Serine Protease Activity Research Articles

Regeneration of the midgut during larval stages of the red flour beetle Tribolium castaneum (Herbst, 1797) upon fasting

The beetle Tribolium castaneum feeds on amylaceous grains, being considered a pest of stored products which causes severe losses to the agricultural productive chain worldwide. The success of this insect as a pest relies, besides other abilities, on the endurance capacity of its developmental stages to survive inhospitable environmental conditions, such as the transition between food reservoirs. Being unable to fly, T. castaneum larvae must remain viable upon starvation and the preservation of the intestinal tract is pivotal in this process. To investigate the remodeling of the midgut of T. castanum larvae, a model of fasting, followed by refeeding was established. The histological profiles of midguts from normally fed insects, from larvae starved for up to 120 h, as well as of refed individuals, were compared. Furthermore, the activity of cysteine and serine proteases and α-amylase was also analyzed under these conditions. During the fasting, the midgut epithelium progressively lost most of its spatial organization, which was recovered 72 h after refeeding. During the fasting, the activity of α-amylase decreased drastically, while serine and cysteine proteinases suffered more discrete reductions. Upon refeeding, α-amylase and serine protease activities increased gradually and reached prior levels in 72 h, while cysteine proteases showed only a small rise, not recovering to previous levels. Overall, from a histological and enzymatic perspective, the intestinal epithelium of larvae of T. castaneum undergoes an extensive remodeling to become almost completely reorganized after 72 h of refeeding. It is reasonable that the observed tissue reconstruction and digestion recovery relies heavily on the nidi of intestinal stem cells. The resilience of T. castaneum larvae upon fasting may explain, at least in part, the remarkable success of this species as a storage pest and should be considered in the design of larvae-based pest control strategies.

Mefloquine causes selective mast cell apoptosis in cutaneous mastocytosis lesions by a secretory granule-mediated pathway.

Mastocytosis is a KIT-related myeloproliferative disease characterised by abnormal expansion of neoplastic mast cells (MC) in the skin or virtually any other organ system. The cutaneous form of adult-onset mastocytosis is almost invariably combined with indolent systemic involvement for which curative therapy is yet not available. Here we evaluated a concept of depleting cutaneous MCs in mastocytosis lesions ex vivo by targeting their secretory granules. Skin biopsies from mastocytosis patients were incubated with or without mefloquine, an antimalarial drug known to penetrate into acidic organelles such as MC secretory granules. Mefloquine reduced the number of dermal MCs without affecting keratinocyte proliferation or epidermal gross morphology at drug concentrations up to 40 μM. Flow cytometric analysis of purified dermal MCs showed that mefloquine-induced cell death was mainly due to apoptosis and accompanied by caspase-3 activation. However, caspase inhibition provided only partial protection against mefloquine-induced cell death, indicating predominantly caspase-independent apoptosis. Further assessments revealed that mefloquine caused an elevation of granule pH and a corresponding decrease in cytosolic pH, suggesting drug-induced granule permeabilisation. Extensive damage to the MC secretory granules was confirmed by transmission electron microscopy analysis. Further, blockade of granule acidification or serine protease activity prior to mefloquine treatment protected MCs from apoptosis, indicating that granule acidity and granule-localised serine proteases play major roles in the execution of mefloquine-induced cell death. Altogether, these findings reveal that mefloquine induces selective apoptosis of MCs by targeting their secretory granules and suggest that the drug may potentially extend its range of medical applications.

