Characterization of a robust serine protease from Bacillus subtilis K‐1

Process apt microbial proteases due to their wide range industrial applications have become the focus of intense scientific research during recent years. Considering the hostile process milieu, the proteases intended for application must be robust enough to withstand the extremes of temperature and pH, and presence of organic solvents and other potential enzyme inhibitors. Current study presents the characterization of a robust protease from a previously isolated bacterium Bacillus subtilis K‐1 (BSK‐1). Purification of BSK‐1 protease (5.21‐fold) was achieved to homogeneity by salt (ammonium sulfate) precipitation, and ion‐exchange (diethyl‐aminoethyl‐sephadex) and size exclusion chromatography (Sephadex G‐100). Molecular weight of BSK‐1 protease was determined by SDS‐PAGE analysis (42 kDa). Though the optimum temperature and pH for BSK‐1 protease activity was 50 °C and 10, respectively, but, the protease exhibited remarkable activity and stability over elevated temperatures (60–80 °C) and a broad pH range (pH 7–11). Protease showed resistance towards several organic solvents/other potential enzyme inhibitors. Drastic activity loss in presence of phenylmethylsulfonyl fluoride indicated that the enzyme is a serine protease. Kinetic parameters (K_m and V_max) for BSK‐1 protease were found to be 0.14 mg ml^?1 and 1176 mg min^?1, respectively. Putative amino acid sequence of BSK‐1 protease (derived from nucleotide sequence of protease gene) suggested that the enzyme belonged to peptidases S8/S53 super family with multidomain of S8. BSK‐1 protease being stable under harsh conditions may serve a model system for understanding the molecular basis of stability, and may help designing novel proteases that are suitable for industrial applications.     

Structural Activation of Pro-inflammatory Human Cytokine IL-23 by Cognate IL-23 Receptor Enables Recruitment of the Shared Receptor IL-12Rβ1

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Murine neutrophils treated with alphaB‐crystallin reduce IL‐12p40 production by dendritic cells

Neutrophils are essential in the fight against invading pathogens. They utilize antimicrobial effector mechanisms, such as phagocytosis, release of proteases and other antimicrobial products, robust oxidative bursts and neutrophil extracellular traps to combat infections. Neutrophils also modulate immune responses through the production of eicosanoids, cytokines and chemokines, as well as via direct communication with other immune cells. This system of high‐intensity offense against pathogens is exquisitely balanced through regulation to limit damage to host tissue. Unfortunately, the control of neutrophils is not failproof. In cases of sterile injury, autoimmunity and even during an infection, neutrophils can cause tissue destruction and become detrimental to the host. For that reason, there is a need to find means to regulate the aberrant activation of these cells. We found that alphaB‐crystallin (αBC), a heat‐shock protein known to have anti‐inflammatory abilities, affects certain properties of mouse neutrophils that subsequently influence the pro‐inflammatory state of antigen‐presenting cells (APCs). More specifically, αBC mediated small but significant increases in the levels of IL‐10 and matrix metalloproteinase 8, and altered hydrogen peroxide secretion by stimulated neutrophils. Further, the heat‐shock protein influenced the communication between neutrophils and dendritic cells by decreasing the production of pro‐inflammatory cytokines, specifically IL‐12p40, by the APCs. αBC could thus contribute to dampening neutrophil inflammatory responses by impacting the effect of neutrophils on other immune cells.     

Microbial-induced meprin β cleavage in MUC2 mucin and a functional CFTR channel are required to release anchored small intestinal mucus

