Serine Protease Research Articles

A sphingolipid rheostat controls apoptosis versus apical cell extrusion as alternative tumour-suppressive mechanisms

Evasion of cell death is a hallmark of cancer, and consequently the induction of cell death is a common strategy in cancer treatment. However, the molecular mechanisms regulating different types of cell death are poorly understood. We have formerly shown that in the epidermis of hypomorphic zebrafish hai1a mutant embryos, pre-neoplastic transformations of keratinocytes caused by unrestrained activity of the type II transmembrane serine protease Matriptase-1 heal spontaneously. This healing is driven by Matriptase-dependent increased sphingosine kinase (SphK) activity and sphingosine-1-phosphate (S1P)-mediated keratinocyte loss via apical cell extrusion. In contrast, amorphic hai1afr26 mutants with even higher Matriptase-1 and SphK activity die within a few days. Here we show that this lethality is not due to epidermal carcinogenesis, but to aberrant tp53-independent apoptosis of keratinocytes caused by increased levels of pro-apoptotic C16 ceramides, sphingolipid counterparts to S1P within the sphingolipid rheostat, which severely compromises the epidermal barrier. Mathematical modelling of sphingolipid rheostat homeostasis, combined with in vivo manipulations of components of the rheostat or the ceramide de novo synthesis pathway, indicate that this unexpected overproduction of ceramides is caused by a negative feedback loop sensing ceramide levels and controlling ceramide replenishment via de novo synthesis. Therefore, despite their initial decrease due to increased conversion to S1P, ceramides eventually reach cell death-inducing levels, making transformed pre-neoplastic keratinocytes die even before they are extruded, thereby abrogating the normally barrier-preserving mode of apical live cell extrusion. Our results offer an in vivo perspective of the dynamics of sphingolipid homeostasis and its relevance for epithelial cell survival versus cell death, linking apical cell extrusion and apoptosis. Implications for human carcinomas and their treatments are discussed.

Protease activated receptor 2 as a novel druggable target for the treatment of metabolic dysfunction-associated fatty liver disease and cancer

Metabolic dysfunction-associated fatty liver disease (MAFLD) is spreading worldwide, largely due to unhealthy lifestyles that contribute to the rise in diabetes, metabolic syndrome, and obesity. In this situation, the progression of injury to metabolic steatohepatitis can evolve to cirrhosis and, eventually, to hepatocellular carcinoma (HCC). It is well known that serine protease enzymes with different functions in cellular homeostasis act as signaling molecules that regulate liver inflammation by activating the protease-activated receptors (PARs) family members, expressed on the cellular plasma membrane. Among them, PAR2 plays a central role in the activation of signaling pathways in response to changes in the extracellular microenvironment. Experimental data have provided evidence that PAR2 is involved not only in inflammatory response but also in insulin resistance, lipid metabolism, and cancer. The major aims of this narrative review are addressed to assess PAR2 involvement in inflammation, metabolism, and liver disease progression and to explore possible therapeutic strategies, based on PAR2 inhibition, in order to prevent its biological effects in the context of MAFLD and cancer.

Protein biomarker signatures of preeclampsia - a longitudinal 5000-multiplex proteomics study

We aimed to explore novel biomarker candidates and biomarker signatures of late-onset preeclampsia (LOPE) by profiling samples collected in a longitudinal discovery cohort with a high-throughput proteomics platform. Using the Somalogic 5000-plex platform, we analyzed proteins in plasma samples collected at three visits (gestational weeks (GW) 12–19, 20–26 and 28–34 in 35 women with LOPE (birth ≥ 34 GW) and 70 healthy pregnant women). To identify biomarker signatures, we combined Elastic Net with Stability Selection for stable variable selection and validated their predictive performance in a validation cohort. The biomarker signature with the highest predictive performance (AUC 0.88 (95% CI 0.85–0.97)) was identified in the last trimester of pregnancy (GW 28–34) and included the Fatty acid amid hydrolase 2 (FAAH2), HtrA serine peptidase 1 (HTRA1) and Interleukin-17 receptor C (IL17RC) together with sFLT1 and maternal age, BMI and nulliparity. Our biomarker signature showed increased or similar predictive performance to the sFLT1/PGF-ratio within our data set, and we were able to validate the biomarker signature in a validation cohort (AUC ≥ 0.90). Further validation of these candidates should be performed using another protein quantification platform in an independent cohort where the negative and positive predictive values can be validly calculated.

