Serine Protease Inhibitor Research Articles

N-glycosylation in the SERPIN domain of C1-Esterase Inhibitor in hereditary angioedema.

Hereditary angioedema is an autosomal dominant disorder caused by defects in C1-esterase inhibitor (C1-INH), resulting in poorly controlled activation of the kallikrein-kinin system and bradykinin overproduction. C1-INH is a heavily glycosylated protein in the serine protease inhibitor (SERPIN) family, yet the role of these glycosylation sites remains unclear. To elucidate the functional impact of N-glycosylation in the SERPIN domain of C1-INH, we engineered four sets consisting of 26 variants at or near the N-linked sequon (NXS/T). Among these, six are reported in HAE patients and five are known C1-INH variants without accessible clinical histories. We systematically evaluated their expression, structure and functional activity with C1¯s, FXIIa and kallikrein. Our findings showed that of the eleven reported variants, seven are deleterious. Deleting N at the three naturally occurring N-linked sequons (N238, N253 and N352) results in pathologic consequences. Altering these sites by substituting N to A disrupts N-linked sugar attachment but preserves protein expression or function. Further, an additional N-linked sugar generated at N272 impairs C1-INH function. These findings highlight the importance of N-linked sequons in modulating the expression and function of C1-INH. Insights gained from identifying the pathological consequences of N-glycan variants should assist in defining more tailored therapy.

Effects of Trichinella spiralis and its serine protease inhibitors on intestinal mucosal barrier function

Trichinella spiralis (T. spiralis) is a highly pathogenic zoonotic nematode that poses significant public health risks and causes substantial economic losses. Understanding its invasion mechanisms is crucial. This study explored how the serine protease inhibitors (SPIs) secreted by T. spiralis affect the host’s intestinal epithelial barrier. Furthermore, the effects of T. spiralis infection on the jejunal barrier function in mice were investigated. The histopathological analysis indicated significant damage to the jejunum, which peaked at day 7 post-infection (dpi). The results of RT-qPCR and western blotting revealed marked reductions in tight junction proteins (ZO-1, occludin, claudin-3), mucins (MUC-1, MUC-2), and anti-inflammatory cytokines (TGF-β, IL-10) from 1 to 15 dpi. There was also increased expression of Toll-like receptors (TLR-1, TLR-2, TLR-4) and pro-inflammatory cytokines (TNF-α, IL-1β). Recombinant SPIs (rKaSPI, rAdSPI) were purified, co-cultured with intestinal epithelial cells (IPECs), and used in mouse models. The protein expression changes in IPECs and mice were consistent with those in T. spiralis-infected tissues. Both SPIs caused the down-regulation of ZO-1, occludin, claudin-3, MUC-1, MUC-2, TGF-β, and IL-10 while up-regulating TLR-4 and pro-inflammatory cytokines. As a result, the intestinal barrier was disrupted, and inflammation was exacerbated. Notably, rAdSPI had a more pronounced effect. In summary, T. spiralis infection caused significant jejunal damage and disrupted the intestinal barrier. T. spiralis-secreted SPIs, especially serpin-type serine protease inhibitors (AdSPI), were pivotal in facilitating invasion by compromising the host’s intestinal barrier and promoting inflammation. This study provides insights into T. spiralis invasion mechanisms and the potential targets for trichinellosis prevention and control.

The viral serpin SPI-1 directly inhibits the host cell serine protease FAM111A.

The host-range mutant of rabbitpox virus (RPXV) with a deletion in the gene encoding the serpin serine protease inhibitor 1 (SPI-1) fails to replicate efficiently in restrictive host cells. Depletion of the host cell serine protease FAM111A restores viral replication in these cells, suggesting that SPI-1 targets FAM111A to facilitate infection. However, direct evidence of SPI-1 inhibiting FAM111A has been lacking. Here, we demonstrate that SPI-1 directly inhibits FAM111A's protease activity in vitro through covalent complex formation, a hallmark of the serpin inhibition mechanism. SPI-1 also exhibits specificity for FAM111A compared to other serine proteases in vitro. Through mutagenesis studies, we identified residues and regions within SPI-1's reactive center loop (RCL) that are critical for FAM111A inhibition and covalent complex formation in vitro, with varying degrees of impact. Notably, these RCL mutations showed a spectrum of effects on SPI-1's ability to support RPXV replication in non-permissive cells, which strongly correlated with their impact on SPI-1's capacity to inhibit FAM111A activity in vitro. Altogether, our study provides direct evidence that SPI-1 inhibits FAM111A protease activity, highlighting FAM111A's antiviral role and its significance as a target of SPI-1 during orthopoxvirus infection.

