Serine Protease Activity Research Articles

S195A is a Catalytically Inactive Mutant of the Protease Domain of the Urokinase-Type Plasminogen Activator (uPA)

Serine Proteases are enzymes that hydrolyze peptide bonds in proteins. The activity of some proteins can be regulated by their cleavage by serine proteases. Coagulation and fibrinolysis are regulated through the subsequent activation of serine proteases, one of which is the Urokinase-type Plasminogen-Activator (uPA). The uPA is a serine protease that plays an important role in fibrinolysis, tissue remodeling, angiogenesis and cancer metastasis. Studying the biophysical behavior of this protein becomes a challenge when the experiments require high concentration of the protein and/or long incubation times because it degrades itself over time. Therefore, designing and preparing a catalytically inactive mutant of uPA that biophysically behaves similarly to wild-type is a key first step for many biophysical experiments. The active site serine 195 is responsible for the nucleophilic attack on the substrate carbonyl group. Our hypothesis was that mutating Ser195 to an alanine would inactivate the protein without altering its dynamic behavior. We mutated Ser195 in the protease domain of the human uPA (huPA) to alanine, expressed it in inclusion bodies in E. coli, refolded it and purified it. We compared the dynamics of the S195A mutant to wildtype huPA protease by Hydrogen-Deuterium Exchange Mass Spectrometry (HDXMS). We compared these results to the Ser195Met mutant and showed that the S195A mutation causes less differences in the loops surrounding the active site. These results show that whereas in thrombin the Ser195Met mutant is the best choice for a catalytically dead mutant, in huPA, the best choice is the Ser195Ala mutant.

Multifaceted Analyses of Epidermal Serine Protease Activity in Patients with Atopic Dermatitis.

The serine proteases kallikrein-related peptidase (KLK) 5 and KLK7 cleave cell adhesion molecules in the epidermis. Aberrant epidermal serine protease activity is thought to play an important role in the pathogenesis of atopic dermatitis (AD). We collected the stratum corneum (SC) from healthy individuals (n = 46) and AD patients (n = 63) by tape stripping and then measuring the trypsin- and chymotrypsin-like serine protease activity. We also analyzed the p.D386N and p.E420K of SPINK5 variants and loss-of-function mutations of FLG in the AD patients. The serine protease activity in the SC was increased not only in AD lesions but also in non-lesions of AD patients. We found, generally, that there was a positive correlation between the serine protease activity in the SC and the total serum immunoglobulin E (IgE) levels, serum thymus and activation-regulated chemokine (TARC) levels, and peripheral blood eosinophil counts. Moreover, the p.D386N or p.E420K in SPINK5 and FLG mutations were not significantly associated with the SC’s serine protease activity. Epidermal serine protease activity was increased even in non-lesions of AD patients. Such activity was found to correlate with a number of biomarkers of AD. Further investigations of serine proteases might provide new treatments and prophylaxis for AD.

Synergy between plasminogen activator inhibitor-1, α-synuclein, and neuroinflammation in Parkinson’s disease

Parkinson’s disease (PD) is a progressive degenerative nervous system disorder and is the second most common neurodegenerative disorder in the elderly population. The disease originates from the loss of dopamine-producing neurons in the substantia nigra in the brain, resulting in unregulated activity of the basal ganglia. Αlpha-synuclein (α-syn) is a protein found to aggregate in the substantia nigra region of patients with PD, forming Lewy Body inclusions; its aggregation may contribute to neuronal cell death in PD. This work hypothesizes about the synergistic relationship between α-syn aggregation and neuroinflammation to up-regulate expression of the serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1). The protease, plasmin, has been shown to cleave extracellular α-syn (including its monomeric, oligomeric, and fibrillary forms), resulting in less aggregation and Lewy Body formation. The zymogen plasminogen is converted to its active serine protease form, plasmin, either by tissue plasminogen activator (tPA) or by urokinase plasminogen activator (uPA) bound to urokinase receptor (uPAR). Both tPA and uPA/uPAR are inhibited by PAI-1. Thus, when PAI-1 levels increase, less plasmin is generated, which would lead to reduced proteolysis of α-syn. Expression of PAI-1 is increased both in inflammatory environments and in the presence of extracellular α-syn aggregates. This scenario suggests a pathological amplification loop: increased extracellular α-syn aggregation activates an inflammatory response from microglia and astrocytes, increasing PAI-1 levels, and decreasing the generation of plasmin. With reduced plasmin, less α-syn can be cleaved, and aggregation continues, sustaining the pathological process. Understanding this putative pathogenic loop could provide insight into the means by which neurodegeneration progresses in PD, and it may offer possible novel therapeutic strategies.