Crystallization and crystallographic studies of human serine protease inhibitor (serpin) B9.

Serine protease inhibitor B9 (serpin B9, also known as protease inhibitor 9 or PI9) plays a critical role in regulating the immune response by specifically inhibiting granzyme B, a serine protease found in cytotoxic T lymphocytes and natural killer cells. Despite its potential as an anticancer drug target, the structural details of serpin B9 have remained elusive until now. In this study, a cleaved form of recombinant human serpin B9 was successfully prepared and crystallized. The crystals belonged to space group P212121, with unit-cell parameters a = 68.51, b = 82.32, c = 101.17 Å, and an X-ray diffraction data set was collected at 1.9 Å resolution. The structure shows that serpin B9 adopts a relaxed conformation, with its cleaved reactive-centre loop inserted into the central β-sheet. Unlike other serpins, serpin B9 shows significant structural deviations around helix D, with a larger surface cavity, which could serve as a promising target for small-molecule inhibitors.

The diversity of AMPA receptor inhibition mechanisms among amidine-containing compounds

Amidine-containing compounds are primarily known as antiprotozoal agents (pentamidine, diminazene, furamidine) or as serine protease inhibitors (nafamostat, sepimostat, camostat, gabexate). DAPI is widely recognized as a fluorescent DNA stain. Recently, it has been shown that these compounds also act as NMDA receptor inhibitors. In this study, we examined the activity of these compounds and analyzed the mechanisms of action in relation to another important class of ionotropic glutamate receptors–calcium-permeable AMPA receptors (CP-AMPARs) and calcium-impermeable AMPA receptors (CI-AMPARs) – using the whole-cell patch-clamp method on isolated male Wistar rat brain neurons. Gabexate and camostat were found to be inactive. Other compounds preferentially inhibited calcium-permeable AMPA receptors with IC50 values of 30–60 µM. DAPI and furamidine were also active against CI-AMPARs with IC50s of 50–60 μM, while others showed poor activity. All active compounds acted as channel blockers, which are able for permeating into the cytoplasm on both CP- and CI-AMPARs. Specifically, sepimostat showed trapping in the closed CP-AMPAR channel. Furamidine and DAPI demonstrated a voltage-independent action on CI-AMPARs, indicating binding to an additional superficial site. While the majority of compounds inhibited glutamate-activated steady-state currents as well as kainate-activated currents on CI-AMPARs, pentamidine significantly potentiated glutamate-induced steady-state responses. The potentiating effect of pentamidine resembles the action of the positive allosteric modulator cyclothiazide although the exact binding site remains unclear. Thus, this study, together with our previous research on NMDA receptors, provides a comprehensive overview of this novel group of ionotropic glutamate receptors inhibitors with a complex pharmacological profile, remarkable diversity of effects and mechanisms of action.

Auranofin is active against Histoplasma capsulatum and reduces the expression of virulence-related genes.

Auranofin is an approved anti-rheumatic drug that has a broad-range inhibitory action against several microorganisms, including human pathogenic fungi. The auranofin activity against Histoplasma capsulatum, the dimorphic fungus that causes histoplasmosis, has not been properly addressed. Since there are few therapeutic options for this life-threatening systemic mycosis, this study evaluated the effects of auranofin on H. capsulatum growth and expression of virulence factors. Minimal inhibitory and fungicidal concentrations (MIC and MFC, respectively) of auranofin against 15 H. capsulatum strains with distinct genetic backgrounds were determined using the yeast form of the fungus and a microdilution protocol. Auranofin activity was also assessed on a macrophage model of infection and on a Tenebrio molitor invertebrate animal model. Expression of virulence-related genes was compared between auranofin treated and untreated H. capsulatum yeast cells using a quantitative PCR assay. Auranofin affected the growth of different strains of H. capsulatum, with MIC and MFC values ranging from 1.25 to 5.0 μM and from 2.5 to >10 μM, respectively. Auranofin was able to kill intracellular H. capsulatum yeast cells and conferred protection against the fungus in the experimental animal model of infection. Moreover, the expression of catalase A, HSP70, superoxide dismutase, thioredoxin reductase, serine proteinase, cytochrome C peroxidase, histone 2B, formamidase, metallopeptidase, Y20 and YPS3 proteins were reduced after six hours of auranofin treatment. CONCLUSIONS/SIGNIFICANCE: Auranofin is fungicidal against H. capsulatum and reduces the expression of several virulence-related genes, which makes this anti-rheumatic drug a good candidate for new medicines against histoplasmosis.

