Extracellular Serine Protease Research Articles

The extracellular serine protease from Staphylococcus epidermidis elicits a type 2-biased immune response in atopic dermatitis patients.

Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease with skin barrier defects and a misdirected type 2 immune response against harmless antigens. The skin microbiome in AD is characterized by a reduction in microbial diversity with a dominance of staphylococci, including Staphylococcus epidermidis (S. epidermidis). To assess whether S. epidermidis antigens play a role in AD, we screened for candidate allergens and studied the T cell and humoral immune response against the extracellular serine protease (Esp). To identify candidate allergens, we analyzed the binding of human serum IgG4, as a surrogate of IgE, to S. epidermidis extracellular proteins using 2-dimensional immunoblotting and mass spectrometry. We then measured serum IgE and IgG1 binding to recombinant Esp by ELISA in healthy and AD individuals. We also stimulated T cells from AD patients and control subjects with Esp and measured the secreted cytokines. Finally, we analyzed the proteolytic activity of Esp against IL-33 and determined the cleavage sites by mass spectrometry. We identified Esp as the dominant candidate allergen of S. epidermidis. Esp-specific IgE was present in human serum; AD patients had higher concentrations than controls. T cells reacting to Esp were detectable in both AD patients and healthy controls. The T cell response in healthy adults was characterized by IL-17, IL-22, IFN-γ, and IL-10, whereas the AD patients' T cells lacked IL-17 production and released only low amounts of IL-22, IFN-γ, and IL-10. In contrast, Th2 cytokine release was higher in T cells from AD patients than from healthy controls. Mature Esp cleaved and activated the alarmin IL-33. The extracellular serine protease Esp of S. epidermidis can activate IL-33. As an antigen, Esp elicits a type 2-biased antibody and T cell response in AD patients. This suggests that S. epidermidis can aggravate AD through the allergenic properties of Esp.

Abstract 5199: Protease-activated CDCP1 as a marker for aggressive primary prostate cancer and castration resistance

Abstract CUB domain-containing protein 1 (CDCP1) is a type 1 transmembrane protein overexpressed in many cancers, including prostate cancer. CDCP1 has been associated with tumor progression, metastasis, drug resistance and poor prognosis in prostate cancer, making it a potential prognostic biomarker and therapeutic target. It has been shown to be overexpressed in a subset of castration resistant prostate cancers (CRPC) and cooperate with the loss of tumor suppressor PTEN to promote metastatic dissemination. Extracellular serine proteases activate CDCP1 by cleaving it into an N-terminal and C-terminal fragments. Proteolytic cleavage has been associated with the tumor-promoting functions of CDCP1. We studied the expression of full-length and cleaved form of CDCP1 in grade group 2-4 primary prostate cancer tissues and CRPCs using immunohistochemistry. Total CDCP1 staining was observed in 60% of primary prostate cancer samples, while the staining of cleaved CDCP1 was somewhat less frequently observed (45%). The results will be further analyzed regarding the PTEN status and for association with survival of the patients. In conclusion, so far, our results have indicated that cleaved CDCP1, which likely represents activated CDCP1, is detectable in GG 2-4 prostate cancers. Citation Format: Ruusu-Maaria Merivirta, Timo-Pekka Lehto, Antti Rannikko, Tuomas Mirtti, Hannu Koistinen. Protease-activated CDCP1 as a marker for aggressive primary prostate cancer and castration resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5199.

Overexpression of Motopsin, an Extracellular Serine Protease Related to Intellectual Disability, Promotes Adult Neurogenesis and Neuronal Responsiveness in the Dentate Gyrus.

