Serine Protease Inhibitor Family Research Articles

Glial-Cell-Line-Derived Neurotrophic Factor Promotes Glioblastoma Cell Migration and Invasion via the SMAD2/3-SERPINE1-Signaling Axis.

Glial-cell-line-derived neurotrophic factor (GDNF) is highly expressed and is involved in the malignant phenotype in glioblastomas (GBMs). However, uncovering its underlying mechanism for promoting GBM progression is still a challenging work. In this study, we found that serine protease inhibitor family E member 1 (SERPINE1) was a potential downstream gene of GDNF. Further experiments confirmed that SERPINE1 was highly expressed in GBM tissues and cells, and its levels of expression and secretion were enhanced by exogenous GDNF. SERPINE1 knockdown inhibited the migration and invasion of GBM cells promoted by GDNF. Mechanistically, GDNF increased SERPINE1 by promoting the phosphorylation of SMAD2/3. In vivo experiments demonstrated that GDNF facilitated GBM growth and the expressions of proteins related to migration and invasion via SERPINE1. Collectively, our findings revealed that GDNF upregulated SERPINE1 via the SMAD2/3-signaling pathway, thereby accelerating GBM cell migration and invasion. The present work presents a new mechanism of GDNF, supporting GBM development.

Assessment of Serum Insulin and VASPIN Levels Among Type 2 Diabetes Mellitus Patients with or Without Obesity: A Cross-sectional Analytical Study.

A collection of diverse conditions together referred to as diabetes mellitus frequently manifest as periods of hyperglycemia and glucose intolerance, which can be caused by insufficient insulin, improper insulin action, or both. A medical condition known as obesity is the accumulation of excess body fat to the point that it may be harmful to one's health. It has been determined that the adipokine vaspin (visceral adipose tissue-derived serpin A12) belongs to the family of serine protease inhibitors. Insulin, a polypeptide hormone, is released by the pancreatic beta cells. This hormone is both anti-catabolic and anabolic. Insulin decreases blood glucose levels by preventing its synthesis and encouraging its storage and use. In total, 125 male and female participants of various ages participated in the current study. They included 25 healthy controls, 50 non-obese and 50 obese non-insulin-dependent diabetes mellitus patients, who visited the MDM Hospital's outpatient clinic in Jodhpur, Rajasthan. Commercially available reagents and kits were utilized for the analysis of serum insulin and vaspin samples. Standard statistical tools were utilized to evaluate the parameters. When results were compared with healthy participants and non-obese NIDDM subjects, obese NIDDM subjects showed statistically significant higher fasting serum insulin and serum vaspin levels (P < 0.0001). Our study's conclusions demonstrated a strong correlation between blood insulin and vaspin levels and diabetes and obesity. Vaspin has a positive impact on insulin resistance, obesity, type 2 diabetes, and metabolic syndrome. It also enhances glucose tolerance and insulin sensitivity, reducing diabetes complications. This novel biomarker has the potential to enhance diabetes mellitus outcomes by facilitating prompt diagnosis, improved management, and reduction of complications.

Clinical outcomes of antithrombin III supplementation in an overt disseminated intravascular coagulation: a longitudinal single-institutional experience and retrospective analysis.