Human TMPRSS2 non-catalytic ectodomain and SARS-CoV-2 S2' subunit interaction mediated SARS-CoV-2 endocytosis: a model proposal with virtual screening for potential drug molecules to inhibit this interaction

This study proposes a novel model for integration of SARS-CoV-2 into host cell via endocytosis as a possible alternative to the prevailing direct fusion model. It is known that the SARS-CoV-2 spike protein undergoes proteolytic cleavage at S1-S2 cleavage site and the cleaved S2 domain is primed by the activated serine protease domain (SPD) of humanTMPRSS2 to become S2'. The activated SPD of TMPRSS2 is formed after it is cleaved by autocatalysis from the membrane bound non-catalytic ectodomain (hNECD) comprising of LDLRA CLASS-I repeat and a SRCR domain. It is known that the SRCR domains as well as LDLRA repeat harboring proteins mediate endocytosis of viruses and certain ligands. Based on this, we put forward a hypothesis that the exposed hNECD binds to the S2' as both are at an interaction proximity soon after S2 is processed by the SPD and this interaction may lead to the endocytosis of virus. Based on this hypothesis we have modelled the hNECD structure, followed by docking studies with the known 3D structure of S2'. The interaction interface of hNECD with S2’ was further used for virtual screening of FDA-approved drug molecules and Indian medicinal plant-based compounds. We also mapped the known mutations of concern and mutations of interest on interaction interface of S2’ and found that none of the known mutations map onto the interaction interface. This indicates that targeting the interaction between the hNECD of TMPRSS2 and S2’ may serve as an attractive therapeutic target. Communicated by Ramaswamy H. Sarma

Effects of deheading and rinsing pretreatment on the quality of white leg shrimp (Litopenaeus vannamei) surimi based on endogenous proteases

In this study, the effects of different pretreatments on the quality of white leg shrimp surimi were investigated based on shrimp endogenous proteases. The results showed that removing the head and rinsing significantly (P < 0.05) improved the gel strength, texture, whiteness, water distribution, and microstructure of the shrimp surimi gels. Headless shrimp surimi (HSS) had higher salt-soluble protein and lower water-soluble protein than whole shrimp surimi (WSS). The shrimp heads had high cathepsin B, L, D, and serine protease activities. Electrophoretic analysis revealed significant degradation of the myofibrillar proteins in the WSS during cold storage and thermal gelation. Moreover, the myosin heavy chains almost disappeared after thermal gelation, and new bands appeared at about 270 kDa and 100 kDa. However, rinsing reduced the endogenous proteases, water-soluble proteins, and concentrated salt-soluble proteins in the shrimp surimi; thus, the quality of the shrimp surimi gel improved after rinsing. These results suggest that the quality of the surimi gel was damaged by the endogenous proteases, and that removal of the shrimp heads and rinsing significantly (P < 0.05) improved the quality of the shrimp surimi gel. The gel properties of WSS were similar after the second rinse to those of unrinsed HSS. The choice of headless shrimp or whole shrimp as the raw material for production needs to be comprehensively considered according to the planned cost and the quality required for the shrimp surimi product. The recommended number of rinses is 1–2.

Chronic Kidney Disease Is Associated With Increased Cardiac Corin Expression But Decreased Proatrial Natriuretic Peptide Conversion Activity.

Background Chronic kidney disease (CKD) is associated with an increased risk of cardiovascular disease. Corin converts proatrial natriuretic peptide into its active form after being activated by PCSK6 (proprotein convertase subtilisin/kexin type 6) protease. It remains unknown whether the PCSK6/corin/atrial natriuretic peptide pathway plays a role in CKD-induced cardiomyopathy. Methods and Results Serum corin, left ventricular mass index, and corin-left ventricular mass index correlation were compared between outpatients with versus without CKD. Cardiac corin expression and activity as well as serum corin were compared between 5/6 nephrectomy CKD animal models and sham controls. The effects of indoxyl sulfate, a uremic toxin, on cardiomyocytes were examined in vitro in H9c2 cells. A total of 543 patients were enrolled in this study. Serum corin levels were elevated in patients with CKD compared with levels in patients without CKD. Serum corin levels correlated negatively with left ventricular mass index in participants without CKD, but not in patients with CKD. Compared with sham controls, CKD mice had higher serum corin levels and increased cardiac expression of corin but reduced cardiac corin conversion activity. Indoxyl sulfate stimulated corin expression while suppressing serine protease activity in H9c2 cardiomyoblasts. Lower PCSK6 expression in CKD mouse hearts and indoxyl sulfate-treated H9c2 cardiomyoblasts may explain, at least partly, the observed CKD-associated reduction in corin activity. Conclusions In CKD, cardiac and serum levels of corin are increased, yet corin activity is suppressed. The latter may be attributable to reduced PCSK6 expression. These findings suggest that corin dysfunction may play a significant role in the pathogenesis of CKD-associated cardiomyopathy.