Motopsin, a serine protease encoded by PRSS12, is secreted by neuronal cells into the synaptic clefts in an activity-dependent manner, where it induces synaptogenesis by modulating Na+/K+-ATPase activity. In humans, motopsin deficiency leads to severe intellectual disability and, in mice, it disturbs spatial memory and social behavior. In this study, we investigated mice that overexpressed motopsin in the forebrain using the Tet-Off system (DTG-OE mice). The elevated agrin cleavage or the reduced Na+/K+-ATPase activity was not detected. However, motopsin overexpression led to a reduction in spine density in hippocampal CA1 basal dendrites. While motopsin overexpression decreased the ratio of mature mushroom spines in the DG, it increased the ratio of immature thin spines in CA1 apical dendrites. Female DTG-OE mice showed elevated locomotor activity in their home cages. DTG-OE mice showed aberrant behaviors, such as delayed latency to the target hole in the Barnes maze test and prolonged duration of sniffing objects in the novel object recognition test (NOR), although they retained memory comparable to that of TRE-motopsin littermates, which normally express motopsin. After NOR, c-Fos-positive cells increased in the dentate gyrus (DG) of DTG-OE mice compared with that of DTG-SO littermates, in which motopsin overexpression was suppressed by the administration of doxycycline, and TRE-motopsin littermates. Notably, the numbers of doublecortin- and 5-bromo-2'-deoxyuridine-labeled cells significantly increased in the DG of DTG-OE mice, suggesting increased adult neurogenesis. Importantly, our results revealed a new function in addition to modulating neuronal responsiveness and spine morphology in the DG: the regulation of neurogenesis.

Pathogenicity assessment of Shiga toxin-producing Escherichia coli strains isolated from wild birds in a major agricultural region in California.

Shiga toxin-producing Escherichia coli (STEC) consists of diverse strains differing in genetic make-up and virulence potential. To better understand the pathogenicity potential of STEC carried by the wildlife, three STEC and one E. coli strains isolated from wild birds near a major agricultural region in California were selected for comparative pathogenomic analyses. Three American crow (Corvus brachyrhynchos) strains, RM9088, RM9513, and RM10410, belonging to phylogroup A with serotypes O109:H48, O9:H30, and O113:H4, respectively, and a red-winged blackbird (Agelaius phoeniceus) strain RM14516 in phylogroup D with serotype O17:H18, were examined. Shiga toxin genes were identified in RM9088 (stx1a), RM10410 (stx1a + stx2d), and RM14516 (stx2a). Unlike STEC O157:H7 strain EDL933, none of the avian STEC strains harbored the pathogenicity islands OI-122, OI-57, and the locus of enterocyte effacement, therefore the type III secretion system biogenesis genes and related effector genes were absent in the three avian STEC genomes. Interestingly, all avian STEC strains exhibited greater (RM9088 and RM14516) or comparable (RM10410) cytotoxicity levels compared with EDL933. Comparative pathogenomic analyses revealed that RM9088 harbored numerous genes encoding toxins, toxins delivery systems, and adherence factors, including heat-labile enterotoxin, serine protease autotransporter toxin Pic, type VI secretion systems, protein adhesin Paa, fimbrial adhesin K88, and colonization factor antigen I. RM9088 also harbored a 36-Kb high pathogenicity island, which is related to iron acquisition and pathogenicity in Yersinia spp. Strain RM14516 carried an acid fitness island like the one in EDL933, containing a nine gene cluster involved in iron acquisition. Genes encoding extracellular serine protease EspP, subtilase cytotoxin, F1C fimbriae, and inverse autotransporter adhesin IatC were only detected in RM14516, and genes encoding serine protease autotransporter EspI and P fimbriae were only identified in RM10410. Although all curli genes were present in avian STEC strains, production of curli fimbriae was only detected for RM9088 and RM14516. Consistently, strong, moderate, and little biofilms were observed for RM9088, RM14516, and RM10410, respectively. Our study revealed novel combinations of virulence factors in two avian strains, which exhibited high level of cytotoxicity and strong biofilm formation. Comparative pathogenomics is powerful in assessing pathogenicity and health risk of STEC strains.

Viral SERPINS-A Family of Highly Potent Immune-Modulating Therapeutic Proteins.