7243 COVID-19 Associated Primary Autoimmune Adrenal Insufficiency: A New Clinical Entity

Abstract Disclosure: J. Naredo Rojas: None. B. Udegbe: None. I. Remba-Shapiro: None. S.L. Stockman: None. L.B. Nachtigall: None. Background: The adrenal glands express angiotensin converting enzyme and transmembrane serine protease 2 receptors for SARS-CoV-2 protein S, facilitating viral entry. Cortisol levels are decreased in COVID-19 infection in correlation with disease severity. A number of potential mechanisms could relate autoimmune adrenal insufficiency (AI) to COVID-19, but whether or not SARS-CoV-2 infection plays a direct role in the autoimmune pathogenesis of primary AI is unknown. Furthermore, while single cases have been reported, autoimmune-mediated primary adrenal insufficiency following COVID-19 illness has not been formally studied. Purpose: To determine the association and clinical presentation of primary autoimmune adrenal insufficiency in patients with COVID-19 disease. Methods: A retrospective chart review was performed on patients presenting to Massachusetts General Hospital (MGH) with primary AI in the setting of recent COVID-19 illness. Review of the literature was performed to identify additional cases. Clinical, biochemical and radiographic characteristics, and interval between the diagnosis of COVID-19 and primary AI diagnosis were evaluated. Criteria for inclusion were biochemical confirmation of AI, clinical signs and/or symptoms of adrenal insufficiency within 6 months of COVID-19 illness, and evidence of autoimmune etiology of AI. Results: Five novel cases of COVID-19 associated primary autoimmune-mediated adrenal insufficiency from our Center were identified. Review of the literature yielded five additional cases. This series combines our Center’s experience, and the cases reported to date, including a total of 10 patients, 6 men and 4 women. All men (6/6) and 2 women (2/4) were < 30 years old. Patients demonstrated clinical evidence of adrenal insufficiency in a median time frame of 14 days (IQR 8.5-23.3) after SARS-CoV-2 infection. Vomiting, hypotension, and hyponatremia were present in all (N=10). Other common symptoms were nausea (90%), fatigue (70%), hyperpigmentation (70%) and hyperkalemia (50%). Nine (90%) had a positive past medical and/or family history of autoimmune disease. Nine (90%) had positive 21-hydroxylase antibodies. The median serum morning cortisol level was 1.1μg/dL with an IQR of 0.7-3.0 μg/dL. The mean ACTH level was 1235.2 pg/mL with a SD of 580.1 pg/mL. The mean ACTH was 19.3 x ULN with a SD of 10.0. Adrenal imaging did not show hemorrhage or necrosis but showed atrophy in 50%. Conclusions: This series reveals that autoimmune-mediated primary adrenal insufficiency may be diagnosed in the aftermath of COVID-19 illness. Age under 30, family history and/or past medical history of autoimmune disease may be associated. Future prospective studies are needed to determine causality. Presentation: 6/1/2024