Antithrombin is a small plasma glycoprotein synthesized in the liver that belongs to the serpin family of serine protease inhibitors and inactivates several enzymes in the coagulation pathway. It plays a leading major factor on coagulation pathway, therefore administration of antithrombin is essential to treat serious clinical conditions such as disseminated intravascular coagulation (DIC). Despite the theoretical benefits of antithrombin supplementation, the optimal antithrombin activity for heparin efficacy and the benefits of antithrombin supplementation in various disease entities are not yet fully understood. The strict administration guidelines on antithrombin III in cases of DIC by the National Health Insurance Service and the Ministry of Food and Drug Safety complied as follows: antithrombin levels below 20 mg/dL in adults; antithrombin activity below 70% of normal in adults; total administration period of antithrombin must be carefully limited to within maximum 3 days, and the total administration dose must be below 7,000 international unit (IU), (loading dose, 1,000 IU in 1 hour: maintenance dose, 500 IU every 6 hours for 3 days). We identified 76 eligible for analysis according to the above-mentioned criteria in our institution (male/female, 59/17). Forty-four were identified to the non-survivor group and 32 patients were recognized as the survivor group. The baseline parameters in the non-survivor and survivor groups were comparable with no significant differences in age (66.5±18.1 vs. 66.0±16.2 years, P=0.90), sex (32/12 vs. 27/5, P=0.35), hospital length of stay (31.1±34.5 vs. 31.2±26.1 days, P=0.99), sequential organ failure assessment (SOFA) (7.3±2.5 vs. 6.6±2.0, P=0.22), simplified acute physiology score II (SAPS II) (46.0±8.8 vs. 43.5±9.2, P=0.23), cause for DIC (P=0.95), and underlying disease (P=0.38). The levels of antithrombin III on the day just before the administration significantly lower in the non-survivor groups than in the survivor groups (50.1%±13.6% vs. 57.6%±12.5%, P=0.01). The hemoglobin level in the 2nd day and 7th day after antithrombin III administration was significantly different between the non-survivor and survivor groups (9.9±1.9 vs. 11.0±2.0 g/dL, P=0.01, and 9.4±1.8 vs. 10.5±1.6 g/dL, P=0.006). The antithrombin III levels on the day of administration [area under the curve (AUC) =0.672] demonstrated significantly better prediction of mortality than the A antithrombin III levels on 1st day (AUC =0.552), the 2nd day (AUC =0.624), and 7th day (AUC =0.593). Our study suggests that the antithrombin administration may be effective tools for DIC treatment, and may be more positively considered, especially in the cases of DIC, which is a frequent complication of septic shock, sepsis, and other critical disease entities and which is associated with a high level of mortality. Furthermore, our study also suggests that the total doses and periods of antithrombin administration, which recommended by national guidelines, may be insufficient, therefore prolongation of period and increase of total dose of antithrombin supplement might be necessary.

Synergy between PEDF and Doxorubicin in Breast Cancer Cells: Effects on Metastatic and Metabolic Pathways.

Pigment epithelium-derived factor (PEDF), a serine protease inhibitor (Serpin) family member, shows promise in inhibiting tumour growth. In our study, we explored the effects of PEDF on the efficacy of the frontline chemotherapy agent doxorubicin (Dox) in BC cells. We found that Dox+PEDF treatment significantly reduced glucose uptake in MDA-MB-231 cells compared to the control (p = 0.0005), PEDF (p = 0.0137), and Dox (p = 0.0171) alone but paradoxically increased it in MCF-7 cells. Our findings further revealed that PEDF, Dox, and Dox+PEDF substantially hindered tumour cell migration from tumour spheroids, with Dox+PEDF showing the most significant impact (p < 0.0001). We also observed notable decreases in the expression of metastatic markers (uPAR, uPA, CXCR4, MT1-MMP, TNF-α) across all treatment groups (p < 0.0001) in both cell lines. When it comes to metabolic pathways, PEDF increased phosphorylated IRS-1 (p-IRS1) levels in MDA-MB-231 and MCF-7 (p < 0.0001), while Dox decreased it, and the combination led to an increase. In MDA-MB-231 cells, treatment with PEDF, Dox, and the combination led to a notable decrease in both phosphorylated AKT (p-AKT) and total AKT levels. In MCF-7, while PEDF, Dox, and their combination led to a reduction in p-AKT, total levels of AKT increased in the presence of Dox and Dox+PEDF. Combining PEDF with Dox enhances the targeting of metastatic and metabolic pathways in breast cancer cell lines. This synergy, marked by PEDF's increasing roles in cancer control, may pave the way for more effective cancer treatments.

Alpha 1 antitrypsin deficiency - etiology, symptoms in various organs, diagnosis, treatment, prognosis