Identification of an exosite at the neutrophil elastase/alpha-1-antitrypsin interface.

Neutrophil elastase (NE) is released by activated neutrophils during an inflammatory response and exerts proteolytic activity on elastin and other extracellular matrix components. This protease is rapidly inhibited by the plasma serine protease inhibitor alpha-1-antitrypsin (AAT), and the importance of this protective activity on lung tissue is highlighted by the development of early onset emphysema in individuals with AAT deficiency. As a serpin, AAT presents a surface-exposed reactive centre loop (RCL) whose sequence mirrors the target protease specificity. Following binding of NE in a 'Michaelis' encounter complex, cleavage of the RCL results in an irreversible complex between the two molecules. Here, the structure of the AAT-NE encounter complex was studied by molecular dynamics, mutagenesis and enzyme kinetics. Exploration of the geometry of interaction between the two molecules revealed the possibility that the interaction interface extends beyond the RCL; a persistent feature of the simulations was the interaction between a region located upstream of β-strand 4C of AAT, comprising three acidic residues (Asp202, Glu199 and Glu204), and Arg147 of NE. Mutation of the acidic residues to either alanine or serine, or a D202R substitution, resulted in a reduced rate of association between recombinant AAT and NE. Addition of salt to the buffer had little effect for these mutants but substantially reduced the rate of interaction of the wild-type protein. These data are consistent with a role for this acidic region on AAT as an exosite that contributes to an optimal interaction with its physiological protease target.

Microsporidian Nosema bombycis secretes serine protease inhibitor to suppress host cell apoptosis via Caspase BmICE.

Microsporidia are a group of intracellular pathogens that actively manipulate host cell biological processes to facilitate their intracellular niche. Apoptosis is an important defense mechanism by which host cell control intracellular pathogens. Microsporidia modulating host cell apoptosis has been reported previously, however the molecular mechanism is not yet clear. In this report, we describe that the microsporidia Nosema bombycis inhibits apoptosis of Bombyx mori cells through a secreted protein NbSPN14, which is a serine protease inhibitor (Serpin). An immunofluorescent assay demonstrated that upon infection with N. bombycis, NbSPN14 was initially found in the B. mori cell cytoplasm and then became enriched in the host cell nucleus. Overexpression and RNA-interference (RNAi) of NbSPN14 in B. mori' embryo cell confirmed that NbSPN14 inhibited host cells apoptosis. Immunofluorescent and Co-IP assays verified the co-localization and interaction of NbSPN14 with the BmICE, the Caspase 3 homolog in B. mori. Knocking out of BmICE or mutating the BmICE-interacting P1 site of NbSPN14, eliminated the localization of NbSPN14 into the host nucleus and prevented the apoptosis-inhibiting effect of NbSPN14, which also proved that the interaction between BmICE and NbSPN14 occurred in host cytoplasm and the NbSPN14 translocation into host cell nucleus is depends on BmICE. These data elucidate that N. bombycis secretory protein NbSPN14 inhibits host cell apoptosis by directly inhibiting the Caspase protease BmICE, which provides an important insight for understanding pathogen-host interactions and a potential therapeutic target for N. bombycis proliferation.

Genome Annotation.