P087 Biomarkers of elastin degradation differentiate between clinically inactive and active disease in ulcerative colitis patients

Abstract Background In ulcerative colitis (UC), the state of chronic inflammation results in increased matrix metalloprotease (MMP) and serine protease activity, which effectively leads to a higher degree of intestinal tissue remodelling, including components of the extracellular matrix (ECM). One of these components is elastin a matrix protein of the interstitial matrix in the lamina propria and submucosa, providing tissue resilience and elasticity. As such, we investigated whether elastin degradation in UC patients was associated with disease activity and severity, potentially enabling patient differentiation based on elastin degradation. Methods Twenty-nine UC patients and 29 healthy donors were included in the study. Disease activity was determined according to the partial Mayo score (pMayo >1) and the Mayo Endoscopic Score (MES). Disease severity and extension was assessed using the Montreal classification. Disease severity was additionally assessed using the Trulove and Witt’s (TW) clinical score. The biomarkers of elastin degradation included: MMP-7 (ELM-7) cathepsin-G (EL-CG) and proteinase-3 (ELP-3), measured in serum by ELISA. One-way ANOVA (Kruskal–Wallis) correcting for the false discovery rate were applied for the statistical analysis. Results TW: ELP-3 levels in moderate-to-severe UC patients were significantly elevated in comparison with HD (p < 0.001). Partial Mayo: EL-CG levels in patients with active UC were significantly elevated in comparison with HD (p < 0.01), and UC patients in remission (p < 0.01). ELP-3 levels were likewise significantly elevated in active UC patients compared with HD (p < 0.001), and UC patients in remission (p < 0.01). Montreal classification: ELM-7 was significantly elevated in active UC compared with HD (p < 0.05), and UC patients in remission (p < 0.05). EL-CG were also significantly elevated in active UC compared with HD (p < 0.05), and UC patients in remission (p < 0.05). ELP-3 was significantly elevated in active UC compared with HD (p < 0.01). According to the MES score, ELP-3 levels in moderate-to-severe UC patients were significantly elevated in comparison to HD (p < 0.01). Conclusion The data presented in this study demonstrate an association between biomarkers of proteolytic elastin degradation and disease activity in UC patients especially the protease-3-derived biomarker, ELP-3, showed significant association with active UC in all the clinical scoring systems as well as the MES score. Utilising these minimally invasive elastin degradation biomarkers could serve as surrogate markers for monitoring of disease activity and potentially aid the differentiation of patients with an active disease from patients in remission or with a lower disease activity for UC.

P101 Biomarkers of elastin degradation are associated with clinical and biochemically active disease in Crohn’s disease