Introduction: Alpha-1 antitrypsin (AAT) is a glycoprotein produced by liver, belonging to the serine protease inhibitor family. Alpha-1 antitrypsin deficiency (AATD) is very common autosomal recessive genetic disease caused by point mutation in SERPINA1 gene. Mutations in the alpha-1 antitrypsin gene lead to production of misfolded AAT resulting in impaired release into the blood. This disorder leads to destruction of connecting tissue especially in lungs and to accumulation of retarded protein in the liver. Purpose: Most studies addressing AAT deficiency focus on presenting symptoms related to the lungs and liver. We want to take a broader look at this issue, so we have closely examined scientific reports on the presentation of the disease in organs other than the lungs and liver. The goal is to gather holistic knowledge about the disease to enhance awareness and treatment. Material and methods: In our paper, we endeavored to address the issue of AAT deficiency comprehensively. We explored symptoms with an emphasis on organs beyond the liver and lungs. We also delved into the etiology, diagnosis, treatment, and prognosis of this disease. Discussion: The clinical symptoms of alpha 1 antitrypsin deficiency extending beyond the liver and lungs remain inadequately described. We know that AAT deficiency can lead to excessive destruction of connective tissue in any organ, not just the lungs and liver. Unfortunately, this condition continues to go undiagnosed, and the number of scientific publications on symptoms from other organs is too limited. This affects the insufficient attention given by doctors to tissue destruction in organs other than the lungs and liver.

The versatile role of Serpina3c in physiological and pathological processes: a review of recent studies.

Murine Serpina3c belongs to the family of serine protease inhibitors (Serpins), clade "A" and its human homologue is SerpinA3. Serpina3c is involved in some physiological processes, including insulin secretion and adipogenesis. In the pathophysiological process, the deletion of Serpina3c leads to more severe metabolic disorders, such as aggravated non-alcoholic fatty liver disease (NAFLD), insulin resistance and obesity. In addition, Serpina3c can improve atherosclerosis and regulate cardiac remodeling after myocardial infarction. Many of these processes are directly or indirectly mediated by its inhibition of serine protease activity. Although its function has not been fully revealed, recent studies have shown its potential research value. Here, we aimed to summarize recent studies to provide a clearer view of the biological roles and the underlying mechanisms of Serpina3c.

Kallistatin as an inhibitory protein against colorectal cancer cells through binding to LRP6

Kallistatin (KL) is a member of the serine proteinase inhibitor (serpin) family regulating oxidative stress, vascular relaxation, inflammation, angiogenesis, cell proliferation, and invasion. The heparin-binding site of Kallistatin has an important role in the interaction with LRP6 leading to the blockade of the Wnt signaling pathway. In this study, we aimed to explore the structural basis of the Kallistatin-LRP6E1E4 complex using in silico approaches and evaluating the anti-proliferative, apoptotic, and cell cycle arrest activities of Kallistatin in colon cancer lines. The molecular docking showed Kallistatin could bind to the LRP6E3E4 much stronger than LRP6E1E2. The Kallistatin-LRP6E1E2 and Kallistatin-LRP6E3E4 complexes were stable during Molecular Dynamics (MD) simulation. The Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) showed that the Kallistatin-LRP6E3E4 has a higher binding affinity compared to Kallistatin-LRP6E1E2. Kallistatin induced higher cytotoxicity and apoptosis in HCT116 compared to the SW480 cell line. This protein-induced cell-cycle arrest in both cell lines at the G1 phase. The B-catenin, cyclin D1, and c-Myc expression levels were decreased in response to treatment with Kallistatin in both cell lines while the LRP6 expression level was decreased in the HCT116 cell line. Kallistatin has a greater effect on the HCT116 cell line compared to the SW480 cell line. Kallistatin can be used as a cytotoxic and apoptotic-inducing agent in colorectal cancer cell lines.

Characterization of antithrombin isoforms and latent forms by ion exchange chromatography coupled to mass spectrometry

Antithrombin is a key protein of the coagulation system belonging to the serine protease inhibitor family. Antithrombin preparations are used as a therapeutic treatment for patients with decreased antithrombin activity. Elucidating the structural features of this protein is an important part of the control strategy to assure a high quality. This study presents an ion exchange chromatographic method coupled to mass spectrometry capable of characterizing antithrombin post-translational modifications such as N-glycosylation, phosphorylation or deamidation. Furthermore, the method was successfully used to evidence irreversible/inactive conformers of antithrombin which are commonly observed for serine protease inhibitors and referred to as latent forms.