The hallmark of genome sequencing projects is to provide genetic information on a species with functional annotations of genes and proteins. This process heavily relies on genome annotation based on homology detections from previously known genomic data. The rapid advancement of genome sequencing technologies has made genome sequencing affordable and effective in terms of the time frame for the generation of genomic data. Hence, genome sequencing has become a common practice. The annotation and characterization of newly sequenced genomes are crucial factors for the success of any biological experiment based on genomic data. The proteogenomic sector requires annotated genome further characterization of proteomic-based studies, and these are coupled with genomic and RNA-seq data. This chapter describes the genome annotation process from scratch genome sequencing to general genome annotation and specialized genome annotation using BLAST, BLAT2GO (now OMICSBOX), PANNZER, gene ontology (GO), and KEGG. It also covers different processes like repeat identification and masking, gene prediction, genome-wide annotation process, and RNA-seq protocol. It also focuses on genes of interest such as genes associated with BGCs (biosynthetic gene clusters), carbohydrate-active enzymes (CAZymes), serpins (serine protease inhibitors), membrane transporters, and toxins. Manual annotation is also a critical step for at least some groups of genes, which are often critical for the species in consideration. This chapter also briefly describes the phylogenetic and phylogenomic processes required during genome annotation.

Modeling and interaction study of alcohol oxidase and ProteaseA in methylotrophic yeast C. boidinii: insights from In-silico analysis

Flavin adenine nucleotide (FAD)-dependent oxidoreductase enzyme Alcohol oxidase (AOX) facilitates the growth of methylotrophic yeast C. boidinii by catabolizing methanol, producing formaldehyde and hydrogen peroxide. Vacuolar Protease-A (PrA) from C. boidinii is responsible for the proteolytic activity of AOX. However, no experimental structures for these enzymes have been reported. This in-silico study involves modeling and interaction analysis of AOX and PrA. A protein-protein interaction study shows that Thr75, Gly74, Arg72, Tyr73, and Met289 amino acids of PrA have shown interaction with AOX. These residues may be crucial for AOX proteolysis. An in-silico study predicts that serine protease inhibitors bind to specific amino acids, potentially obstructing PrA’s degradable activity on AOX. PrA does not interact with the FAD binding sites in AOX. Instead, it interacts with AOX at sites (Ser337, Ala34, and Tyr343) where AOX monomers interact, hindering octamer formation the active form of AOX. During simulation, strong dynamics in PrA were found in the loop regions of the structure, as observed in the complexes. This in-silico work aims to corroborate the experimental research, which lacks structural studies on the proteolysis process.

Integrative multi-omics analysis reveals the contribution of neoVTX genes to venom diversity of Synanceia verrucosa

BackgroundAnimal venom systems are considered as valuable model for investigating the molecular mechanisms underlying phenotypic evolution. Stonefish are the most venomous and dangerous fish because of severe human envenomation and occasionally fatalities, whereas the genomic background of their venom has not been fully explored compared with that in other venomous animals.ResultsIn this study, we followed modern venomic pipelines to decode the Synanceia verrucosa venom components. A catalog of 478 toxin genes was annotated based on our assembled chromosome-level genome. Integrative analysis of the high-quality genome, the transcriptome of the venom gland, and the proteome of crude venom revealed mechanisms underlying the venom complexity in S. verrucosa. Six tandem-duplicated neoVTX subunit genes were identified as the major source for the neoVTX protein production. Further isoform sequencing revealed massive alternative splicing events with a total of 411 isoforms demonstrated by the six genes, which further contributed to the venom diversity. We then characterized 12 dominantly expressed toxin genes in the venom gland, and 11 of which were evidenced to produce the venom protein components, with the neoVTX proteins as the most abundant. Other major venom proteins included a presumed CRVP, Kuntiz-type serine protease inhibitor, calglandulin protein, and hyaluronidase. Besides, a few of highly abundant non-toxin proteins were also characterized and they were hypothesized to function in housekeeping or hemostasis maintaining roles in the venom gland. Notably, gastrotropin like non-toxin proteins were the second highest abundant proteins in the venom, which have not been reported in other venomous animals and contribute to the unique venom properties of S. verrucosa.ConclusionsThe results identified the major venom composition of S. verrucosa, and highlighted the contribution of neoVTX genes to the diversity of venom composition through tandem-duplication and alternative splicing. The diverse neoVTX proteins in the venom as lethal particles are important for understanding the adaptive evolution of S. verrucosa. Further functional studies are encouraged to exploit the venom components of S. verrucosa for pharmaceutical innovation.