Abstract Background In Crohn’s disease (CD), the extensive and potentially transmural inflammation results in increased activity of both matrix metalloproteases (MMPs) and serine proteases, causing a higher degree of intestinal tissue remodelling. This increased proteolytic activity could potentially cause degradation and loss of function of mechanical and functional matrix proteins, such as elastin. Therefore, we sought to investigate the association between biomarkers of elastin degradation and the disease activity in CD patients. Methods Seventy-two CD patients and 29 healthy donors (HD) were included in the study. Disease activity was determined according to the Crohn’s disease activity index score (CDAI >150) and/or a faecal calprotectin (fCALP >250). Additionally, CD patients were endoscopically assessed according to the simple endoscopic score (SES) for CD. Different protease derived biomarkers of elastin degradation: protease-3 (ELP-3), MMP-7 (ELM-7) and cathepsin-G (EL-CG) was measured in serum by ELISA. One-way ANOVA (Kruskal–Wallis) was applied for the statistical analysis. Results The levels of ELP-3 was significantly elevated in active CD when compared with the HD (p < 0.001), and inactive CD (p < 0.01). Levels of EL-G were significantly elevated when comparing active CD and HD (p < 0.05), with the same result observed for the levels of EL-CG when comparing active CD and the HD (p < 0.05). Endoscopically, ELP-3 was shown significantly elevated in moderate–to-severe CD patients when compared with the HD (p < 0.01). Conclusion In this study, measurements of the elastin degradation markers were capable of differentiating between CD patients with either a clinically active or biochemically active disease, with the biomarker levels being significantly highest in the patients with an active disease. This was also the case when assessing endoscopic disease activity, where the protease-3-derived biomarker levels were highest in patients of moderate-to-severe disease activity. As such, the data provide indications of the beneficial use of these serum biomarkers as additional disease activity assessment tools for CD patients.

In Silico Study of Alkaline Serine Protease and Production Optimization in Bacillus sp. Khoz1 Closed Bacillus safensis Isolated from Honey

Protease is a kind of enzyme with different applications in medicine and industry. The limitation of the enzymes in industry is lack of tolerance for harsh conditions (high temperature, pH, and low water condition). This study intends to evaluate in silico study and screen the alkaline serine protease producing bacteria from honey and optimize the production of low water active alkaline serine protease. Plackett–Burman method was applied to improve effective factors on protease production, including pH, inoculum size, temperature, time, soybean protein, KH2PO4, MgSO4, CaCl2, NaCl, Glycerin and glucose concentration. Further, the Box–Behnken method was used to optimize the significant level of selected factors. Besides, in silico study was utilized to create the enzyme structure and investigate the stability in harsh conditions. The results showed that the highest protease production belongs to Bacillus sp. Khoz1 closed Bacillus safensis, Also temperature, glucose and soybean protein concentration were three significant factors for protease production and the optimized level for them were 35 °C, 0.5 g/l, and 38.32 g/l, respectively. The microorganism was able to produce protease until 98.36 U/ml and it was 98.68% similar to B. safensis. The stability of microbial alkaline serine protease was also determined in high pH and temperature conditions. The best stable condition for isolated protease was pH 9 and temperature at 50 °C. The in silico data showed that this protease has similar 3D structure to Bacillus subtilis Subtilisin E and highly charge amino acids on its surface were that caused this enzyme still activate and stable in low water condition.

Assessing the stability of historical and desiccated snake venoms from a medically important Ecuadorian collection

Bothrops asper and Bothrops atrox are important venomous snakes from Ecuador responsible for the most of ophidic accidents, which in the past were treated with a national polyvant antivenom. For years, the venom pools were collected and stored at room temperature in a laboratory. Taking into account the controversial ability of desiccated samples to retain their biological effects and enzymatic activities, we investigated the biochemical and toxicological properties of venoms after years of storage. The proteomic profiles of historical venoms analyzed by high-performance liquid chromatography and electrophoresis are very similar. The fresh batches of venom were more lethal than those stored for years, just as the initial and current LD50 values of these samples changed. Significant differences were showed in the myotoxic and hemorrhagic activity of some venom pools, while no significant statistical differences were found for the edema activity. The enzymatic assays revealed a variation in proteolytic activity on azocasein and phospholipase A2 activity, and low differences were reported for thrombin-like serine protease activity. The maintenance of the proteomic profile and certain toxicological activities convert this venom library in a valuable source for research purposes. Nonetheless, the significative reduction of toxicological activities, such as hemorrhagic activity not feasible using these samples for the antivenom production.