8636 Characterization Of The Role Of Neutrophils In Promoting Progression Of Lymphangioleiomyomatosis

Abstract Disclosure: T.R. Henson: None. B. Minor: None. S.R. Hammes: None. Lymphangioleiomyomatosis (LAM) is a rare, multisystemic disease that is associated with cystic lung destruction. LAM is caused by small smooth muscle cell-like tumors throughout the lung that contain deleterious mutations in the tuberous sclerosis complex genes TSC1 or TSC2. This attenuates the activity of the mammalian target of rapamycin complex 1 (mTORC1) causing increased proliferation of the smooth muscle cells. LAM is a sexually dimorphic disease that primarily affects genetic females. Clinical observations implicate the role of estrogen in disease progression, as it is exacerbated during pregnancy and by use of exogenous estrogens in birth control. LAM tumor-derived cells lines have shown that direct estrogen stimulation of cell proliferation is modest compared to the dramatic in vivo effects of estradiol (E2) stimulation in a uterine-specific TSC2-null mouse model. Creation of a monoclonal cell line from these mice resulted in the generation of the LTM3 cell line, which also demonstrates low response to direct estrogen stimulation in vitro. This indicates that direct E2 stimulation may act upon other cell types in the tumor microenvironment. Immunophenotyping of uterine-specific TSC2-null mice has shown that the tumor microenvironment is enriched with myeloid cells specifically neutrophils. Functional analysis of neutrophil suppression in vivo has shown a reduction in myometrial growth in the uterine-specific TSC2-null mice, indicating a key role for neutrophil-mediated LAM tumor growth. Altered transcriptional regulation of neutrophils further highlights the importance of neutrophils in LAM progression. Bulk messenger RNA sequencing revealed E2 dependent increase in the neutrophil derived serine protease neutrophil elastase (NE). NE in vitro has been shown to increase migration, invasion, and proliferation of LAM cell lines, while inhibition in vivo has shown a reduction to the myometrial growth. Previously published data by the lab has shown that E2 increases tumor induced expansion of neutrophils in the bone marrow. Isolation and purification of Neutrophils from mouse bone marrow stimulated with tumor conditioned media from the LTM3 cells has shown signs of activation via upregulated gene expression. The focus of the current research has been the characterization of bone marrow derived neutrophils. Currently working with an in vitro system to study the proliferation of LTM3 and the rat leiomyoma (ELT3) cell lines when they are co-cultured with neutrophils. Understanding the mechanism by which neutrophils are activated and interact with LAM cells can give understanding to disease progression may provide insight into future treatment options. Presentation: 6/3/2024

Resolving the Structure of a Guanine Quadruplex in TMPRSS2 Messenger RNA by Circular Dichroism and Molecular Modeling.

The presence of a guanine quadruplex in the opening reading frame of the messenger RNA coding for the transmembrane serine protease 2 (TMPRSS2) may pave the way to original anticancer and host-oriented antiviral strategy. Indeed, TMPRSS2 in addition to being overexpressed in different cancer types, is also related to the infection of respiratory viruses, including SARS-CoV-2, by promoting the cellular and viral membrane fusion through its proteolytic activity. The design of selective ligands targeting TMPRSS2 messenger RNA requires a detailed knowledge, at atomic level, of its structure. Therefore, we have used an original experimental-computational protocol to predict the first resolved structure of the parallel guanine quadruplex secondary structure in the RNA of TMPRSS2, which shows a rigid core flanked by a flexible loop. This represents the first atomic scale structure of the guanine quadruplex structure present in TMPRSS2 messenger RNA.

Genome resequencing and genome-wide polymorphisms in mosquito vectors Aedes aegypti and Aedes albopictus from south India

Aedes aegypti and Aedes albopictus mosquitoes spread major vector-borne viral diseases in tropical and sub-tropical regions of the globe. In this study, we sequenced the genome of Indian Ae. aegypti and Ae. albopictus and mapped to their reference genomes. Comparative genomics were performed between our strain and the reference strains. A total of 14,416,484 single nucleotide polymorphisms (SNPs) and 156,487 insertions and deletions (InDels) were found in Ae. aegypti, and 28,940,433 SNPs and 188,987 InDels in Ae. albopictus. Particular emphasis was given to gene families involved in mosquito digestion, development, and innate immunity, which could be putative candidates for vector control. Serine protease cascades and their inhibitors called serpins, play a central role in these processes. We extracted high-impact variants in genes associated with serine proteases and serpins. This study reports for the first time a high coverage genome sequence data of an Indian Ae. albopictus mosquito. The results from this study will provide insights into Indian Aedes specific polymorphisms and the evolution of immune related genes in mosquitoes, which can serve as a resource for future comparative genomics and those pursuing the development of targeted biopesticides for effective mosquito control strategies.