Regulation of epidermal barrier function and pathogenesis of psoriasis by serine protease inhibitors.

Serine protease inhibitors (Serpins) are a protein superfamily of protease inhibitors that are thought to play a role in the regulation of inflammation, immunity, tumorigenesis, coagulation, blood pressure and cancer metastasis. Serpins is enriched in the skin and play a vital role in modulating the epidermal barrier and maintaining skin homeostasis. Psoriasis is a chronic inflammatory immune-mediated skin disease. At present, most serpins focus on the pathogenesis of psoriasis vulgaris. Only a small number, such as the mutation of SerpinA1/A3/B3, are involved in the pathogenesis of GPP. SerpinA12 and SerpinG1 are significantly elevated in the serum of patients with psoriatic arthritis, but their specific mechanism of action in psoriatic arthritis has not been reported. Some Serpins, including SerpinA12, SerpinB2/B3/B7, play multiple roles in skin barrier function and pathogenesis of psoriasis. The decrease in the expression of SerpinA12, SerpinB7 deficiency and increase in expression of SerpinB3/4 in the skin can promote inflammation and poor differentiation of keratinocyte, with damaged skin barrier. Pso p27, derived from SerpinB3/B4, is an autoantigen that can enhance immune response in psoriasis. SerpinB2 plays a role in maintaining epidermal barrier integrity and inhibiting keratinocyte proliferation. Here we briefly introduce the structure, functional characteristics, expression and distribution of serpins in skin and focus on the regulation of serpins in the epidermal barrier function and the pathogenic role of serpins in psoriasis.

Clinical practice guidelines for management of disseminated intravascular coagulation in Japan 2024. Part 1: sepsis.

The Japanese Society on Thrombosis and Hemostasis (JSTH) published the first-ever disseminated intravascular coagulation (DIC) guidelines in 2009. Fifteen years later, the JSTH developed new guidelines covering DIC associated with various underlying conditions. These guidelines were developed in accordance with the GRADE system to determine the strength of the recommendations and certainty of the evidence. This article was drafted as Part 1 of an overall DIC guideline covering various underlying conditions, with sepsis as the subject. In this section, seven key clinical issues (questions) are set. Question 1, regarding DIC diagnosis, introduces several diagnostic criteria, such as the JAAM-2, ISTH overt, SIC, and JSTH DIC criteria and recommends choosing the appropriate diagnostic criteria for DIC based on an understanding of their diagnostic properties. For pharmacotherapy in DIC patients with sepsis, we recommend the administration of antithrombin (Question 2) and recombinant thrombomodulin (Question 3) (both GRADE 1B). However, we do not make a clear recommendation regarding the administration of heparin (Question 6) and serine protease inhibitors (Question 7) because of the lack of evidence. Combination therapy, order of administration, and other administration methods for antithrombin and recombinant thrombomodulin are proposed as important future research questions (Questions 4 and 5).

Clinical practice guidelines for management of disseminated intravascular coagulation in Japan 2024. Part 2: hematologic malignancy.

Disseminated intravascular coagulation (DIC) associated with hematologic malignancies, particularly acute promyelocytic leukemia (APL), is characterized by marked fibrinolytic activation, which leads to severe bleeding complications. Therefore, appropriate diagnosis and management of DIC are crucial for preventing bleeding-related mortality. However, to date, no clinical guidelines have specifically addressed hematologic malignancy-associated DIC. Therefore, we developed diagnostic and management algorithms for DIC based on a systematic literature review. Notably, these guidelines recommend using the JSTH DIC diagnostic criteria (2017 version) or the former Ministry of Health and Welfare DIC diagnostic criteria (1983 version) to diagnose DIC. Furthermore, in the management of DIC, it is essential to treat the underlying disease through transfusion of platelet concentrates and fresh frozen plasma, if necessary. A systematic review of antifibrinolytic and anticoagulant therapies concluded that tranexamic acid therapy is not strongly recommended for patients with APL undergoing treatment with all-trans retinoic acid (Grade 1C). The use of recombinant thrombomodulin is weakly recommended (Grade 2B), whereas the use of other anticoagulants, including heparin and serine protease inhibitors, is weakly not recommended (Grade 2C). Therefore, we hope that these guidelines will help physicians find the best possible solutions in clinical practice.