Activity-based protein profiling reveals active serine proteases that drive malignancy of human ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) is an understudied poor prognosis subtype of ovarian cancer lacking in effective targeted therapies. Efforts to define molecular drivers of OCCC malignancy may lead to new therapeutic targets and approaches. Among potential targets are secreted proteases, enzymes which in many cancers serve as key drivers of malignant progression. Here, we found that inhibitors of trypsin-like serine proteases suppressed malignant phenotypes of OCCC cell lines. To identify the proteases responsible for malignancy in OCCC, we employed activity-based protein profiling to directly analyze enzyme activity. We developed an activity-based probe featuring an arginine diphenylphosphonate warhead to detect active serine proteases of trypsin-like specificity and a biotin handle to facilitate affinity purification of labeled proteases. Using this probe, we identified active trypsin-like serine proteases within the complex proteomes secreted by OCCC cell lines, including two proteases in common, tissue plasminogen activator and urokinase-type plasminogen activator. Further interrogation of these proteases showed that both were involved in cancer cell invasion and proliferation of OCCC cells and were also detected in in vivo models of OCCC. We conclude the detection of tissue plasminogen activator and urokinase-type plasminogen activator as catalytically active proteases and significant drivers of the malignant phenotype may point to these enzymes as targets for new therapeutic strategies in OCCC. Our activity-based probe and profiling methodology will also serve as a valuable tool for detection of active trypsin-like serine proteases in models of other cancers and other diseases.

Dibenzyl (benzo [d] thiazol-2-yl (hydroxy) methyl) phosphonate (DBTMP) showing anti-S. aureus and anti-biofilm properties by elevating activities of serine protease (SspA) and cysteine protease staphopain B (SspB).

Staphylococcus aureus biofilms are the pathogenic factor in the spread of infection and are more pronounced in multidrug-resistant strains of S. aureus, where high expression of proteases is observed. Among various proteases, Serine protease (SspA) and cysteine protease Staphopain B (SspB) are known to play a key role in the biofilm formation and removal of biofilms. In earlier studies, we have reported Dibenzyl (benzo [d] thiazol-2-yl (hydroxy) methyl) phosphonate (DBTMP) exhibits anti-S. aureus and anti-biofilm properties by elevating the expression of the protease. In this study, the effect of DBTMP on the activities of SspA, and SspB of S. aureus was evaluated. The SspA and SspB genes of S. aureus ATCC12600 were sequenced (Genbank accession numbers: MZ456982 and MW574006). In S. aureus active SspA is formed by proteolytic cleavage of immature SspA, to get this mature SspA (mSspA), we have PCR amplified the mSspA sequence from the SspA gene. The mSspA and SspB genes were cloned, expressed, and characterized. The pure recombinant proteins rSspB and rmSspA exhibited a single band in SDS-PAGE with a molecular weight of 40 and 30KD, respectively. The activities of rmSspA and rSspB are 32.33 and 35.45Units/mL correspondingly. DBTMP elevated the activities of rmSspA and rSspB by docking with respective enzymes. This compound disrupted the biofilms formed by the multidrug-resistant strains of S. aureus and further prevented biofilm formation. These findings explain that DBTMP possesses anti-S. aureus and anti-biofilm features.