Serine protease inhibitors, SERPINS, are a highly conserved family of proteins that regulate serine proteases in the central coagulation and immune pathways, representing 2-10% of circulating proteins in the blood. Serine proteases form cascades of sequentially activated enzymes that direct thrombosis (clot formation) and thrombolysis (clot dissolution), complement activation in immune responses and also programmed cell death (apoptosis). Virus-derived serpins have co-evolved with mammalian proteases and serpins, developing into highly effective inhibitors of mammalian proteolytic pathways. Through interacting with extracellular and intracellular serine and cysteine proteases, viral serpins provide a new class of highly active virus-derived coagulation-, immune-, and apoptosis-modulating drug candidates. Viral serpins have unique characteristics: (1) function at micrograms per kilogram doses; (2) selectivity in targeting sites of protease activation; (3) minimal side effects at active concentrations; and (4) the demonstrated capacity to be modified, or fine-tuned, for altered protease targeting. To date, the virus-derived serpin class of biologics has proven effective in a wide range of animal models and in one clinical trial in patients with unstable coronary disease. Here, we outline the known viral serpins and review prior studies with viral serpins, considering their potential for application as new sources for immune-, coagulation-, and apoptosis-modulating therapeutics.

Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

Prostasin and matriptase are extracellular membrane serine proteases with opposing effects in solid epithelial tumors. Matriptase is an oncoprotein that promotes tumor initiation and progression, and prostasin is a tumor suppressor that reduces tumor invasion and metastasis. Previous studies have shown that a subgroup of Burkitt lymphoma have high levels of ectopic matriptase expression but no prostasin. Reducing the matriptase level via small interfering RNAs in B lymphoma cells impeded tumor xenograft growth in mice. Here, we report a novel approach to matriptase regulation in B cancer cells by prostasin via exosomes to initiate a prostasin-matriptase protease activation cascade. The activation and shedding of matriptase were monitored by measuring its quantity and trypsin-like serine protease activity in conditioned media. Sustained activation of the protease cascade in the cells was achieved by the stable expression of prostasin. The B cancer cells with prostasin expression presented phenotypes consistent with its tumor suppressor role, such as reduced growth and increased apoptosis. Prostasin exosomes could be developed as an agent to initiate the prostasin-matriptase cascade for treating B lymphoma with further studies in animal models.

An Empirical Cross-Sectional Study to Identify the Olfactory and Gustatory Effects of SARS-CoV-2 – A Social Analysis

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is a glycan-coated coronavirus with an exposed receptor-binding domain (RBD), capable of binding to the angiotensin-converting enzyme-2 (ACE2) receptors and entering the cell through the cleavage of the spike proteins by the extracellular host enzyme Transmembrane serine protease 2 (TMPRSS2). The severity of the infection is directly associated with loss of taste (ageusia) and loss of smell, varying from hyposmia (reduced perception of smell) to anosmia (complete loss of smell). The mechanisms underlying the independent symptoms remain inconclusive; however, have been linked to sustentacular cell infection, changes in the olfactory bulb and subcortical regions of the brain, defects in the respiratory centres of the central nervous system and cerebral glucose abnormalities. The survey for this cross-sectional study was conducted in 2021 and involved 126 individuals who were exposed to or tested positive for COVID-19, through the Reverse transcription polymerase chain reaction (RT-PCR). 6.3% of covid-negative and 34.9% covid-positive individuals exposed to COVID-19, experienced anosmia and ageusia. This study aims to identify patterns in symptoms presented when an individual is infected with COVID-19; focusing on the correlation between anosmia and ageusia and associated visual impairments or cognitive difficulties. The understanding of varied symptom combinations observed amongst individuals differing in age, sex, comorbidities or vaccination status could aid doctors and healthcare practitioners to provide a prognosis specific to the subset to which the patients belong, depending on their symptom combinations and severity for the same.

Damage sensing mediated by serine proteases Hayan and Persephone for Toll pathway activation in apoptosis-deficient flies.