Heparin Resistance in Patients Receiving Extracorporeal Membrane Oxygenation: A Review.

Heparin resistance (HR) in patients on extracorporeal membrane oxygenation (ECMO) exacerbates bleeding and thrombogenesis. Thus far, there is no universal definition of what this condition entails and no unified strategy for assessing heparin's efficacy in ECMO patients. The most frequent discrepancy when it comes to defining HR is the difference in the reported doses: units per day (U/d) or per kilogram per hour (U/kg/h). Another disagreement arises with regard to the various methods of measuring unfractionated heparin (UFH) efficacy. Due to numerous processes that begin with ECMO initiation, including protein layer formation on the surface of circuits, the recruitment of immune cells, the activation of complement and contact activation systems, and platelets, assessing pure antithrombin consumption is complicated. Moreover, there is an alternative anticoagulation procedure performed by a serine protease inhibitor named heparin cofactor II, which could also contribute to heparin consumption. Considering simultaneously launched processes of inflammation and thrombogenesis in response to contact with artificial surfaces on ECMO, we listed the possible mechanisms contributing to additional antithrombin consumption. The effect of the flow on the platelets' activation and von Willebrand factor (vWF) assembly was also described. We reviewed the scientific literature from PubMed and Embase to identify possible definitions of heparin resistance during ECMO treatment among pediatric and adult cohorts. We identified 13 records describing different approaches to assessing HR and described our vision of delineating HR on ECMO.

Vaspin inhibits ferroptosis: A new hope for treating myocardial ischemia–reperfusion injury

Objective: Myocardial ischemia–reperfusion injury (MIRI) is a critical pathological basis for cardiovascular diseases. In recent years, the effect of ferroptosis on MIRI has attracted extensive attention. Vaspin, an adipose tissue-derived serine protease inhibitor, has multiple biological functions, including anti-inflammatory and antioxidant effects. This study aims to investigate the molecular mechanism by which vaspin alleviates MIRI by regulating hypoxia-inducible factor-1α (HIF-1α) and ferroptosis signaling pathways. Material and Methods: A mouse model of myocardial ischemia/reperfusion (I/R) and a hypoxia/reoxygenation (H/R) model was used to evaluate the protective effects of vaspin on MIRI. The mechanism by which ferroptosis is modulated by the vaspin/HIF-1α signaling pathway was investigated by constructing a vaspin overexpression adenoviral vector. Myocardial infarct size and histological changes were assessed using triphenyltetrazolium chloride and hematoxylin–eosin staining. Ferroptosis-related proteins were detected by Western blot assay, and apoptosis and reactive oxygen species levels were analyzed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. Iron content in myocardial tissue and cells was measured by enzyme-linked immunosorbent assay. Results: Myocardial I/R increased myocardial infarct size and serum lactate dehydrogenase (LDH) levels compared with the control group, indicating severe myocardial injury. Western blot results showed that MIRI reduced endogenous vaspin and HIF-1α levels and inhibited glutathione peroxidase 4. In vivo and in vitro vaspin overexpression treatment reduced infarct size, decreased LDH levels, inhibited ferroptosis pathway activity, and alleviated oxidative stress levels in myocardial tissues. In the H/R model, vaspin overexpression upregulated HIF-1α, inhibited ferroptosis markers, and reduced apoptosis and iron deposition. However, inhibiting HIF-1α reversed the cardioprotective and anti-ferroptotic effects of vaspin. Conclusion: Vaspin inhibits ferroptosis and upregulates the HIF-1α signaling pathway to mitigate myocardial I/R injury. The vaspin/HIF-1α pathway could be a potential target for MIRI prevention and treatment and offers fresh perspectives on ischemic heart disease management. Vaspin could be a novel cardioprotective agent that plays a significant role in the prevention and treatment of cardiovascular diseases.

