generated and transported for strain growth. The keratinase has been applied for soybean hydrolysis, which generates 2234 peptides and 559.93 mg/L17 amino acids. Therefore, the keratinases, inducing from the poultry waste, have great potential to be further applied for producing bioaccessible peptides and amino acids for feed industry.

ITRAQ-based quantitative proteomic analysis of the antibacterial mechanism of silver nanoparticles against multidrug-resistant Streptococcus suis.

The increase in antibiotic resistance of bacteria has become a major concern in clinical treatment. Silver nanoparticles (AgNPs) have significant antibacterial effects against Streptococcus suis. Therefore, this study aimed to investigate the antibacterial activity and mechanism of action of AgNPs against multidrug-resistant S. suis. The effect of AgNPs on the morphology of multidrug-resistant S. suis was observed using scanning electron microscopy (SEM). Differentially expressed proteins were analyzed by iTRAQ quantitative proteomics, and the production of reactive oxygen species (ROS) was assayed by H2DCF-DA staining. SEM showed that AgNPs disrupted the normal morphology of multidrug-resistant S. suis and the integrity of the biofilm structure. Quantitative proteomic analysis revealed that a large number of cell wall synthesis-related proteins, such as penicillin-binding protein and some cell cycle proteins, such as the cell division protein FtsZ and chromosomal replication initiator protein DnaA, were downregulated after treatment with 25 μg/mL AgNPs. Significant changes were also observed in the expression of the antioxidant enzymes glutathione reductase, alkyl hydroperoxides-like protein, α/β superfamily hydrolases/acyltransferases, and glutathione disulfide reductases. ROS production in S. suis positively correlated with AgNP concentration. The potential antibacterial mechanism of AgNPs may involve disrupting the normal morphology of bacteria by inhibiting the synthesis of cell wall peptidoglycans and inhibiting the growth of bacteria by inhibiting the cell division protein FtsZ and Chromosomal replication initiator protein DnaA. High oxidative stress may be a significant cause of bacterial death. The potential mechanism by which AgNPs inhibit S. suis biofilm formation may involve affecting bacterial adhesion and interfering with the quorum sensing system.

The neutrophil oxidant hypothiocyanous acid causes a thiol-specific stress response and an oxidative shift of the bacillithiol redox potential in Staphylococcus aureus.

During infections, Staphylococcus aureus is exposed to hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN), which are produced by the neutrophil myeloperoxidase as potent antimicrobial killing agents. In this work, we applied RNAseq transcriptomics, Brx-roGFP2 biosensor measurements, and phenotype analyses to investigate the stress responses and defense mechanisms of S. aureus COL toward HOSCN stress. Based on the RNAseq transcriptome profile, HOSCN exerts strong thiol-specific oxidative, electrophile, and metal stress responses as well as protein damage in S. aureus, which is indicated by the strong induction of the HypR, TetR1, PerR, QsrR, MhqR, CstR, CsoR, CzrA, AgrA, HrcA, and CtsR regulons. Phenotype analyses of various mutants in HOSCN-responsive genes revealed that the HOSCN reductase MerA conferred the highest resistance toward HOSCN stress in S. aureus COL, whereas the QsrR and MhqR electrophile stress regulons do not contribute to protection. Brx-roGFP2 biosensor measurements and bacillithiol (BSH)-specific Western blot analyses revealed a strong oxidative shift of the bacillithiol redox potential (EBSH) and increased S-bacillithiolations in S. aureus, indicating that BSH is oxidized to bacillithiol disulfide (BSSB) under HOSCN stress. While the ΔmerA mutant was delayed in recovery of the reduced EBSH, overproduction of MerA in the ΔhypR mutant enabled faster recovery of EBSH due to efficient HOSCN detoxification. Moreover, both MerA and BSH were shown to contribute to HOSCN resistance in growth assays. In summary, HOSCN provokes a thiol-specific oxidative, electrophile, and metal stress response, an oxidative shift in EBSH and increased S-bacillithiolation in S. aureus.IMPORTANCEStaphylococcus aureus colonizes the skin and the airways but can also lead to life-threatening systemic and chronic infections. During colonization and phagocytosis by immune cells, S. aureus encounters the thiol-reactive oxidant HOSCN. The understanding of the adaptation mechanisms of S. aureus toward HOSCN stress is important to identify novel drug targets to combat multi-resistant S. aureus isolates. As a defense mechanism, S. aureus uses the flavin disulfide reductase MerA, which functions as HOSCN reductase and protects against HOSCN stress. Moreover, MerA homologs have conserved functions in HOSCN detoxification in other bacteria, including intestinal and respiratory pathogens. In this work, we studied the comprehensive thiol-reactive mode of action of HOSCN and its effect on the reversible shift of the EBSH to discover new defense mechanisms against the neutrophil oxidant. These findings provide new leads for future drug design to fight the pathogen at the sites of colonization and infections.