A-231 A modified Sampson LDL-C equation to improve concordance with the reference method at low levels of LDL-C

Abstract Background Patients at high risk for Atherosclerotic Cardiovascular Disease (ASCVD), who do not achieve adequate lowering of LDL-cholesterol (LDL-C) with maximally tolerated statins or who are statin-intolerant, are often treated with proprotein convertase subtilisin/kexin (type 9) serine protease inhibitor (PCSK9-inh) therapy. The LDL-C reference method, Beta-Quantitation (BQ), is not routinely performed in these patients. Because low values are measured less accurately with current methods, a variety of equations have been developed to calculate LDL-C from the standard lipid panel test results (total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C)). We further modified the Sampson LDL-C (S-LDL-C) equation by a novel approach by combining logistic regression (LR) and linear regression to minimize errors when compared to BQ for low LDL-C. Methods A dataset of BQ results from Mayo Medical Labs was divided randomly into a training set (TG ≤ 1500 mg/dL, TC ≤ 1050 mg/dL, n=19,639) and a validation set (TG ≤ 800 mg/dL, BQ-LDL-C ≤ 130 mg/dL, n=12,910). The training dataset was analyzed by LR, using BQ-LDL-C to classify patients as being above or below the 70 mg/dL cut-point. Dependent variables in the LR model were the S-LDL-C, HDL-C, TC, TG and an interaction term (TG x NonHDL-C). The sum of the exponential terms in the trained LR equation for the dependent variables were then fitted by least squares linear regression to match BQ-LDL-C to develop the modified S-LDL-C equation (mS-LDL-C). We used the validation dataset to compare Friedewald (F-LDL-C), extended Martin (eM-LDL-C), Sampson (S-LDL-C) and modified Sampson (mS-LDL-C) equations by Cohen’s kappa coefficient for agreement with BQ-LDL-C and normalized Matthew’s correlation coefficient (MCC), which considers any imbalances in the number of false high versus false low results. Results The mS-LDL-C equation (mS-LDL-C = (TC/0.9423 - HDL-C/0.9582 - TG/9.397 - ((TGxNonHDL-C)/1305) + TG2/8569) - 5.688), primarily differs from the S-LDL-C equation in the value of the coefficients that include TG as a variable. For S-LDL-C ≤100 mg/dL, mS-LDL-C had the best concordance with BQ-LDL-C in terms of the total percent of patients misclassified as either high or low (mS-LDL-C: 21.4%, S-LDL-C: 24.4%, eM-LDL-C: 25.4% F-LDL-C: 30.8%). mS-LDL-C also had the best kappa score when compared to BQ (mS-LDL-C: 0.833, S-LDL-C: 0.810, eM-LDL-C: 0.789, F-LDL-C: 0.743). MCC was also the highest for mS-LDL-C (mS-LDL-C: 0.916, S-LDL-C: 0.906, eM-LDL-C: 0.894, F-LDL-C: 0.879). Finally, mS-LDL-C also showed the best concordance with BQ-LDL-C when an alternative lower LDL-C therapeutic decision cut-point of 55 mg/dL is used (kappa of 0.766 versus 0.724) but showed an increasingly poorer performance than S-LDL-C for higher LDL-C whenever it exceeded 150 mg/dL. Conclusions A strategy to maximize the overall accuracy of estimated LDL-C could provide an automatic reflex in a laboratory information system to the mS-LDL-C equation whenever S-LDL-C is <100 mg/dL. By doing so, the use of the more accurate mS-LDL-C equation for low LDL-C should improve PCSK9-inh therapeutic decision making over the other LDL-C equations.

Homology-based identification and structural analysis of Pangasius hypophthalmus Annexins and Serine proteases to search molecules for wound healing applications

Chronic wounds and burns are a worldwide healthcare problem that erodes patients’ well-being and healthcare systems. This silent and costly epidemic requires new, cost-efficient solutions to improve patients’ physical and economic welfare. Eschar-degrading vegetal and bacterial proteases have been utilized as a solution. However, these proteins are evolutionarily far from those present in human wound healing. Serine protease (SP) and annexin (ANX) proteins interact within the skin healing process. A homology-based identification pipeline can help in discovering selective human SP and ANX analogs in the epithelial tissue of the fast-healing species, Pangasius hypophthalmus. In the present work, we found 14 candidates for RT-PCR in P. hypophthalmus using homology inference. The genetically detected candidates were then structurally and sequentially analyzed to understand their possible relation to SPs and ANXs involved in human wound healing. A total of six TBLASTN/BLASTX candidates (four SPs and two ANXs) were detected in P. hypophthalmus skin. Structural analysis revealed that all SP candidates resembled human KLK4, KLK5, KLK6, and KLK8, whereas all ANX only resembled human ANXA4. Structure and sequence analysis revealed high conservation of ANX Ca2+ binding sites (GDXD) and SP catalytic triad (HDS) motifs. In addition, structural analysis revealed that SP substrate selectivity position 186 was the main difference between human KLK5 and P. hypophthalmus SPs. These findings may allow the proposal and testing of more selective formulations, broadening treatments beyond debridement.