Infrared Spectral Patterns of Thyroglobulin Bearing Thyroiditogenic Epitopes.

Thyroglobulin is a major autoantigen to which autoimmune response, destroying the thyroid gland in Hashimoto's thyroiditis, is directed. To detect a pathological autoimmune response to thyroglobulin, as well as the successful induction of experimental autoimmune thyroiditis, thyroglobulin carrying thyroiditogenic epitopes is necessary. It is not known which features of thyroglobulin structure determine the presence of thyroiditogenic epitopes and can serve as markers of their presence. We compared structure of thyroglobulin bearing thyroiditogenic epitopes (freshly isolated thyroglobulin) and thyroglobulin which had lost thyroiditogenic epitopes (lyophilized thyroglobulin). Fourier-transform infrared (FTIR) spectroscopy was used to elucidate the structure of thyroglobulin. The markers indicating the presence of thyroiditogenic epitopes on thyroglobulin are the vibrations of diiodotyrosine, monoiodotyrosine/diiodotyrosine relation in the range of 0.24-0.43 (95% confidence interval) and relatively high (> 32%) α-helix content. The loss of thyroiditogenic epitopes on thyroglobulin is associated with a weakening or complete disappearance of diiodotyrosine oscillations and a decrease in the proportion of α-helices in secondary structure. Thyroglobulin extracted with phenylmethylsulfonyl fluoride (PMSF) added is characterized by the same relatively high monoiodotyrosine/diiodotyrosine relation and low proportion of alpha helices as thyroglobulin without thyroiditogenic epitopes. Therefore, serine protease inhibitor PMSF is not suitable for extraction of native thyroglobulin bearing thyroiditogenic epitopes. FTIR spectroscopy can be used to detect thyroiditogenic epitopes on thyroglobulin.

Intracellular autoactivation and surface location of hepsin, TMPRSS2, and TMPRSS13

AimsHepsin (HPN), a Type II transmembrane serine protease (TTSP), is involved in hepatocyte metabolism and various diseases. It undergoes autoactivation on the surface of human hepatoma cells, a mechanism not observed in other cell types. This study aims to explore HPN activation and surface expression in endometrial epithelial cells. Materials and methodsWe studied HPN zymogen activation and cell surface expression in human embryonic kidney 293 and endometrial epithelial AN3CA and Ishikawa cells using site-directed mutagenesis, Western blotting, flow cytometry, and immunostaining. Treatments with brefeldin A (BFA) and monensin, along with co-transfection assays, were employed to assess HPN activation and expression before reaching the cell surface. We also analyzed the activation and expression of TMPRSS2 and TMPRSS13 and examined the effect of the serine protease inhibitor HAI-1 on these proteases. Key findingsHPN zymogen autoactivates in the endoplasmic reticulum (ER) and Golgi apparatus. Its active form reduces cell surface expression through trans-autodegradation, a mechanism also applicable to in TMPRSS2 and TMPRSS13. Additionally, HAI-1 interacts with these TTSPs in different ways: it inhibits HPN activation and stabilizes its cell-surface expression; it inhibits TMPRSS2 activation without affecting its cell-surface expression; and it facilitates TMPRSS13 activation, protecting it from degradation and stabilizing its cell surface expression. SignificanceThese results revealed an intracellular autoactivation and expression mechanism of HPN, TMPRSS2, and TMPRSS13, differing from the extracellular activated TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation, potentially aiding in treating TTSP-related endometrial diseases.