Activity of serine proteases from Fasciola hepatica eggs in relation to pH and temperature

This study was conducted to analyse the serine protease of Fasciola hepatica eggs by specific substrates and inhibitors, and investigation of the effects of pH and temperature on proteases’ activity and stability. Adult worms were isolated from infected livers. After homogenisation, their protein levels were measured with the Bradford method. Total proteolytic activity of the Fasciola hepatica extract was evaluated with azocasein substrate at pH values from 2 to 12. N-benzoyl–arginine–p-nitroanilide (BApNA) trypsin and N-succinyl-alanine-alanine-prolin-phenylalanine-p-nitroanilide (SAAPFpNA) chymotrypsin substrates were used to measure specific protease activities. The effect of protease inhibitors phenylmethane sulfonyl fluoride (PMSF), pepsin, and ethylenediaminetetraacetic acid (EDTA) on these enzymes was evaluated. Estimation of optimum temperature and pH was performed in the temperature range of 10–90 °C and pH values from 2–12. The optimum pH activities for trypsin and chymotrypsin were at alkaline pH and for total proteolytic activity at acidic pH. The results using protease inhibitors showed that the eggs had serine protease activity. The optimum temperature activity of trypsin and chymotrypsin was 50 °C. These proteases were stable up to 40 °C. Due to the importance of pH and temperature in life cycle of Fasciola hepatica, these findings can be used for induction of some modifications in pH and preventing the activity of the enzyme for decrement of the efficacy of embryonic development and egg hatching of this zoonotic parasite.

Overhydration Measured by Bioimpedance Spectroscopy and Urinary Serine Protease Activity Are Risk Factors for Progression of Chronic Kidney Disease

Background: Overhydration (OH) is common in chronic kidney disease (CKD) and might be related to the excretion of urinary serine proteases. Progression of CKD is associated with proteinuria; however, the interrelations of urinary serine proteases, OH, and progression of CKD remain unclear. Methods: In n = 179 patients with stable nondialysis-dependent CKD of all stages, OH was measured using bioimpedance spectroscopy (Body Composition Monitor; Fresenius), and urinary serine protease activity was determined using the peptide substrate S-2302. After a median follow-up of 5.9 (IQR: 3.9–6.5) years, progression to end-stage renal disease (ESRD) was analyzed retrospectively. Results: OH correlated with baseline MDRD-eGFR, urinary albumin creatinine ratio (ACR), and urinary aprotinin-sensitive serine protease activity. Progression to ESRD occurred in n = 33 patients (19%) and correlated with OH and urinary serine protease activity as well as MDRD-eGFR and ACR. Patients were divided into 2 groups determined by cutoff values from receiver operating characteristics for MDRD-eGFR (32 mL/min/1.73 m²), ACR (43 mg/g creatinine), urinary serine protease activity (0.9 RU/g creatinine), and OH (1 L/1.73 m²). Across these cutoff values, Kaplan-Meier curves for renal survival showed significant separations of the groups. In Cox regression adjusted for MDRD-eGFR, ACR, P-NT-pro-BNP, systolic blood pressure, and diabetes mellitus, patients with OH >1 L/1.73 m² had a 3.32 (95% CI: 1.26–8.76)-fold higher risk for progression to ESRD. Conclusions: Our results corroborate that OH detected by bioimpedance spectroscopy in CKD patients is an independent risk factor for progression to ESRD in addition to GFR and albuminuria. Urinary serine protease activity is associated with OH and progression of CKD and provides a possible underlying mechanism.

Structural insights into the role of the N-terminus in the activation and function of extracellular serine protease from Staphylococcus epidermidis.