Antitumor Activity of Serine Enzyme and Crude Extracts of The Earthworm Lumbricus terrestris Against A549 Human Lung Cancer Cell Line

Background: Numerous disorders have been successfully treated with earthworms, and this has been documented in ancient texts. These invertebrates are a rich natural source of biological and pharmacological compounds with antioxidant and anticancer properties. The present study aims to isolate and partially purify serine protease from the earthworm and evaluate the cytotoxic and antitumor activities of serine protease and crude extracts of the earthworm. Methods: Lumbricus terrestris earthworms were collected and processed to prepare crude extracts, isolate, and partially purify them. The anti-tumour activity's serine enzyme was evaluated by MTT assay and Live/Dead Cell Viability Assay using A549 and NHF cell lines. Results: When the concentration was doubled, it hurt the viability of A549 and NHF cells. The IC50 values for A549cell lines were194.3 and 22.57 μg/ml for crude and serine protease, respectively. Also, the crude extract does not show cytotoxicity against the normal cell line (NHF cells). The IC50 values 391,78.61 mg/ ml, respectively, using Live/Dead Cell Viability Assay, demonstrate that an increased proportion of apoptotic cell deaths in A549 cells after treatment with crude as well as enzyme there is a significant increase in the mean count of dead cells after being treated with enzyme as well as crude the mean count was 172.7 3;127.5 in cells treated with serine enzyme and crude extract, respectively, whereas in the untreated cells, it was 33.1 reflects a significant difference when compared with treated cells with serine enzyme and crud extracts (P<0.001). Conclusion: These findings revealed that Serin enzyme and crude extract of Lumbricus terrestris possesses excellent antitumor activity.

C1q/MASP Complexes-Hybrid Complexes of Classical and Lectin Pathway Proteins Are Found in the Circulation.

Complement pathways, traditionally regarded as separate entities invitro, are increasingly noted for cross-communication and bypass mechanisms. Among these, the MBL/ficolin/CL-associated serine protease (MASP)-3, a component of lectin pathway pattern recognition molecules, has shown the ability to process critical substrates such as pro-factor D and insulin growth factor binding protein-5. Given shared features between lectin pathway pattern recognition molecules and C1q from the classical pathway, we hypothesized that C1q might be a viable invivo binding partner for the MASPs. We used microscale thermophoresis, ELISA, and immunoprecipitation assays to detect C1q/MASP complexes and functionally assessed the complexes through enzymatic cleavage assays. C1q/MASP-3 complexes were detected in human serum and correlated well with MASP-3 serum levels in healthy individuals. The binding affinity between MASP-3 and C1q invitro was in the nanomolar range, and the interaction was calcium-dependent, as demonstrated by their dissociation in the presence of EDTA. Furthermore, most of the circulating C1q-bound MASP-3 was activated. Based on immunoprecipitation, also C1q/MASP-2 complexes appeared to be present in serum. Finally, C1q/MASP-2 and C1q/MASP-3 invitro complexes were able to cleave C4 and pro-factor D, respectively. Our study reveals the existence of C1q/MASP complexes in the circulation of healthy individuals, and both C1q/MASP-2 and C1q/MASP-3 complexes display proteolytic activity. Hence, this study uncovers a crosstalk route between complement pathways not previously described.