The mechanisms by which the innate immune system senses damage have been extensively explored in multicellular organisms. In Drosophila, various types of tissue damage, including epidermal injury, tumor formation, cell competition, and apoptosis deficiency, induce sterile activation of the Toll pathway, a process that requires the use of extracellular serine protease (SP) cascades. Upon infection, the SP Spätzle (Spz)-processing enzyme (SPE) cleaves and activates the Toll ligand Spz downstream of two paralogous SPs, Hayan and Persephone (Psh). However, upon tissue damage, it is not fully understood which SPs establish Spz activation cascades nor what damage-associated molecules can activate SPs. In this study, using newly generated uncleavable spz mutant flies, we revealed that Spz cleavage is required for the sterile activation of the Toll pathway, which is induced by apoptosis-deficient damage of wing epidermal cells in adult Drosophila. Proteomic analysis of hemolymph, followed by experiments with Drosophila Schneider 2 (S2) cells, revealed that among hemolymph SPs, both SPE and Melanization Protease 1 (MP1) have high capacities to cleave Spz. Additionally, in S2 cells, MP1 acts downstream of Hayan and Psh in a similar manner to SPE. Using genetic analysis, we found that the upstream SPs Hayan and Psh contributes to the sterile activation of the Toll pathway. While SPE/MP1 double mutants show more impairment of Toll activation upon infection than SPE single mutants, Toll activation is not eliminated in these apoptosis-deficient flies. This suggests that Hayan and Psh sense necrotic damage, inducing Spz cleavage by SPs other than SPE and MP1. Furthermore, hydrogen peroxide, a representative damage-associated molecule, activates the Psh-Spz cascade in S2 cells overexpressing Psh. Considering that reactive oxygen species (ROS) were detected in apoptosis-deficient wings, our findings highlight the importance of ROS as signaling molecules that induce the activation of SPs such as Psh in response to damage.

Extracellular matrix-related and serine protease proteins in the amniotic fluid of women with early preterm labor: Association with spontaneous preterm birth, intra-amniotic inflammation, and microbial invasion of the amniotic cavity.

We aimed to determine whether altered levels of various extracellular matrix (ECM)-related and serine protease proteins in the amniotic fluid (AF) are associated with imminent spontaneous preterm birth (SPTB; ≤7 days) and intra-amniotic inflammation and/or microbial invasion of the amniotic cavity (IAI/MIAC) in women with early preterm labor (PTL). This retrospective cohort study included 252 women with singleton pregnancies undergoing transabdominal amniocentesis who demonstrated PTL (24-31weeks). The AF was cultured for microorganism detection to characterize MIAC. IL-6 concentrations were determined in the AF samples to identify IAI (≥2.6ng/mL). The following mediators were measured in the AF samples using ELISA: kallistatin, lumican, MMP-2, SPARC, TGFBI, and uPA. Kallistatin, MMP-2, TGFBI, and uPA levels were significantly higher and SPARC and lumican levels were significantly lower in the AF of women who spontaneously delivered within 7 days than in the AF of those who delivered after 7 days; the levels of the first five mediators were independent of baseline clinical variables. In the multivariate analysis, elevated levels of kallistatin, MMP-2, TGFBI, and uPA and low levels of lumican and SPARC in the AF were significantly associated with IAI/MIAC and MIAC, even after adjusting for the gestational age at sampling. The areas under the curves of the aforementioned biomarkers ranged from 0.58 to 0.87 for the diagnoses of each of the corresponding endpoints. ECM-related (SPARC, TGFBI, lumican, and MMP-2) and serine protease (kallistatin and uPA) proteins in the AF are involved in preterm parturition and regulation of intra-amniotic inflammatory/infectious responses in PTL.