Extracellular serine protease (Esp) from Staphylococcus epidermidis is a glutamyl endopeptidase that inhibits the growth and formation of S. aureus biofilms. Previously, crystal structures of the matured and active Esp have been determined. Interestingly, many of the staphylococcal glutamyl endopeptidase zymogens, including V8 from Staphylococcus aureus and Esp from S. epidermidis, contain unusually long pro-peptide segments; however, their function is not known. With the aim of elucidating the function of these pro-peptide segments, crystal structures of the Esp zymogen (Pro-Esp) and its variants were determined. It was observed that the N-terminus of the Pro-Esp crystal structure is flexible and is not associated with the main body of the enzyme, unlike in the known active Esp structure. In addition, the loops that border the putative substrate-binding pocket of Pro-Esp are flexible and disordered; the structural components that are responsible for enzyme specificity and efficiency in serine proteases are disordered in Pro-Esp. However, the N-terminal locked Pro-Esp variants exhibit a rigid substrate-binding pocket similar to the active Esp structure and regain activity. These structural studies highlight the role of the N-terminus in stabilizing the structural components responsible for the activity and specificity of staphylococcal glutamyl endopeptidases.

ПРОТЕОЛІТИЧНА АКТИВНІСТЬ ПЛАЗМИ КРОВІ ТА ПЕЧІНКИ ЩУРІВ ЗА ХРОНІЧНОЇ АЛКОГОЛЬНОЇ ІНТОКСИКАЦІЇ

Today, the problem of alcohol abuse is quite relevant around the world. According to the World Health Organization, alcohol abuse is one of the three causes of premature death and is one of the main etiological factors that that cause the development of chronic diseases of the liver and other organs. The severity of clinical manifestations and the prognosis of further dynamics of ongoing alcohol intoxication can be investigated by studying the composition of proteins in tissues, as it is known that this pathology has a pronounced protein dystrophy and active proteolysis. Although much is known today about the effects of alcohol on proteolytic systems, many questions still need to be explored, as proteomic studies can be used to find biomarkers and therapeutic targets for ethanol abuse. The aim of the study was: to investigate the proteolytic activity of blood plasma and liver of rats in chronic alcohol intoxication at 11 and 21 days of the experiment. The model of chronic alcohol intoxication was modeled on male rats – weight 180-200g by intragastric administration of 30% ethyl alcohol solution for 10 days on an empty stomach, at the rate of 2 ml per 100 g of animal weight. Total proteolytic activity, activity of metalloproteinases and serine proteinases were determined by the method of determining caseinolytic activity. The protein concentration was determined by the Bradford method. Statistical processing of the study results was performed by conventional methods of variation statistics. It was found that on the 11th day from the beginning of ethanol administration to rats, there was an increase in total proteolytic activity and activity of metetalloproteinases in blood plasma and liver.

Nafamostat mesylate attenuates the pathophysiologic sequelae of neurovascular ischemia.

Nafamostat mesylate, an apparent soi-disant panacea of sorts, is widely used to anticoagulate patients undergoing hemodialysis or cardiopulmonary bypass, mitigate the inflammatory response in patients diagnosed with acute pancreatitis, and reverse the coagulopathy of patients experiencing the commonly preterminal disseminated intravascular coagulation in the Far East. The serine protease inhibitor nafamostat mesylate exhibits significant neuroprotective effects in the setting of neurovascular ischemia. Nafamostat mesylate generates neuroprotective effects by attenuating the enzymatic activity of serine proteases, neuroinflammatory signaling cascades, and the endoplasmic reticulum stress responses, downregulating excitotoxic transient receptor membrane channel subfamily 7 cationic currents, modulating the activity of intracellular signal transduction pathways, and supporting neuronal survival (brain-derived neurotrophic factor/TrkB/ERK1/2/CREB, nuclear factor kappa B. The effects collectively reduce neuronal necrosis and apoptosis and prevent ischemia mediated disruption of blood-brain barrier microarchitecture. Investigational clinical applications of these compounds may mitigate ischemic reperfusion injury in patients undergoing cardiac, hepatic, renal, or intestinal transplant, preventing allograft rejection, and treating solid organ malignancies. Neuroprotective effects mediated by nafamostat mesylate support the wise conduct of randomized prospective controlled trials in Western countries to evaluate the clinical utility of this compound.