Small leucine zipper protein negatively regulates liver fibrosis by suppressing the expression of plasminogen activator inhibitor-1

Liver fibrosis, which is caused by viral infection, toxic exposure, and autoimmune diseases, is a chronic liver disease. Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor of tissue-type plasminogen activator (tPA) and urokinase plasminogen activator, which convert plasminogen into plasmin. Therefore, PAI-1 suppresses fibrinolysis by blocking plasmin synthesis and is involved in liver fibrosis via extracellular matrix deposition. Small leucine zipper protein (sLZIP) acts as a transcription factor and plays critical roles in many cellular processes. However, the role of sLZIP in liver fibrosis remains unclear. In this study, we investigated the role of sLZIP in regulating PAI-1 transcription and liver fibrosis. sLZIP knockdown enhanced the expression of PAI-1 at the mRNA and protein levels. sLZIP knockdown also increased PAI-1 secretion and suppressed blood clot lysis by blocking tPA activity. Moreover, conditioned medium derived from sLZIP knockdown cells downregulated the expression of matrix metalloprotease (MMP)-2 and MMP-9 in the presence of tPA in hepatic stellate cells (HSCs). Liver-specific sLZIP knockout mice showed deteriorated liver fibrosis compared to control mice in a bile duct ligation-induced fibrosis model. These findings demonstrate that sLZIP functions as a negative regulator of liver fibrosis by suppressing PAI-1 transcription and HSC activation.

MiR-149-3p targeting TMPRSS4 regulates the sensitivity to cisplatin to inhibit the progression of lung cancer.

Lung cancer cells tend to develop resistance to cisplatin (DDP) during continuous chemotherapy, making it crucial to improve DDP sensitivity to enhance therapeutic outcomes. The levels of miR-149-3p in lung tissues and cells, as well as the biological behaviors of lung cancer cells, were analyzed. H446/DDP and A549/DDP cell lines were established to investigate how miR-149-3p affects lung cancer cells' sensitivity to DDP. Bioinformatics analysis predicted transmembrane serine protease 4 (TMPRSS4) as a downstream target of miR-149-3p, which was subsequently confirmed. Western blot analysis was used to examine proteins related to migration, invasion, apoptosis, and TMPRSS4 expression. Additionally, a subcutaneous graft tumor model in nude mice was created to assess the impact of miR-149-3p on tumor growth. In lung cancer tissues and cells, miR-149-3p expression was reduced, while TMPRSS4 expression was elevated. Overexpression of miR-149-3p inhibited cancer progression, promoted apoptosis, and enhanced the chemosensitivity of lung cancer cells to DDP. Moreover, miR-149-3p negatively regulated TMPRSS4, reducing malignancy-associated characteristics of lung cancer cells and further improving their DDP sensitivity. In vivo, high miR-149-3p expression increased the chemosensitivity of cancer cells. In conclusion, miR-149-3p suppresses the aggressive progression of lung cancer by directly downregulating TMPRSS4 and enhances the responsiveness of lung cancer cells to DDP.

Genotypes of carboxypeptidase A1 and gamma-glutamyltransferase 1 may be useful tools for the diagnosis and the predictor of worrisome features of intraductal papillary mucinous neoplasm in Japan.

This study aimed to clarify whether several single-nucleotide polymorphisms (SNPs)-related chronic pancreatitis such as carboxypeptidase A1 (CPA1), carboxypeptidase B1 (CPB1), Gamma-glutamyltransferase 1 (GGT1), G-protein-coupled receptor Class C Group 6 Member A (GPRC6A), and serine protease inhibitor, Kazal type 1 (SPINK-1) genotypes were associated with clinical characteristics of patients with intraductal papillary mucinous neoplasm (IPMN) and worrisome features of IPMN. We enrolled 100 patients with IPMN and 116 patients as a control. Serum p-amylase, lipase, trypsin, phospholipase A2 (PLA2), and elastase-1 levels were measured. An Olympus EUS (GF-UCT 260) was used to perform endosonography in 100 patients with IPMN. Total EUS score was evaluated using endosonography. DNA was isolated from the duodenal tissue using a commercial system and polymerase chain reaction (PCR) was performed on 7500 Fast PCR System. There were no associations between glucose tolerances, lipid levels and genotypes of CPA1, GGT1, GPRC6A, and SPINK-1 in patients with IPMN. CPA1 genotype was significantly associated with the pathophysiology of IPMN. Then, GGT1 genotype was also significantly associated with EUS total score and the size of cyst more than 20 mm and more than 30 mm as one of worrisome features of IPMN. Genotypes of carboxypeptidase A1 and gamma-glutamyltransferase 1 may be useful tools for the diagnosis and the predictor of worrisome features of IPMN.