Effects of ammonia-N stress on molecular mechanisms associated with immune behavior changes in the haemocytes of Litopenaeus vannamei

High concentration of ammonia-N will inhibit the immune defense of aquatic animals. The neuroendocrine-immune (NEI) regulatory mechanism under ammonia-N stress has been systematically studied, but the final response mechanism of ammonia-N affecting the immune system remains unclear. To investigate the relationship among immune factors of Litopenaeus vannamei (L. vannamei) exposed to 0, 2, 10 and 20 mg/L ammonia-N, the determination of complement components, C-type lectins, proPO system, signal transduction pathway and phagocytosis as well as exocytosis were performed. The results showed that the expressions of complement components including C1q, MBL, ficolin and alpha-2 macroglobulin (A2M) and the complement receptor integrin were decreased significantly in ammonia-N treatment groups at 6,12 and 24 h. C-type lectins and signal transduction factors changed significantly. The decrease of phagocytosis-related genes and phagocytic activity were similar to the changes of complement components, C-type lectins and the signal pathway. The mRNA abundance of exocytosis-related genes was significantly down-regulated under ammonia-N exposure. Correspondingly, significantly changes occurred in the expressions of PPAE and PPO3, immune factors-related genes (Pen3, crustin, stylicins, ALFs and LYC) and inflammatory factors (HSP90, TNFα, IL-16) in haemocytes. Eventually, the serine proteinase activity, PO activity, antibacterial activity and bacteriolytic activity in plasma were decreased significantly. In addition, we speculated that under ammonia-N stress, phagocytosis and exocytosis were affected by complement components, and C-type lectins through intracellular signal transduction pathway. Complement components may involve in the regulation of proPO-activating system to response to ammonia-N stress. This study helped to further understanding the relationship among immune factors of crustacean in response to environmental stress, which implied that when it comes to the decrease of immunity affected by environmental stress, we should not only focus on the mechanism of upstream neuroendocrine response, but also pay attention to the role of immune factors.

Rodent models to study sodium retention in experimental nephrotic syndrome.

Sodium retention and edema are hallmarks of nephrotic syndrome (NS). Different experimental rodent models have been established for simulating NS, however, not all of them feature sodium retention which requires proteinuria to exceed a certain threshold. In rats, puromycin aminonucleoside nephrosis (PAN) is a classic NS model introduced in 1955 that was adopted as doxorubicin-induced nephropathy (DIN) in 129S1/SvImJ mice. In recent years, mice with inducible podocin deletion (Nphs2Δipod ) or podocyte apoptosis (POD-ATTAC) have been developed. In these models, sodium retention is thought to be caused by activation of the epithelial sodium channel (ENaC) in the distal nephron through aberrantly filtered serine proteases or proteasuria. Strikingly, rodent NS models follow an identical chronological time course after the development of proteinuria featuring sodium retention within days and spontaneous reversal thereafter. In DIN and Nphs2Δipod mice, inhibition of ENaC by amiloride or urinary serine protease activity by aprotinin prevents sodium retention, opening up new and promising therapeutic approaches that could be translated into the treatment of nephrotic patients. However, the essential serine protease(s) responsible for ENaC activation is (are) still unknown. With the use of nephrotic rodent models, there is the possibility that this (these) will be identified in the future. This review summarizes the various rodent models used to study experimental nephrotic syndrome and the insights gained from these models with regard to the pathophysiology of sodium retention.

Serine and Cysteine Peptidases: So Similar, Yet Different. How the Active-Site Electrostatics Facilitates Different Reaction Mechanisms.

The catalytic mechanisms of serine and cysteine peptidases are similar: the proton of the nucleophile (serine or cysteine) is transferred to the catalytic histidine, and the nucleophile attacks the substrate for cleavage. However, they differ in an important aspect: cysteine peptidases form a stable ion-pair intermediate in a stepwise mechanism, while serine peptidases follow a concerted mechanism. While it is known that a positive electrostatic potential at the active site of cysteine peptidases stabilizes the cysteine anion in the ion-pair state, the physical basis of the concerted mechanism of serine peptidases is poorly understood. In this work, we use continuum electrostatic analysis and quantum mechanical/molecular mechanical (QM/MM) simulations to demonstrate that a destabilization of an anionic serine by a negative electrostatic potential in combination with a compact active site geometry facilitates a concerted mechanism in serine peptidases. Moreover, we show that an anionic serine would destabilize the protein significantly compared to an anionic cysteine in cysteine peptidases, which underlines the necessity of a concerted mechanism for serine peptidases. On the basis of our calculations on an inactive serine mutant of a natural cysteine peptidase, we show that the energy barrier for the catalytic mechanism can be substantially decreased by introducing a negative electrostatic potential and by reducing the relevant distances indicating that these parameters are essential for the activity of serine peptidases. Our work demonstrates that the concerted mechanism of serine peptidases represents an evolutionary innovative way to perform catalysis without the energetically expensive need to stabilize the anionic serine. In contrast in cysteine peptidases, the anionic cysteine is energetically easily accessible and it is a very efficient nucleophile, making these peptidases mechanistically simple. However, a cysteine is highly oxygen sensitive, which is problematic in an aerobic environment. On the basis of the analysis in this work, we suggest that serine peptidases represent an oxygen-insensitive alternative to cysteine peptidases.