Ферментные препараты бактериальных протеаз для получения белковых гидролизатов без горечи

Casein hydrolysates were obtained using enzyme preparations (EPs) of bacterial proteases, Alcalase, Protamex, Neutrase, and Protosubtilin. The dependence of the hydrolysate bitterness on the decrease of the EP proteolytic activity in the presence of PMSF, an inhibitor of serine proteases, was observed. It was found that the use of Neutrase provided non-bitter hydrolysates, while the proteolytic activity of Neutrase decreased by 44% in the presence of PMSF. A domestic EP of Protosubtilin lost 59% activity in the presence of PMSF and formed bitter hydrolysates. Casein hydrolysates obtained using Alcalase were characterized by the most severe bitterness, probably due to the fact that almost all proteolytic activity in this preparation is represented by a serine protease. The Bacillus subtilis 359 strain with a ratio of neutral and serine protease activities of 52 to 48% was selected from the VNIIPBT Collection of Bacterial Producers of Proteases. The concentrated preparation was obtained from the culture liquid of B. subtilis 359 strain by spray-drying. The preparation obtained hydrolyzed casein similarly to Neutrase and ensured the absence of bitterness in hydrolysates. neutral protease, serine protease, bitterness of hydrolysates, Bacillus subtilis This work was financially supported by a Grant of the Basic Research Program of the Russian Academy of Sciences for 2019-2021 (Project no. 0529-2019-0066).

CGI-58: Versatile Regulator of Intracellular Lipid Droplet Homeostasis.

Comparative gene identification-58 (CGI-58), also known as α/β-hydrolase domain-containing 5 (ABHD5), is a member of a large family of proteins containing an α/β-hydrolase-fold. CGI-58 is well-known as the co-activator of adipose triglyceride lipase (ATGL), which is a key enzyme initiating cytosolic lipid droplet lipolysis. Mutations in either the human CGI-58 or ATGL gene cause an autosomal recessive neutral lipid storage disease, characterized by the excessive accumulation of triglyceride (TAG)-rich lipid droplets in the cytoplasm of almost all cell types. CGI-58, however, has ATGL-independent functions. Distinct phenotypes associated with CGI-58 deficiency commonly include ichthyosis (scaly dry skin), nonalcoholic steatohepatitis, and hepatic fibrosis. Through regulated interactions with multiple protein families, CGI-58 controls many metabolic and signaling pathways, such as lipid and glucose metabolism, energy balance, insulin signaling, inflammatory responses, and thermogenesis. Recent studies have shown that CGI-58 regulates the pathogenesis of common metabolic diseases in a tissue-specific manner. Future studies are needed to molecularly define ATGL-independent functions of CGI-58, including the newly identified serine protease activity of CGI-58. Elucidation of these versatile functions of CGI-58 may uncover fundamental cellular processes governing lipid and energy homeostasis, which may help develop novel approaches that counter against obesity and its associated metabolic sequelae.

Identification and characterization of a serine protease from Bacillus licheniformis W10: A potential antifungal agent

Bacillus licheniformis W10 is a strain of biocontrol bacteria that was obtained from plant rhizosphere screening. In this study, we purified, identified, and carried out bioinformatics analysis of the W10 antifungal protein from Bacillus licheniformis. Mass spectrometry analysis was carried out by passing the antifungal protein through a high-resolution time-of-flight mass spectrometer. Mascot searches of the tandem mass spectrometry data identified this antifungal protein as a serine protease, and the 1347 bp gene encoding this protein was cloned. Bioinformatics analysis of this protein indicated that it contains 448 amino acid residues, has a molecular weight of 48,794.16 Da and an isoelectric point of 6.04, and is a hydrophilic protein. In the secondary and tertiary structure of this protein, the proportion of α-helices and β-folds is similar, and the protein possesses a Peptidase_S8 conserved domain. Using BApNA as a substrate, it was found that the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) can inhibit the W10 antifungal protein. PMSF concurrently reduced the inhibitory effects of the antifungal protein on Botrytis cinerea, showing that the W10 antifungal protein possesses serine protease activity. The W10 antifungal protein has good thermal stability. The study implies potential of this enzyme for biocontrol of fungal plant pathogens.