Metabolomics Profiling Reveals the Role of PEDF in Triple-Negative Breast Cancer Cell MDA-MB-231 under Glycaemic Loading

Pigment epithelium-derived factor (PEDF) is a secreted glycoprotein that belongs to the serine protease inhibitor (serpin) family. An increase in PEDF activity has been shown to be a potent inhibitor of tumour progression and proliferation, suggesting a possible therapeutic target. There is still a great deal to learn about how PEDF controls metabolic pathways in breast cancer and its metastatic form. Given this, the primary purpose of this study was to use a metabolomics approach to gain a better understanding of the mechanisms driving the reprogramming of metabolic events involved in breast cancer pertaining to PEDF under various glycaemic loads. We employed gas chromatography-quadrupole mass spectrometry (GC-Q-MS) to investigate metabolic changes in the triple-negative breast cancer (TNBC) cell line MDA-MB-231 treated with PEDF under glycaemic loading. Multivariate and univariate analyses were carried out as indicative tools via MetaboAnalyst (V.5.0) and R packages to identify the significantly altered metabolites in the MDA-MB-231 cell line after PEDF exposure under glycaemic loading. A total of 61 metabolites were found, of which nine were selected to be distinctively expressed in MDA-MB-231 cells under glycaemic conditions and exhibited differential responses to PEDF (p < 0.05, VIP > 1). Abnormalities in amino acid metabolism pathways were observed. In particular, glutamic acid, glutamine, and phenylalanine showed different levels of expression across different treatment groups. The lactate and glucose-6-phosphate production significantly increased in high-glucose vs. normal conditions while it decreased when the cells were exposed to PEDF, confirming the positive influence on the Warburg effect. The TCA cycle intermediates, including malate and citric acid, showed different patterns of expression. This is an important finding in understanding the link of PEDF with metabolic perturbation in TNBC cells in response to glycaemic conditions. Our findings suggest that PEDF significantly influenced the Warburg effect (as evidenced by the significantly lower levels of lactate), one of the well-known metabolic reprogramming pathways in cancer cells that may be responsive to metabolic-targeted therapeutic strategies. Moreover, our results demonstrated that GC-MS-based metabolomics is an effective tool for identifying metabolic changes in breast cancer cells after glycaemic stress or in response to PEDF treatment.

Computational modeling and molecular mapping of serine protease inhibitor family A5 (SERPINA5) structure, associated with tau expression and Alzheimer's disease.

Serine Protease Inhibitor Family AS (SERPINA5), also known as PCI (Protein C Inhibitor) or PAl3 (plasminogen activator inhibitor 3), is a member of the serine proteinase inhibitor family. Our previous studies have shown that increased expression of the SERPINA5 gene is associated with tau expression in the brain and Alzheimer's disease. We hypothesize selective targeting of SERPINA5 has potential as a therapy for Alzheimer's disease. Since the full-length 3D human SERPINA5 structure is still unknown, we performed computational modeling in silico using Schrodinger Suite 2022-2 (Prime software), AlphaFold 2 and OpenFold (trainable PyTorch reproduction of DeepMind's AlphaFold). Molecular mapping was used to identify binding hot spots, which are regions of the protein surface that can bind small molecules or drug fragments and was calculated by ATLAS (FTMap) software. The best structures with different conformational states were selected and molecular mapping was performed for each of them. The druggability hot spots were determined, then ranked according to their ability to bind small molecules and peptides on the SERPINA5 protein surface. These results will be used in molecular docking, molecular dynamics simulations and biological testing research.

Hypoxia-induced ROS aggravate tumor progression through HIF-1α-SERPINE1 signaling in glioblastoma.

Hypoxia, as an important hallmark of the tumor microenvironment, is a major cause of oxidative stress and plays a central role in various malignant tumors, including glioblastoma. Elevated reactive oxygen species (ROS) in a hypoxic microenvironment promote glioblastoma progression; however, the underlying mechanism has not been clarified. Herein, we found that hypoxia promoted ROS production, and the proliferation, migration, and invasion of glioblastoma cells, while this promotion was restrained by ROS scavengers N-acetyl-L-cysteine (NAC) and diphenyleneiodonium chloride (DPI). Hypoxia-induced ROS activated hypoxia-inducible factor-1α (HIF-1α) signaling, which enhanced cell migration and invasion by epithelial-mesenchymal transition (EMT). Furthermore, the induction of serine protease inhibitor family E member 1 (SERPINE1) was ROS-dependent under hypoxia, and HIF-1α mediated SERPINE1 increase induced by ROS via binding to the SERPINE1 promoter region, thereby facilitating glioblastoma migration and invasion. Taken together, our data revealed that hypoxia-induced ROS reinforce the hypoxic adaptation of glioblastoma by driving the HIF-1α-SERPINE1 signaling pathway, and that targeting ROS may be a promising therapeutic strategy for glioblastoma.