Exploring the Biologically Active Metabolites Produced by Bacillus cereus for Plant Growth Promotion, Heat Stress Tolerance, and Resistance to Bacterial Soft Rot in Arabidopsis

Eight gene clusters responsible for synthesizing bioactive metabolites associated with plant growth promotion were identified in the Bacillus cereus strain D1 (BcD1) genome using the de novo whole-genome assembly method. The two largest gene clusters were responsible for synthesizing volatile organic compounds (VOCs) and encoding extracellular serine proteases. The treatment with BcD1 resulted in an increase in leaf chlorophyll content, plant size, and fresh weight in Arabidopsis seedlings. The BcD1-treated seedlings also accumulated higher levels of lignin and secondary metabolites including glucosinolates, triterpenoids, flavonoids, and phenolic compounds. Antioxidant enzyme activity and DPPH radical scavenging activity were also found to be higher in the treated seedlings as compared with the control. Seedlings pretreated with BcD1 exhibited increased tolerance to heat stress and reduced disease incidence of bacterial soft rot. RNA-seq analysis showed that BcD1 treatment activated Arabidopsis genes for diverse metabolite synthesis, including lignin and glucosinolates, and pathogenesis-related proteins such as serine protease inhibitors and defensin/PDF family proteins. The genes responsible for synthesizing indole acetic acid (IAA), abscisic acid (ABA), and jasmonic acid (JA) were expressed at higher levels, along with WRKY transcription factors involved in stress regulation and MYB54 for secondary cell wall synthesis. This study found that BcD1, a rhizobacterium producing VOCs and serine proteases, is capable of triggering the synthesis of diverse secondary metabolites and antioxidant enzymes in plants as a defense strategy against heat stress and pathogen attack.

Purification and characterization of a high molecular weight serine protease from Microbacterium paraoxydans sp. SKS10.

Alkaline proteases from microbial sources have been found suitable for diverse industrial applications, with serine proteases being the most common enzymes used in the detergent industry. In the present study, we have purified and characterized an extracellular alkaline serine protease from Microbacterium paraoxydans sp. SKS10. The protease was purified using ammonium sulfate precipitation followed by different chromatography techniques (fold purification 6.919). Km and Vmax for the protease were determined to be 0.183mg/mL and 4.904U/mL, respectively. This enzyme is a thermostable high molecular weight (∼109.4kDa) protease which has maximal activity at 60°C, and above pH 10. Inhibitor assays revealed the enzyme to be a serine protease whose activity increased by 2.5-fold in the presence of EDTA. This enzyme remained active in the presence of various metal salts and organic solvents and was compatible with commercially available laundry detergents highlighting its potential for use in the detergent industry.

Abstract 5564: SerpinE2: A potential biomarker for urological cancers

Abstract SerpinE2, also known as protease nexin-1, is an extracellular serine protease inhibitor modulating the activity of serine proteases such as thrombin, plasmin, urokinase-type plasminogen activator and trypsin. Dysregulation between extracellular serine proteases and their cognate inhibitors has been implicated in malignant progression, making serpins promising biomarkers for tumor progression and prognosis. SerpinE2 is upregulated in many cancers and promotes tumor invasion and metastasis. In this study, we analyzed serpinE2 expression by immunohistochemistry on tissue microarrays comprising of samples from prostate, bladder and kidney cancers. The studied samples were clinically hard-to-risk stratify and thus relevant for biomarker studies. We aimed to study the association of serpinE2 expression with survival and clinicopathological features in all three cancers, and evaluate its potential as an immunohistochemical biomarker. In the case of bladder cancer, association of serpinE2 expression with response to neoadjuvant chemotherapy was also assessed. We found that although the strong expression of serpinE2 was related to more aggressive prostate cancers (p = 0.017) and high grade bladder cancers (p = 0.034), it was not associated with relapse, treatment response or survival of prostate and bladder cancer patients. In renal cancer, on the contrary, serpinE2 expression was significantly lower in patients with tumor relapse (p = 0.048) and high expression predicted favorable disease-specific survival (p = 0.013). Our findings help understanding the functional role of serpinE2 in cancer progression and guide the prognostic biomarker research of serpinE2, particularly for prediction of renal cancer-specific survival. Citation Format: Ruusu-Maaria Kovanen, Netta Koskinen, Timo-Pekka Lehto, Antti Rannikko, Tuomas Mirtti, Hannu Koistinen. SerpinE2: A potential biomarker for urological cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5564.