Structural Basis for Nanomolar‐Affinity Inhibition of Neutrophil Serine Protease Activity by the S. aureus EAP Domain Protein, Eap1

Extracellular adherence domain proteins (EAP) are a novel class of innate immune evasion molecules secreted by the human pathogen, Staphylococcus aureus. EAP domains are potent and selective inhibitors of neutrophil elastase (NE) and cathepsin‐G (CG), which are the two most abundant neutrophil serine proteases (NSPs). Previous work from our group has shown that the prototypical EAP domain protein, EapH1, relies on structural plasticity of a single inhibitory site to block activity of NE and CG. However, whether other EAP domain proteins follow similar structure/function relationships remains unknown. To address this limitation, we studied herein the inhibitory properties of Eap1 and determined its structure both free and bound to either NE and CG. Eap1 displayed both dose‐dependent inhibition of NE and CG with Ki values of 0.24 nM +/‐ 0.1 and 8.5 nM +/‐ 1.2, respectively. The structure of EAP1 determined at 1.45 A limiting resolution adopted a compact, b‐grasp fold typical of ~100 residue EAP domain proteins. Whereas the structure of Eap1 bound to CG and determined at 1.95 A revealed an overall inhibitory pose similar to that seen for EapH1, the structure of Eap1 bound to NE and determined at 2.20 A surprisingly showed an inhibitory pose involving a distal region of the EAP domain. Although EAP domains share high levels of sequence and structural homology to one another, our work demonstrates that these high‐affinity inhibitors are capable for forming structurally divergent complexes with two target proteases that themselves share high levels of sequence and structural homology one another.

The Arf-GAP Proteins AoGcs1 and AoGts1 Regulate Mycelial Development, Endocytosis, and Pathogenicity in Arthrobotrys oligospora.

Small GTPases from the ADP-ribosylation factor (Arf) family and their activating proteins (Arf-GAPs) regulate mycelial development, endocytosis, and virulence in fungi. Here, we identified two orthologous Arf-GAP proteins, AoGcs1 and AoGts1, in a typical nematode-trapping fungus Arthrobotrys oligospora. The transcription of Aogcs1 and Aogts1 was highly expressed in the sporulation stage. The deletion of Aogcs1 and Aogts1 caused defects in DNA damage, endocytosis, scavenging of reactive oxygen species, lipid droplet storage, mitochondrial activity, autophagy, serine protease activity, and the response to endoplasmic reticulum stress. The combined effects resulted in slow growth, decreased sporulation capacity, increased susceptibility to chemical stressors and heat shock, and decreased pathogenicity of the mutants compared with the wild-type (WT) strain. Although deletion of Aogcs1 and Aogts1 produced similar phenotfypic traits, their roles varied in conidiation and proteolytic activity. The ΔAogts1 mutant showed a remarkable reduction in conidial yield compared with the WT strain but not in proteolytic activity; in contrast, the ΔAogcs1 mutant showed an increase in proteolytic activity but not in sporulation. In addition, the growth of ΔAogcs1 and ΔAogts1 mutants was promoted by rapamycin, and the ΔAogts1 mutant was sensitive to H-89. Collectively, the ΔAogts1 mutant showed a more remarkable difference compared with the WT strain than the ΔAogcs1 mutant. Our study further illustrates the importance of Arf-GAPs in the growth, development, and pathogenicity of nematode-trapping fungi.