Circulating plasminogen activator inhibitor‐1 (PAI‐1) levels in the preclinical stage of Alzheimer’s disease

AbstractBackgroundPlasminogen activator inhibitor‐1 (PAI‐1), a secreted glycoprotein and member of the serine protease inhibitor family, has been implicated to have a role in Alzheimer’s disease (AD) pathogenesis by regulating amyloid‐beta (Aβ) accumulation (Neurobiol Aging 32:1079,2011; J Alzheimers Dis. 64:447,2018). A recent study found that plasma PAI‐1 was differentially expressed in subjects with Aβ pathology and neurodegeneration (A+TN+) compared to A‐TN‐ subjects, with decreased plasma PAI‐1 levels in clinical AD compared to cognitively normal controls (Alzheimers Dement 17:1452,2021). However, whether circulating PAI‐1 levels are altered during the preclinical stage of AD, where there is Aβ and tau pathology but no significant cognitive impairment, remains unclear.MethodCognitively normal (Clinical Dementia Rating of 0) volunteers from the Healthy Aging &amp; Senile Dementia and Adult Children Study (Missouri, USA) with body mass index &lt; 30, fasting plasma and cerebrospinal fluid (CSF) samples were included in this cross‐sectional study. Preclinical AD was defined by previously established CSF criteria (preclinical AD: 29 men, 26 women; control: 35 men, 65 women). Plasma PAI‐1 levels were measured by a commercially available immunoassay (MilliporeSigma) and log transformed.ResultIn men, plasma PAI‐1 levels were significantly lower in preclinical AD compared to control subjects (p=0.029) and were associated with CSF Aβ42 and p‐tau181 levels (CSF Aβ42: age‐adjusted beta coefficient 0.38, p=0.007; CSF p‐tau181: age‐adjusted beta coefficient ‐0.32, p=0.026) but not CSF tau levels (p&gt;0.05). In women, there was no significant difference in plasma PAI‐1 levels between preclinical AD and control subjects (p&gt;0.05).ConclusionThese studies raise the possibility that, in men, alterations in peripheral PAI‐1 occur during the earliest stages of AD. Studies in larger cohorts will be needed to confirm these observations and to determine their clinical utility and the underlying mechanisms of any sex differences.

Gene cloning and functional study of PmKSPI from Pinctada fucata martensii

Kunitz-type serine protease inhibitors (KSPI) are a family of serine protease inhibitors (SPIs) and are extensively found in animals, plants, and microbes. SPI can inhibit proteases that may be harmful or unwanted to its cells. Here, a four-domain Kunitz-type SPI, PmKSPI, was cloned by RACE in the pearl oyster Pinctada fucata martensii. The full-length cDNA sequence of PmKSPI was 1318 bp, including the 5′ UTR (25 bp), the 3′ UTR (96 bp) and ORF (1197 bp). Homology analysis indicated that PmKSPI had the highest resemblance (30.14%) with its homolog in Crassostrea gigas. Phylogenetic analysis revealed that PmKSPI clustered with homologs in other molluscs. We found that PmKSPI mRNA expression in P. f. martensii was distributed in all six tissues, with the highest level in the mantle, and almost no expression in other tissues. After PAMPs challenge, expression of PmKSPI mRNA in the mantle was significantly up-regulated. The recombinant protein rPmKSPI significantly inhibited the growth of 5 kinds of Gram-negative bacteria but had little effect on Gram-positive bacterial activity. Transmission electron microscopy showed that plasmolysis occurred in two Gram-negative bacteria species when treated with rPmKSPI. rPmKSPI may thus have a bactericidal effect by destroying the bacterial cell membrane or cell walls and releasing its contents. Therefore, our results suggest that PmKSPI is tightly associated with the immunological defence of P. f. martensii.

Expression of Trichinella spiralis serpin Tsp_01570 in Pichia pastoris: a first insight of its biomodulatory activity.