PS-B08-13: PLASMIN INHIBITION ALLEVIATES HYPERTENSION AND PODOCYTE INJURY IN DAHL SALT-SENSITIVE RATS

Objective: The epithelial sodium channel (ENaC) in the kidney plays pivotal roles in blood pressure regulation, and the γ subunit is activated by extracellular serine proteases. In proteinuric renal diseases, plasmin filtered through injured glomeruli proteolytically activates γENaC. In addition, filtered plasmin directly causes podocyte injury. We previously reported that Dahl salt-sensitive (DS) rats fed a high-salt (HS) diet developed severe hypertension and proteinuria together with γENaC activation, and that a synthetic serine protease inhibitor, camostat mesilate, mitigated these changes. However, the role of plasmin in DS rats remains unclear. In this study, we evaluated the relationship between plasmin and hypertension as well as glomerular injury and the effects of plasmin inhibitors in DS rats. Design and method: Five-week-old male DS rats were divided into normal-salt (NS) diet, HS diet, and HS + plasmin inhibitors [tranexamic acid (TA, 2 mg/mL TA in drinking water) and synthetic plasmin inhibitor YO-2 (4 mg/kg/day, intraperitoneal injection)] groups. After systolic blood pressure measurement and 24-h urine collection over time for 5 weeks, the rats were sacrificed for biochemical examination. Results: The HS group displayed severe hypertension and proteinuria together with activation of plasmin in urine and γENaC in the kidney, which were not attenuated by TA. On the other hand, YO-2 mitigated both hypertension and proteinuria [SBP (mmHg): NS, 130.8 ± 7.5; HS, 209.0 ± 14.2; HS+YO-2, 183.8 ± 8.4; Urinary protein (mg/day): NS, 15.4 ± 3.2; HS 259.0 ± 129.3; HS+YO-2, 94.5 ± 31.5]. YO-2 inhibited the attachment of plasmin(ogen) to podocytes and alleviated podocyte injury by reducing glomerular apoptotic cells. Furthermore, YO-2 suppressed the upregulation of protease-activated receptor-1 and phosphorylated ERK1/2 as well as mRNA expression of inflammatory and pro-fibrotic cytokines in the kidney. Conclusion: These results indicate that plasmin plays important roles in the development of salt-sensitive hypertension and glomerular injuries in a rat model of hypertension, and suggest that plasmin inhibition could be a potential therapeutic strategy against salt-sensitive hypertension.

Nafamostat Mesylate in Combination with the Mouse Amino-Terminal Fragment of Urokinase-Human Serum Albumin Improves the Treatment Outcome of Triple-Negative Breast Cancer Therapy.

Triple-negative breast cancer (TNBC) is highly aggressive and causes a higher proportion of metastatic cases. However, therapies directed to specific molecular targets have rarely achieved clinically meaningful improvements in the outcome of TNBC therapy. A urokinase-type plasminogen activator (uPA), one of the best-validated biomarkers of breast cancer, is an extracellular proteolytic serine protease involved in many pathological and physiological processes, including tumor cell invasion and metastasis. Nafamostat mesylate (NM) is a synthetic compound that inhibits various serine proteases and has been used as a therapeutic agent for the treatment of TNBC. Nevertheless, NM has poor specificity for serine proteases and is easy be hydrolyzed; moreover, the inhibitory mechanism of TNBC therapy is unclear. In this study, we combine NM with a macromolecular drug delivery vehicle, mouse amino-terminal fragment of urokinase-human serum albumin (mATF-HSA), to form a complex (mATF-HSA:NM) using the dilution-incubation-purification method. mATF specifically targets uPAR overexpressed on the surface of TNBC cells; moreover, HSA prevents NM from being hydrolyzed by numerous serine proteases. mATF-HSA:NM showed stronger inhibitory effects on the proliferation and metastasis of TNBC in vitro and in vivo without significant cytotoxicity on normal cells and tissues. In addition, we demonstrated that NM mediates metastasis of TNBC cells through inhibition of uPA using a stable uPA knockdown cell line (MDA-MB231 shuPA). Overall, we have developed a macromolecular complex targeted to treat high uPAR-expressing tumor types, and mATF-HSA can potentially be used to load other types of drugs with tumor-targeting specificity for mouse tumor models and is a promising tool to study tumor biology in mouse tumor models.