Anti-Protease Activity Deficient Secretory Leukocyte Protease Inhibitor (SLPI) Exerts Cardioprotective Effect against Myocardial Ischaemia/Reperfusion.

Inhibition of proteases shows therapeutic potential. Our previous studies demonstrated the cardioprotection by the Secretory Leukocyte Protease Inhibitor (SLPI) against myocardial ischaemia/reperfusion (I/R) injury. However, it is unclear whether the cardioprotective effect of SLPI seen in our previous works is due to the inhibition of protease enzymes. Several studies demonstrate that the anti-protease independent activity of SLPI could provide therapeutic benefits. Here, we show for the first time that recombinant protein of anti-protease deficient mutant SLPI (L72K, M73G, L74G) (mt-SLPI) could significantly reduce cell death and intracellular reactive oxygen species (ROS) production against an in vitro simulated I/R injury. Furthermore, post-ischaemic treatment of mt-SLPI is found to significantly reduce infarct size and cardiac biomarkers lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) activity, improve cardiac functions, attenuate I/R induced-p38 MAPK phosphorylation, and reduce apoptotic regulatory protein levels, including Bax, cleaved-Caspase-3 and total Capase-8, in rats subjected to an in vivo I/R injury. Additionally, the beneficial effect of mt-SLPI was not significantly different from the wildtype (wt-SLPI). In summary, SLPI could provide cardioprotection without anti-protease activity, which could be more clinically beneficial in terms of providing cardioprotection without interfering with basal serine protease activity.

Simulations suggest double sodium binding induces unexpected conformational changes in thrombin.

Thrombin is a Na[Formula: see text]-activated serine protease existing in two forms targeted to procoagulant and anticoagulant activities, respectively. There is one Na[Formula: see text]-binding site that has been the focus of the study of the thrombin. However, molecular dynamics (MD) simulations suggest that there might be actually two Na[Formula: see text]-binding sites in thrombin and that Na[Formula: see text] ions can even bind to two sites simultaneously. In this study, we performed 12 independent 2-µs all-atom MD simulations for the wild-type (WT) thrombin and we studied the effects of the different Na[Formula: see text] binding modes on thrombin. From the root-mean-square fluctuations (RMSF) for the [Formula: see text]-carbons, we see that the atomic fluctuations mainly change in the 60s, 170s, and 220s loops, and the connection (residue 167 to 170). The correlation matrices for different binding modes suggest regions that may play an important role in thrombin's allosteric response and provide us a possible allosteric pathway for the sodium binding. Amorim-Hennig (AH) clustering tells us how the structure of the regions of interest changes on sodium binding. Principal component analysis (PCA) shows us how the different regions of thrombin change conformation together with sodium binding. Solvent-accessible surface area (SASA) exposes the conformational change in exosite I and catalytic triad. Finally, we argue that the double binding mode might be an inactive mode and that the kinetic scheme for the Na[Formula: see text] binding to thrombin might be a multiple-step mechanism rather than a 2-step mechanism.

Safety, Tolerability, and Pharmacokinetic Evaluation of Single and Multiple Doses of the Dipeptidyl Peptidase 1 Inhibitor Brensocatib in Healthy Japanese and White Adults.