Identification of two point mutations associated with inherited antithrombin deficiency

BackgroundAntithrombin (AT) is a serine protease inhibitor which exerts its anticoagulant effect through binding to serine residues in the active centers of procoagulant serine proteases. Its deficiency is associated with increased risk of venous thrombosis. We aim to investigate the pathogenic mechanism of two natural mutants (W221C and M284R) in inherited AT deficiency.MethodsWe analyzed 9 unrelated patients with inherited AT deficiency by extracting peripheral blood DNA and sequencing the SERPINC1 gene after amplification by polymerase chain reaction. Enzyme-linked immunosorbent assay and heparin affinity chromatography were used to assess AT secretion and purification efficiency. The mutant AT models were evaluated via computational simulations.ResultsAmong the 9 patients with inherited AT deficiency, 8 patients had type I AT deficiency, and one patient had type II AT deficiency with subtype of reactive site mutation. Seven of them experienced venous thrombotic events and all patients were found genetic mutations including missense (n = 6), deletion (n = 2) and insertion (n = 1). Two point mutations, W221C and M284R, were identified and were hypothesized to affect AT by destabilizing the central β-sheet. Based on immunoassays and heparin purification, the W221C mutant may impair AT secretion, whereas M284R mutant decreased the total AT production (696.8 ± 151.6 ng/ml versus 3833.72 ± 315.4 ng/ml, p = 0.029). Both mutants delayed the peak of AT release in heparin affinity chromatography.ConclusionsOur study demonstrates that two mutations in SERPINC1 gene altered the production and structure of AT by in vitro protein expression and functional studies, including protein secretion and production. These findings enhance our understanding of the genetic basis of AT deficiency and its possible clinical implications.

Spint1 disruption in mouse pancreas leads to glucose intolerance and impaired insulin production involving HEPSIN/MAFA

SPINT1, a membrane-anchored serine protease inhibitor, regulates cascades of pericellular proteolysis while its tissue-specific functions remain incompletely characterized. In this study, we generate Spint1-lacZ knock-in mice and observe Spint1 expression in embryonic pancreatic epithelium. Pancreas-specific Spint1 disruption significantly diminishes islet size and mass, causing glucose intolerance and downregulation of MAFA and insulin. Mechanistically, the serine protease HEPSIN interacts with SPINT1 in β cells, and Hepsin silencing counteracts the downregulation of Mafa and Ins1 caused by Spint1 depletion. Furthermore, we demonstrate a potential interaction between HEPSIN and GLP1R in β cells. Spint1 silencing or Hepsin overexpression reduces GLP1R-related cyclic AMP levels and Mafa expression. Spint1-disrupted mice also exhibit a significant reduction in Exendin-4-induced insulin secretion. Moreover, SPINT1 expression increases in islets of prediabetic humans compared to non-prediabetic groups. The results unveil a role for SPINT1 in β cells, modulating glucose homeostasis and insulin production via HEPSIN/MAFA signaling.

Sea Anemone Kunitz Peptide HCIQ2c1: Structure, Modulation of TRPA1 Channel, and Suppression of Nociceptive Reaction In Vivo.

TRPA1 is a homotetrameric non-selective calcium-permeable channel. It contributes to chemical and temperature sensitivity, acute pain sensation, and development of inflammation. HCIQ2c1 is a peptide from the sea anemone Heteractis magnifica that inhibits serine proteases. Here, we showed that HCIQ2c1 significantly reduces AITC- and capsaicin-induced pain and inflammation in mice. Electrophysiology recordings in Xenopus oocytes expressing rat TRPA1 channel revealed that HCIQ2c1 binds to open TRPA1 and prevents its transition to closed and inhibitor-insensitive 'hyperactivated' states. NMR study of the 15N-labeled recombinant HCIQ2c1 analog described a classical Kunitz-type structure and revealed two dynamic hot-spots (loops responsible for protease binding and regions near the N- and C-termini) that exhibit simultaneous mobility on two timescales (ps-ns and μs-ms). In modelled HCIQ2c1/TRPA1 complex, the peptide interacts simultaneously with one voltage-sensing-like domain and two pore domain fragments from different channel's subunits, and with lipid molecules. The model explains stabilization of the channel in the open conformation and the restriction of 'hyperactivation', which are probably responsible for the observed analgetic activity. HCIQ2c1 is the third peptide ligand of TRPA1 from sea anemones and the first Kunitz-type ligand of this channel. HCIQ2c1 is a prototype of efficient analgesic and anti-inflammatory drugs.