The serine protease plasmin plays detrimental roles in epithelial sodium channel activation and podocyte injury in Dahl salt-sensitive rats.

Salt-sensitive hypertension is associated with poor clinical outcomes. The epithelial sodium channel (ENaC) in the kidney plays pivotal roles in sodium reabsorption and blood pressure regulation, in which its γ subunit is activated by extracellular serine proteases. In proteinuric nephropathies, plasmin filtered through injured glomeruli reportedly activates γENaC in the distal nephron and causes podocyte injury. We previously reported that Dahl salt-sensitive (DS) rats fed a high-salt (HS) diet developed hypertension and proteinuria along with γENaC activation and that a synthetic serine protease inhibitor, camostat mesilate, mitigated these changes. However, the role of plasmin in DS rats remained unclear. In this study, we evaluated the relationship between plasmin and hypertension as well as podocyte injury and the effects of plasmin inhibitors in DS rats. Five-week-old DS rats were divided into normal-salt diet, HS diet, and HS+plasmin inhibitor (either tranexamic acid [TA] or synthetic plasmin inhibitor YO-2) groups. After blood pressure measurement and 24 h urine collection over 5 weeks, rats were sacrificed for biochemical analyses. The HS group displayed severe hypertension and proteinuria together with activation of plasmin in urine and γENaC in the kidney, which was significantly attenuated by YO-2 but not TA. YO-2 inhibited the attachment of plasmin(ogen) to podocytes and alleviated podocyte injury by inhibiting apoptosis and inflammatory/profibrotic cytokines. YO-2 also suppressed upregulation of protease-activated receptor-1 and phosphorylated ERK1/2. These results indicate an important role of plasmin in the development of salt-sensitive hypertension and related podocyte injury, suggesting plasmin inhibition as a potential therapeutic strategy.

IgG Binds Escherichia coli Serine Protease EspP and Protects Mice From E. coli O157:H7 Infection.

Shiga toxin-producing Escherichia coli O157:H7 is a virulent strain causing severe gastrointestinal infection, hemolytic uremic syndrome and death. To date there are no specific therapies to reduce progression of disease. Here we investigated the effect of pooled immunoglobulins (IgG) on the course of disease in a mouse model of intragastric E. coli O157:H7 inoculation. Intraperitoneal administration of murine IgG on day 3, or both on day 3 and 6, post-inoculation improved survival and decreased intestinal and renal pathology. When given on both day 3 and 6 post-inoculation IgG treatment also improved kidney function in infected mice. Murine and human commercially available IgG preparations bound to proteins in culture filtrates from E. coli O157:H7. Bound proteins were extracted from membranes and peptide sequences were identified by mass spectrometry. The findings showed that murine and human IgG bound to E. coli extracellular serine protease P (EspP) in the culture filtrate, via the IgG Fc domain. These results were confirmed using purified recombinant EspP and comparing culture filtrates from the wild-type E. coli O157:H7 strain to a deletion mutant lacking espP. Culture filtrates from wild-type E. coli O157:H7 exhibited enzymatic activity, specifically associated with the presence of EspP and demonstrated as pepsin cleavage, which was reduced in the presence of murine and human IgG. EspP is a virulence factor previously shown to promote colonic cell injury and the uptake of Shiga toxin by intestinal cells. The results presented here suggest that IgG binds to EspP, blocks its enzymatic activity, and protects the host from E. coli O157:H7 infection, even when given post-inoculation.

Overexpression of Motopsin, an Extracellular Serine Protease Related to Intellectual Disability, Elevates Neurogenesis and Neuronal Responsivity in the Dentate Gyrus

Overexpression of Motopsin, an Extracellular Serine Protease Related to Intellectual Disability, Elevates Neurogenesis and Neuronal Responsivity in the Dentate Gyrus

Characterization of the Proteolytic Activity of a Halophilic Aspergillus reticulatus Strain SK1-1 Isolated from a Solar Saltern.