The bacterial serine protease inhibitor ecotin inhibits neutrophil elastase enzymatic activity in cystic fibrosis sputa

Cystic Fibrosis (CF) airway disease is characterized by impaired mucociliary clearance, chronic, polymicrobial infections and robust, neutrophil-dominated inflammation. Pulmonary disease is the leading cause of morbidity and mortality in people with CF and is due to progressive airflow obstruction and ultimately respiratory failure. One of the earliest abnormalities in CF airway disease is the recruitment of neutrophils to the lungs. Neutrophil activation leads to the release of their intracellular content, including neutrophil elastase (NE), that damages lung tissues in CF. Our goal is to characterize a known bacterial NE inhibitor, ecotin, in the CF airway environment. Our results indicate that ecotins cloned from four Gram-negative bacterial species (Campylobacter rectus, Campylobacter showae, Escherichia coli and Pseudomonas aeruginosa) inhibit NE activity in CF sputum samples in a dose-dependent manner. Although we observed differences in the NE-inhibitory activity of the tested ecotins with the Campylobacter homologs being the most effective in NE inhibition in CF sputa, none of the ecotins impaired the ability of human neutrophils to kill major CF respiratory pathogens, P. aeruginosa or S. aureus, in vitro. Overall, we demonstrate that bacterial ecotins inhibit NE activity in CF sputa without compromising bacterial killing by neutrophils.

Antithrombin regulates neutrophil activities through the stimulation of C-type lectin family 1A

It has been suggested that a serine proteinase inhibitor, antithrombin (AT), exerts anti-inflammatory effects on different types of cells, independent of thrombin inhibition. In the present study, we aimed to identify a specific receptor for antithrombin by a screening method using a transmembrane tethered-AT ligand expressed on HEK293T cells together with the co-expression of candidate receptors, followed by the immunoprecipitation of a complex of AT ligand with a receptor. We identified C-type lectin family 1A (CLEC1A) as a receptor for AT. We confirmed the binding of AT to the extracellular domain of CLEC1A using surface plasmon resonance. Recombinant as well as native AT concentration-dependently induced the rounding of purified human neutrophils in shape, associated with the suppression of spontaneous ROS production in vitro but argatroban did not, indicating the independence of AT effects on thrombin inhibition. Native AT maintained the passage of neutrophils through the artificial microcapillaries. Both AT also enhanced the phagocytosis of pHrodo-labeled E.coli and prolonged the viability of the neutrophils. The cellular effects of AT were similar to those of HRG which has the same CLEC1A as a receptor, and were partially inhibited by the addition of anti-CLEC1A antibody to the incubation media. These results suggested that CLEC1A is a novel receptor for AT and the stimulation of CLEC1A by AT at least in part mediates the important functional changes of human neutrophils.

Enhanced gelation of sturgeon frozen surimi via egg white: Exploring the role of endogenous serine protease inhibition

AbstractThe degradation of frozen sturgeon surimi can be attributed to the endogenous serine protease. This study was first to examine the impact of egg whites on the frozen sturgeon surimi's gel properties from the perspective of inhibiting endogenous serine protease. The protease activity of egg whites group (CA + EW group, consisting of 4% egg whites and cryoprotectants) was 45.15% lower than that of cryoprotectants group (CA group, consisting of 3% sucrose, 3% sorbitol, and 0.3% sodium tripolyphosphate) at 12 weeks. From the results of inhibition kinetics, serine protease was inhibited by both anticompetitive and noncompetitive inhibition modes. Molecular docking analysis indicated that egg white achieves this inhibitory effect through ionic interactions and hydrogen bonding with serine protease. However, this inhibitory effect was absent when the freezing period was extended to 24 weeks. Compared with CA group, the CA + EW group exhibited 54.76% and 4.59% increase in gel strength and water‐holding capacity, and 32.42% reduction in cooking loss after 24 weeks of freezing. Egg whites also impeded water migration and enhanced the density and smoothness of the gel microstructure, reducing protein aggregation. These results indicated that egg whites serve a dual function: inhibiting serine protease and filling the gel network within 12 weeks, mitigating protein aggregation and ice crystal formation over 24 weeks. This study elucidated the mechanism underlying the impact of egg white on endogenous serine protease in sturgeon surimi during long‐term freezing, laying a theoretical foundation for the industrialization of sturgeon surimi.