The mucus that covers and protects the epithelium of the intestine is built around its major structural component, the gel-forming MUC2 mucin. The gel-forming mucins have traditionally been assumed to be secreted as nonattached. The colon has a two-layered mucus system where the inner mucus is attached to the epithelium, whereas the small intestine normally has a nonattached mucus. However, the mucus of the small intestine of meprin β-deficient mice was now found to be attached. Meprin β is an endogenous zinc-dependent metalloprotease now shown to cleave the N-terminal region of the MUC2 mucin at two specific sites. When recombinant meprin β was added to the attached mucus of meprin β-deficient mice, the mucus was detached from the epithelium. Similar to meprin β-deficient mice, germ-free mice have attached mucus as they did not shed the membrane-anchored meprin β into the luminal mucus. The ileal mucus of cystic fibrosis (CF) mice with a nonfunctional cystic fibrosis transmembrane conductance regulator (CFTR) channel was recently shown to be attached to the epithelium. Addition of recombinant meprin β to CF mucus did not release the mucus, but further addition of bicarbonate rendered the CF mucus normal, suggesting that MUC2 unfolding exposed the meprin β cleavage sites. Mucus is thus secreted attached to the goblet cells and requires an enzyme, meprin β in the small intestine, to be detached and released into the intestinal lumen. This process regulates mucus properties, can be triggered by bacterial contact, and is nonfunctional in CF due to poor mucin unfolding.The gastrointestinal (GI) tract is protected from self-digestion and microbiota by mucus (1). This mucus is differently organized throughout the GI tract: the stomach and colon have a two-layered mucus system with an inner mucus layer attached to the epithelium and formed by stratified mucin sheets (2). This layer is 50–200 µm thick and impenetrable for bacteria, is constantly renewed by secreted mucins from the goblet cells, and further toward the lumen proteolytically converted into a nonattached and less dense outer mucus layer. This outer layer is the habitat and nutritional source for the commensal bacteria (2). In contrast, the small intestine has only one single mucus type that is not attached to the epithelium (3).The main structural component of the intestinal mucus is the heavily glycosylated polymeric MUC2 mucin which is densely packed inside the goblet cell and after secretion and a 1,000-fold expansion forms flat, net-like structures stacked into stratified mucin sheets in the colon (4). The same MUC2 mucin is processed differently in the small intestine where it appeared less organized but still filled the space between villi (3). This mucus was easily aspirated and penetrable to beads the size of bacteria (3). Bacteria could penetrate, but still the space between the villi was kept free of bacteria in the small intestine due to effective intestinal peristalsis, fast mucus renewal, and a high concentration of antibacterial peptides and proteins (3, 5). In fact, the structure of the small intestinal mucus as a nonattached and less organized layer mimics the situation for the outer colon mucus layer that has been shown to be generated from the inner mucus layer by proteolytic processing of the MUC2 mucin (2).Meprins are zinc-dependent metalloendopeptidases and belong to the astacin family (6). They comprise two homologous enzymes, meprin α and meprin β, where meprin α is the secreted and meprin β the membrane-tethered variant. They can form heterodimers (meprin α and β), homodimers (meprin β), and large oligomers (meprin α), forming one of the largest secreted protease complexes known. Although both enzymes share a common domain structure, they exhibit distinct features in substrate recognition and cleavage specificities. The enzymes can hydrolyze a broad variety of substrates, such as growth factors, peptide hormones, or compounds of the extracellular matrix like procollagen III, fibronectin, and tenascin-C (7–9). The meprin β enzyme is highly expressed in the enterocytes of the small intestine and is thereby localized close to the mucin networks (10).Cystic fibrosis (CF) is a severe disease that affects most of the mucus-producing organs of the body (11). The disease is caused by a nonfunctional cystic fibrosis transmembrane conductance regulator (CFTR) channel that normally mediates passive transport of chloride and bicarbonate ions (12, 13). Although a majority of the clinical CF symptoms can be attributed to stagnant mucus, the more precise link between the lack of CFTR function and mucus properties has been difficult to understand. We recently showed that the small intestinal mucus of CF mice, in contrast to the WT, was attached to the epithelium and impossible to aspirate (14). Although CF mice have only minor lung problems, their intestinal phenotype is similar to the human disease characterized by meconium ileus and distal intestinal obstruction syndrome (DIOS). When the CF mucus was secreted into a solution of ∼100 mM bicarbonate, the mucus was released from its attachment. Mucins are densely packed in the goblet cell granulae due to Ca²⁺ ions and low pH, and the role of bicarbonate is to remove the Ca²⁺ ions and increase the pH to allow for the >1,000-fold mucin expansion (4). When already formed mucus was treated with bicarbonate, the mucus was normalized and possible to aspirate, although its increased mucin density remained largely unaltered (14). This suggested that mucin attachment and expansion might be different phenomena and made us analyze this further.We have now found that meprin β is able to cleave the MUC2 mucin N terminus and that this is involved in the detachment of the mucus of the small intestine in a process controlled by bacteria and a functional CFTR channel. We thus describe a fundamental constitutive mechanism which involves an endogenous protease acting on the mucus network to alter its attached properties.     