Salterns are hypersaline environments that are inhabited by diverse halophilic microorganisms, including fungi. In this study, we isolated a fungal strain SK1-1 from a saltern in the Republic of Korea, which was identified as Asperillus reticulatus. This is the first reported saline-environment-derived A. reticulatus that belongs to the Aspergillus penicillioides clade and encompasses xerophilic fungi. SK1-1 was halophilic, obligately requiring NaCl for growth, with a maximum radial growth of 6%–9% (w/v) NaCl. To facilitate the biotechnological application of halophilic fungi, we screened the SK1-1 strain for proteolytic activity. Proteases have widespread applications in food processing, detergents, textiles, and waste treatment, and halophilic proteases can enable protein degradation in high salt environments. We assessed the proteolytic activity of the extracellular crude enzyme of SK1-1 using azocasein as a substrate. The crude protease exhibited maximum activity at 40–50 °C, pH 9.5–10.5, and in the absence of NaCl. It was also able to retain up to 69% of its maximum activity until 7% NaCl. Protease inhibitor assays showed complete inhibition of the proteolytic activity of crude enzymes by Pefabloc® SC. Our data suggest that the halophilic A. reticulatus strain SK1-1 produces an extracellular alkaline serine protease.

Tenebrio molitor Spätzle 1b Is Required to Confer Antibacterial Defense Against Gram-Negative Bacteria by Regulation of Antimicrobial Peptides.

Innate immunity is the ultimate line of defense against invading pathogens in insects. Unlike in the mammalian model, in the insect model, invading pathogens are recognized by extracellular receptors, which activate the Toll signaling pathway through an extracellular serine protease cascade. In the Toll-NF-κB pathway, the extracellular spätzle protein acts as a downstream ligand for Toll receptors in insects. In this study, we identified a novel Spätzle isoform (TmSpz1b) from RNA sequencing database of Tenebrio molitor. TmSpz1b was bioinformatically analyzed, and functionally characterized for the antimicrobial function by RNA interference (RNAi). The 702 bp open reading frame of TmSpz1b encoded a putative protein of 233 amino acid residues. A conserved cystine-knot domain with seven cysteine residues in TmSpz1b was involved in three disulfide bridges and the formation of a spätzle dimer. TmSpz1b was mostly expressed in the hemocytes of T. molitor late instar larvae. The mRNA expression of TmSpz1b was highly induced in the hemocytes after Escherichia coli, Staphylococcus aureus, and Candida albicans stimulation of T. molitor larvae. TmSpz1b silenced larvae were significantly more susceptible to E. coli infection. In addition, RNAi-based functional assay characterized TmSpz1b to be involved in the positive regulation of antimicrobial peptide genes in hemocytes and fat bodies. Further, the TmDorX2 transcripts were downregulated in TmSpz1b silenced individuals upon E. coli challenge suggesting the relationship to Toll signaling pathway. These results indicate that TmSpz1b is involved in the T. molitor innate immunity, causes the sequestration of Gram-negative bacteria by the regulatory action of antimicrobial peptides, and enhances the survival of T. molitor larvae.

Pneumococcal Extracellular Serine Proteases: Molecular Analysis and Impact on Colonization and Disease.

The pathobiont Streptococcus pneumoniae causes life-threatening diseases, including pneumonia, sepsis, meningitis, or non-invasive infections such as otitis media. Serine proteases are enzymes that have been emerged during evolution as one of the most abundant and functionally diverse group of proteins in eukaryotic and prokaryotic organisms. S. pneumoniae expresses up to four extracellular serine proteases belonging to the category of trypsin-like or subtilisin-like family proteins: HtrA, SFP, PrtA, and CbpG. These serine proteases have recently received increasing attention because of their immunogenicity and pivotal role in the interaction with host proteins. This review is summarizing and focusing on the molecular and functional analysis of pneumococcal serine proteases, thereby discussing their contribution to pathogenesis.