Cardiac-Specific Overexpression of Catalytically Inactive Corin Reduces Edema, Contractile Dysfunction, and Death in Mice with Dilated Cardiomyopathy.

Humans with dilated cardiomyopathy (DCM) and heart failure (HF) develop low levels of corin, a multi-domain, cardiac-selective serine protease involved in natriuretic peptide cleavage and sodium and water regulation. However, experimental restoration of corin levels markedly attenuates HF progression. To determine whether the beneficial effects of corin in HF require catalytic activity, we engineered cardiac overexpression of an enzymatically inactive corin transgene (corin-Tg(i)). On a wild-type (WT) background, corin-Tg(i) had no evident phenotypic effects. However, in a well-established genetic model of DCM, corin-Tg(i)/DCM mice had increased survival (p < 0.01 to 0.001) vs. littermate corin-WT/DCM controls. Pleural effusion (p < 0.01), lung edema (p < 0.05), systemic extracellular free water (p < 0.01), and heart weight were decreased (p < 0.01) in corin-Tg(i)/DCM vs. corin-WT/DCM mice. Cardiac ejection fraction and fractional shortening improved (p < 0.01), while ventricular dilation decreased (p < 0.0001) in corin-Tg(i)/DCM mice. Plasma atrial natriuretic peptide, cyclic guanosine monophosphate, and neprilysin were significantly decreased. Cardiac phosphorylated glycogen synthase kinase-3β (pSer9-GSK3β) levels were increased in corin(i)-Tg/DCM mice (p < 0.01). In summary, catalytically inactive corin-Tg(i) decreased fluid retention, improved contractile function, decreased HF biomarkers, and diminished cardiac GSK3β activity. Thus, the protective effects of cardiac corin on HF progression and survival in experimental DCM do not require the serine protease activity of the molecule.

Production and characterization of a novel alkaline protease from a newly isolated Neurospora crassa through solid-state fermentation

Microbial proteases are widely used to prepare protein hydrolysates with health-promoting biopeptides. Here, a newly isolated strain of Neurospora crassa (named as CGMCC3088) was used to produce proteases through solid-state fermentation of okara as the substrate. The optimal fermentation conditions are: okara, 10 g; water, 21 mL; initial pH, 5.0; incubation temperature, 30 °C; inoculation amount, 2 mL; fermentation time, 72 h, with a corresponding protease activity of 1959.82 U/g. The protease was further purified by ammonium sulphate precipitation, followed by ion-exchange chromatography on DEAE-Sepharose and Sephadex G-75. The molecular weight of the protease was 30 kDa, and further mass spectrometry analysis clearly indicated that it was a novel protease. The protease had the optimal activity at 55 °C and pH 9. The enzyme activity was partially inhibited by SDS and metal ions, whereas little affected by organic solvents. The protease was completely inactivated by phenylmethylsulfonyl fluoride, indicating its dominant serine protease activity. The enzyme preferably hydrolyzed casein, and kinetic analysis showed that its Km and Vmax were 2.18 mg/mL and 36.36 μg/mL/min, respectively. Therefore, Neurospora crassa CGMCC3088 has the potential to produce a novel organic solvent-stable alkaline protease, which may be applied to the preparation of bioactive ingredients.

ITRAQ-Based Quantitative Proteomic Analysis of Digestive Juice across the First 48 Hours of the Fifth Instar in Silkworm Larvae.