Brensocatib, an investigational first‐in‐class, small‐molecule, orally bioavailable, selective, and reversible dipeptidyl peptidase 1 inhibitor that blocks activation of neutrophil serine proteases, is currently under clinical development for the treatment of bronchiectasis and other chronic inflammatory diseases. In a 2‐part phase 1 study, the safety, tolerability, and pharmacokinetics of brensocatib were evaluated in healthy Japanese and White adults. In part A, participants received single and multiple once‐daily doses of brensocatib (10, 25, or 40 mg) or placebo after an overnight fast. In part B, participants received a single oral dose of brensocatib 40 mg on days 1 and 8, with or without food in a crossover fashion. Following a single dose and at steady state, brensocatib exposure was dose dependent, with low to moderate interindividual variability; systemic exposure between Japanese and White participants was similar. Elimination half‐life of brensocatib ranged from 22 to 28 hours, resulting in ≈2‐fold accumulation in maximum plasma concentration and area under the plasma concentration–time curve at steady state. In both ethnic groups, the presence of food slightly delayed brensocatib absorption with time to maximum plasma concentration increased by 0.7 to 1.7 hours, but it had no significant effect on brensocatib exposure (maximum plasma concentration and area under the plasma concentration–time curve). Brensocatib was well tolerated in Japanese and White participants. The most frequently reported treatment‐emergent adverse events were headache and skin exfoliation. No clinically significant vital signs, laboratory abnormalities, or evidence of renal toxicity were observed. The results from this study demonstrate that brensocatib can be administered with or without food and that dose adjustment is unnecessary for Japanese patients when receiving brensocatib treatment.

Novel Detection Method for Evaluating the Activity of an Alkaline Serine Protease from Bacillus clausii.

Until now, the detection methods for serine proteases have been quite time-consuming or cannot indicate the "real" protease activity. Here, a rapid and simple method for determining the "real" activity of serine proteases toward AAPX (a kind of mixed polypeptide substrates, with X representing 20 standard amino acids) was developed. This AAPX method has high reliability, sensitivity, and repeatability and can be used for detecting the serine protease activity spectrophotometrically. Additionally, the site-directed saturation mutagenesis library of alkaline serine protease PRO (BcPRO) from Bacillus clausii was screened with this AAPX method. Three beneficial mutants S99R, S99H, and S99W were identified, and S99W displayed the highest activity. In comparison to wild-type BcPRO, S99W exhibited enhanced catalytic performance toward eight AAPX monomers, and the molecular dynamics simulation revealed the mechanism responsible for its improved activity toward AAPM. Consequently, this work provides an efficient method for detecting, characterizing, mining, and high-throughput screening of serine proteases.

Chicken cathelicidin-2 promotes IL-1β secretion via the NLRP3 inflammasome pathway and serine proteases activity in LPS-primed murine neutrophils

Cathelicidins have antimicrobial and immunomodulatory activities. Previous studies have shown that chicken cathelicidin-2 (CATH-2) exerts strong anti-inflammatory activity through LPS neutralization. However, it is still unclear whether other intracellular signaling pathways are involved in CATH-2 immunomodulation. Therefore, the CATH-2-meadiated immune response was investigated in LPS-primed neutrophils. Firstly, inflammatory cytokines release was determined in LPS-primed neutrophils. The results showed that CATH-2 significantly promoted secretion of IL-1β and IL-1α while IL-6 and TNF-α were not affected. IL-1β is the key indicator of inflammasome activation. Next, NLRP3 inflammasome signaling pathway was explored using neutrophils of Nlrp3−/−, Asc−/− and Casp1−/− mice and the results showed that the CATH-2-enhanced IL-1β release was completely abrogated, indicating it is NLRP3-dependent. Moreover, CATH-2 significantly induced activation of caspase-1 and gasdermin D (GSDMD) but did not affect LPS-induced mRNA expression of IL-1β and NLRP3, demonstrating that CATH-2 serves as the second signal activating the NLRP3 inflammasome. Furthermore, CATH-2-mediated IL-1β secretion and caspase-1 activation is dependent on potassium efflux but independent of P2X7R. In addition, other signaling pathways including JNK, ERK and SyK were investigated using different inhibitors and the results showed that these signaling pathway inhibitors partially attenuated CATH-2-enhanced IL-1β secretion, especially the JNK inhibitor. Finally, the role of serine protease in CATH-2-mediated NLRP3 inflammasome activation was investigated in neutrophils and the results showed that serine protease activity is involved in CATH-2-enhanced IL-1β secretion and caspase-1 activation. In conclusion, after LPS priming in neutrophils, CATH-2 can be an agonist of the NLRP3 inflammasome. Our study increases the understanding on immunomodulatory effects of chicken cathelicidins and provides new insight on chicken cathelicidins-mediated immune response.