Effects of electrical stimulation in C2C12 muscle constructs

Electrical stimulation affects the deposition of extracellular matrices and cellular differentiation. Type I collagen is one of the most abundant extracellular matrix proteins; however, not much is known about the effects of electrical stimulation on collagen type I deposition in C2C12 cells. Thus, we studied the effects of electrical voltage and stimulation frequency in 3D cultured C2C12 muscle cells in terms of metabolic activity, type I collagen deposition and cell morphology. Electrically excitable C2C12 muscle cells were seeded in collagen scaffolds and stimulated with rectangular signals of voltage (2, 5, 7 V) and frequency (1, 2 Hz), using parallel carbon electrodes spaced 1 cm apart. Metabolic activity was quantified by the glucose:lactate concentration ratio in the medium. Apoptotic activity was assessed by TUNEL staining and changes in collagen deposition were identified by immunohistology. The ultrastructure of the tissue was examined by TEM. Glucose and lactate analysis indicated that all groups had similar metabolic activity. TUNEL stain showed no significant difference in apoptotic damage induced by electrical stimulation compared to the control. Samples stimulated at 2 Hz exhibited reduced collagen deposition compared to the control and 1 Hz stimulated samples. Muscle-protein marker desmin was highly expressed in constructs stimulated with 1 Hz/5 V sample. TEM revealed that the stimulated samples developed highly organized sarcomeres, which coincided with improved contractile properties in the 1 Hz/5 V- and 2 Hz/5 V-stimulated groups. Our data implicate that a specific electrical frequency may modulate type I collagen accumulation and a specific voltage may affect the differentiation of muscle sarcomeres in excitable cells.     

Vitamin B12 and hepatic enzyme serum levels correlate with interleukin-6 in alcohol-dependent individuals without liver disease

Alcohol abuse is a major cause of liver cirrhosis as well as chronic liver disease. The aim of the present study was to investigate the possible correlation, between liver dysfunction biological markers and vitamin B12, with interleukin-6, in the serum of alcohol-dependent individuals without liver disease (AWLD). In a sample of 43 alcohol abusing/dependent subjects (33 males and 10 females) treated on an inpatient basis according to a standard detoxification protocol, the serum activities of the hepatic enzymes (ASAT, ALAT, gamma-GT), as well as the concentration of B12 and IL-6, were determined on admission. A strong positive correlation has been observed between IL-6 and B12, ASAT, ALAT, and gamma-GT at the beginning of the detoxification period. The results confirmed that in alcohol-dependent individuals, the median serum concentration of IL-6, before the beginning of the treatment, had a significant positive correlation with the liver dysfunction biological markers and B12. In conclusion, IL-6 might be used as an additional diagnostic marker for the degree of liver dysfunction in alcohol dependent individuals.     

Stankiewicz-Isidor syndrome: expanding the clinical and molecular phenotype

PurposeHaploinsufficiency of PSMD12 has been reported in individuals with neurodevelopmental phenotypes, including developmental delay/intellectual disability (DD/ID), facial dysmorphism, and congenital malformations, defined as Stankiewicz-Isidor syndrome (STISS). Investigations showed that pathogenic variants in PSMD12 perturb intracellular protein homeostasis. Our objective was to further explore the clinical and molecular phenotypic spectrum of STISS.MethodsWe report 24 additional unrelated patients with STISS with various truncating single nucleotide variants or copy-number variant deletions involving PSMD12. We explore disease etiology by assessing patient cells and CRISPR/Cas9-engineered cell clones for various cellular pathways and inflammatory status.ResultsThe expressivity of most clinical features in STISS is highly variable. In addition to previously reported DD/ID, speech delay, cardiac and renal anomalies, we also confirmed preaxial hand abnormalities as a feature of this syndrome. Of note, 2 patients also showed chilblains resembling signs observed in interferonopathy. Remarkably, our data show that STISS patient cells exhibit a profound remodeling of the mTORC1 and mitophagy pathways with an induction of type I interferon-stimulated genes.ConclusionWe refine the phenotype of STISS and show that it can be clinically recognizable and biochemically diagnosed by a type I interferon gene signature.