Prognostic and diagnostic value of SPINK mRNAs expression in head and neck squamous cell carcinoma based on genome-wide analysis

Aim: Head and neck squamous cell carcinoma (HNSC) is a major contributor to the global cancer burden. The serine protease inhibitor Kazal-type (SPINK) gene family has been linked to various cancers. This study explores the prognostic value of SPINK genes in predicting overall survival (OS) in HNSC patients. Methods: We analyzed RNA sequencing and clinical data from 504 cancer and 44 non-cancer samples from the TCGA database. Differential expression and functional enrichment analyses gene ontology and Kyoto encyclopedia of genes and genomes (GO and KEGG) were performed using clusterProfiler. Protein-protein interaction (PPI) networks were built with STRING and visualized. Immune infiltration was evaluated using single-sample Gene Set Enrichment Analysis (ssGSEA). Survival analysis utilized Kaplan-Meier curves and Cox regression models. Results: Our results showed that SPINK5, SPINK7, SPINK8, SPINK9, and SPINK14 were significantly overexpressed in normal tissues compared to carcinoma tissues, whereas SPINK1, SPINK4, and SPINK6 showed higher expression in carcinoma tissues. Correlation analysis revealed significant relationships among SPINK family members. GO and KEGG analyses highlighted their involvement in processes such as negative regulation of peptidase activity and serine-type endopeptidase inhibitor activity. PPI network analysis indicated close interactions between several SPINK proteins and other relevant proteins. Immune infiltration analysis showed that NK cells and Th2 cells were negatively correlated with SPINK genes, while mast cells and neutrophils were positively correlated. Survival analysis revealed that high mRNA expression levels of SPINK1, SPINK5, and SPINK6 were significantly associated with OS in HNSC patients. Receiver operating characteristic (ROC) curve analysis indicated that these genes have diagnostic value. We developed a nomogram model that combines tumor stage and SPINK gene expression providing a predictive tool for patient prognosis. Conclusions: This study elucidates the multifaceted roles of the SPINK gene family in HNSC. These findings offer valuable insights into their potential as diagnostic biomarkers and therapeutic targets.

Serine protease inhibitor AEBSF(4-(2-aminoethyl)-benzenesulfonyl fluoride) decreased ischemic brain injury through inhibiting endoplasmic reticulum stress, oxidative stress, and autophagy in rats

4-(2-Aminoethyl)-benzenesulfonyl fluoride (AEBSF) is a serine protease inhibitor that may alleviate endoplasmic reticulum (ER) stress, a significant contributing factor to cerebral ischemia/reperfusion injury. The molecular crosstalk between ER stress, oxidative stress and autophagy represents a vicious cycle that can be pharmacologically targeted to minimize neuronal death after acute injuries to the central nervous system. However, the neuroprotective effects of AEBSF in the context of cerebral ischemia/reperfusion injury remain unknown. In this study,we reported the neuroprotective effect of AEBSF against cerebral ischemia/reperfusion injury and explored the mechanisms involved, particularly its role in reducing ER stress, oxidative stress and autophagy. Rats were pretreated with AEBSF or a vehicle before a 90-minute middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion. Our results demonstrate that AEBSF treatment reduced infarct volume and improved neurological function compared to vehicle treated rats after 24 h of reperfusion. Furthermore,AEBSF treatment decreased the expression of caspase-3, suggesting a decrease in neuronal apoptosis. Additionally, AEBSF treatment lowered levels of key ER stress biomarkers, including glucose-regulated protein 78 (GRP78), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), and CCAAT-enhancer-binding protein homologous protein (CHOP), while the levels of inositol-requiring enzyme 1α (IRE1α) remained unchanged. AEBSF also decreased the oxidative stress biomarker neuronal nitric oxide synthase (nNOS) and its related molecule pro-MMP-9. Importantly, treatment with AEBSF reversed the trends of autophagy biomarker LC3B II/α-tubulin, Beclin1, and SQSTM1 at 24 h after reperfusion. In conclusion, AEBSF significantly mitigates ischemic brain damage and promotes neurological recovery by inhibiting ER stress, oxidative stress, and autophagy, highlighting its potential as a therapeutic option for ischemic stroke.