Exploring the activity and inhibition of urokinase-type plasminogen activator (UPA)

Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that activates plasminogen into the active serine protease, plasmin. Studying the catalytic activity of uPA gives insight to plasmin’s involvement in anticoagulation, cell adhesion, and degradation of the extracellular matrix, among others. The uPA’s catalytic activity is influenced by the orientation of the catalytic triad and the substrate binding affinity at the specificity pocket. This enzyme consists of an Amino-Terminal Fragment (ATF), a protease domain, and a disordered linker which connects the ATF and the protease domain.

Blocking serine protease activity prevents semenogelin degradation leading to hyperviscous semen in humans.

Prostate-specific antigen (PSA) is a prostate-specific serine protease enzyme that hydrolyzes gel-forming proteins (semenogelins) and changes the semen from gel-like to watery viscosity, a process called semen liquefaction. Highly viscous semen and abnormal liquefaction reduce sperm motility and contribute to infertility. Previously, we showed that nonspecific serine protease inhibitor (AEBSF) prevented proteolytic degradation of semenogelin in mice. However, it is unclear whether similar effect could be recapitulated in fresh human ejaculates. Therefore, in this study we evaluated the effect of AEBSF on the degradation of semenogelin (SEMG1) and its subsequent impact on semen liquefaction and sperm motility in fresh semen ejaculates collected from healthy men. We found that AEBSF showed a dual contraceptive action where it effectively 1) prevented degradation of SEMG1 resulting in viscous semen and 2) decreased sperm motility in human semen samples. However, the impact of AEBSF on sperm motility and viability could be due to its inhibitory activity toward other serine proteases or simply due to its toxicity. Therefore, to determine whether inhibition of PSA activity alone could disrupt SEMG1 degradation and contribute to hyperviscous semen, a neutralizing PSA antibody was used. We found that PSA antibody effectively prevented SEMG1 degradation with a subtle impact on sperm motility. These findings suggest that the target inhibition of PSA activity can prevent proteolytic degradation of SEMG1 and block liquefaction process, resulting in hyperviscous semen. As it is currently unknown if blocking semen liquefaction alone could prevent pregnancy, it needs further extensive studies before drawing any translational conclusions.

Activation of multiple proteolysis systems contributes to acute cadmium cytotoxicity.

Cadmium exhibits both toxic and carcinogenic effects, and its cytotoxicity is linked to various cellular pathways, such as oxidative stress, ubiquitin-proteasome, and p53-mediated response pathways. The molecular mechanism(s) underlying cadmium cytotoxicity appears to be complex, but remains largely unclear. Here, we examined the effects of cadmium on the protein catabolism using two surrogate markers, DNA topoisomerases I and II alpha and its contribution to cytotoxicity. We have found that cadmium exposure induced time- and concentration-dependent decreases in the protein level of surrogate markers and therefore suggest that cadmium may be involved in proteolysis system activation. A pharmacological study further revealed the novel role(s) of these proteolytic activities and reactive oxygen species (ROS) in the cadmium-induced acute toxicity: (i) Proteasome inhibition only partially relieved the cadmium-induced proteolysis of topoisomerases; (ii) Moreover, we report for the first time that the activation of metalloproteases, serine proteases, and cysteine proteases contributes to the acute cadmium cytotoxicity; (iii) Consistent with the notion that both ROS generation and proteolysis system activation contribute to the cadmium-induced proteolysis and cytotoxicity, the scavenger N-acetylcysteine and aforementioned protease inhibition not only reduced the cadmium-induced topoisomerase degradation but also alleviated the cadmium-induced cell killing. Taken together, acute cadmium exposure may activate multiple proteolytic systems and ROS formation, subsequently leading to intracellular damage and cytotoxicity. Thus, our results provide a novel insight into potential action mechanism(s) by which cadmium exerts its cytotoxic effect and suggest potential strategies to prevent cadmium-associated acute toxicity.