The silkworm is an oligophagous insect for which mulberry leaves are the sole diet. The nutrients needed for vital activities of the egg, pupal, and adult stages, and the proteins formed in the cocoon, are all derived from the larval stages. The silkworm feeds and grows quickly during the larval stages. In particular, the amount of leaf ingested and digested quickly increases from the ecdysis to the gluttonous stage in the fifth instar period. In this study, we used the iTRAQ proteomic technique to identify and analyze silkworm larval digestive juice proteins during this period. A total of 227 proteins were successfully identified. These were primarily serine protease activity, esterase activity, binding, and serine protease inhibitors, which were mainly involved in the digestion and overcoming the detrimental effects of mulberry leaves. Moreover, 30 genes of the identified proteins were expressed specifically in the midgut. Temporal proteomic analysis of digestive juice revealed developmental dynamic features related to molecular mechanisms of the principal functions of digesting, resisting pathogens, and overruling the inhibitory effects of mulberry leaves protease inhibitors (PIs) with a dynamic strategy, although overruling the inhibitory effects has not yet been confirmed by previous study. These findings will help address the potential functions of digestive juice in silkworm larvae.

Nomenclature of factor XI and the contact system

Nomenclature of factor XI and the contact system

Cofactor and Cofactor Mimetics

Many natural enzymes need the assistance of protein cofactors to catalyze chemical reactions at a physiologically relevant speed and several of the enzymes that make up for the coagulation cascade are no exception in this regard. Notably, activated factors VII, IX and X display relatively poor enzymatic activity towards their respective macromolecular substrates. The reason for their low proteolytic activity originates from a number of structural restrictions. For instance, not all enzymes are capable to efficiently fold their new amino-terminus into the active site pocket, leaving the catalytic triad immature. Furthermore, serine protease activation is often associated with a reduced plasticity of the protease domain, which improves their proteolytic activity. Nevertheless, some enzymes still require additional stabilization to reduce flexibility of their protease domain. Protein cofactors are designed to optimize the proteolytic activity of such serine proteases, and can improve the catalytic efficiency of these enzymes by one-thousand to one-million fold. The allosteric changes induced by these protein cofactors are specific to each cofactor/enzyme pair. When focusing on the cofactor role of Factor VIIIa (FVIIIa; which stimulates the catalytic activity of factor IXa; FIXa), several aspects are of importance. First, FVIIIa has high affinity for phosphatidylserine-containing phospholipid-membranes, favoring formation of the FVIIIa/FIXa complex at the membrane surface. Being assembled at the membrane surface limits their movements to two dimensions, and enforces the affinity between both proteins. Second, the interactions between FVIIIa and FIXa involve an extended protein surface, which includes interactions between the FVIIIa light chain and FIXa light chain as well as between the FVIIIa A2 domain and the FIXa protease domain. Due to this extended interactive surface, the complex mimics a staked tree, in which FVIIIa orients the FIXa active site at the appropriate distance from the membrane surface. Moreover, binding of the FVIIIa A2 domain to FIXa surface loops reduces flexibility of the protease domain, and it is likely that allosteric changes induced by the A2-domain optimize the conformation of the active site region. Finally, FVIIIa provides also a binding site for the substrate FX. This not only allows FVIIa to function as a molecular bridge between enzyme and substrate, but also helps to align the FX activation peptide with the FIXa active site. This multistep process by which FVIII acts as a cofactor for FIXa may help us to understand how other non-FVIII molecules can be used to stimulate FIXa activity. Several molecular entities have been reported that are enhancing FIXa activity, including short synthetic peptides, monoclonal antibodies and, perhaps best known at this moment, bispecific antibodies that bind both FIXa and FX. Given the complex molecular structure that FVIIIa has and needs to stimulate FIXa activity, it is of interest to reflect on how this translates to the non-FVIII molecules in terms of regulation and potential cofactor activity. Differences in regulation and activity are of particular relevance for laboratory monitoring of these molecules and in the therapeutic setting. Knowing these limitations will help us to optimize the therapeutic application of non-FVIII molecules. Disclosures Lenting: Spark Therapeutics: Honoraria; Catalyst Biosciences: Honoraria; Sobi: Honoraria; Shire/Takeda: Honoraria; NovoNordisk: Honoraria; Biotest: Honoraria; LFB: Honoraria; Roche: Honoraria; laelaps therapeutics: Equity Ownership.