Serpins represent one of the most diverse families of serine protease inhibitors. Despite their complexity, they are virtually found in all organisms and play an important role in homeostasis processes such as blood coagulation, inflammation, fibrinolysis, immune responses, chromatin condensation, tumor suppression, and apoptosis. There has recently been particular interest in studying serpin functions in infection and inflammation, especially since more serpins from parasites have been identified and characterized. Among helminths, Trichinella spiralis is one of the few parasites with an extremely strong ability to induce host immune suppression. Previous studies show that serpins are present in Trichinella and are expressed differentially at different parasite stages. More interesting, there is evidence of a recombinant serpin from Trichinella pseudospiralis that alters macrophage polarization in vitro. This finding could be relevant to comprehend the modulation process of the immune response. We expressed Tsp_01570, a putative serpin gene from Trichinella spiralis, in the eukaryotic system Pichia pastoris SMD1168H and evaluated its presence at different parasite stages, finding the serine protease inhibitor in the crude extract of adult worms. The effect of recombinant serpin on THP-1 cells was tested by quantification of IL-12p40, TNF-α, IL-4, and IL-10 cytokines released by ELISA. We also evaluated the expression of the M1 markers, CCR7 and CD86, and the M2 markers, CD163 and CD206, by immunofluorescence staining. This study represents the first insight in elucidating the importance of serpin Tsp_01570 as a potential molecular modulator.

Serpin-loaded extracellular vesicles promote tissue repair in a mouse model of impaired wound healing

Chronic metabolic diseases such as diabetes are characterized by delayed wound healing and a dysregulation of the inflammatory phase of wound repair. Our study focuses on changes in the payload of extracellular vesicles (EVs) communicating between immune cells and stromal cells in the wound bed, which regulate the rate of wound closure. Adoptive transfer of EVs from genetically defined mouse models are used here to demonstrate a functional and molecular basis for differences in the pro-reparative biological activity of diabetic (db/db) vs. wildtype EVs in wound healing. We identify several members of the Serpin family of serine protease inhibitors that are absent in db/db EVs, then we overexpress Serpin A1, F2 and G1 in EVs to evaluate their effect on wound healing in db/db mice. Serpins have an important role in regulating levels of elastase, plasmin and complement factors that coordinate immune cell signaling in full thickness wounds in a diabetic model. Here, we establish a novel therapeutic approach by engineering the payload of EVs based on proteomic analysis. Serpin-loaded EVs were used to rescue the Serpin deficiency identified by proteomics and promote wound healing in db/db mice, as well as evaluated how EVs affected extracellular matrix remodeling and the resolution of tissue injury. Therefore, we propose that the identification of EV payloads that are downregulated in diabetic wounds can be systematically analyzed for their functional activity and potential as a therapeutic, based on whether their re-expression in engineered EVs restores normal kinetics of tissue repair in chronic wounds.Graphical

Grass carp SERPINA1 inhibits GCRV infection through degrading CF2.

SERPINA1, a member of the serine protease inhibitor family, plays a role in viral infection and inflammation by regulating the activities of serine and cysteine proteases. To date, there have been no reports on the immune function of SERPINA1 in fishes. In this study, we first cloned the serpina1 gene of grass carp (Ctenopharyngodon idellus) and found that it could respond rapidly to the infection of Grass carp reovirus (GCRV), and overexpression of serpina1 could enhance the antiviral response of CIK cells. A polyclonal antibody of SERPINA1 was prepared, and the protein interacting with SERPINA1 was screened by CoIP/MS in grass carp hepatopancreas tissue. It was found that SERPINA1 interacted with coagulation factor 2 (CF2) and could degrade it in a dose-dependent manner. In addition, overexpression of cf2 contributed to the infection of GCRV in CIK cells, whereas co-expression of serpina1 and cf2 in grass carp reduced the copy number of GCRV in cells. The results showed that grass carp SERPINA1 could inhibit GCRV infection by degrading CF2. This study proposes that SERPINA1 can inhibit viral infection through interaction with the coagulation factor, providing new insights into the molecular mechanism of SERPINA1’s antiviral function.

Native metabolomics identifies the rivulariapeptolide family of protease inhibitors

The identity and biological activity of most metabolites still remain unknown. A bottleneck in the exploration of metabolite structures and pharmaceutical activities is the compound purification needed for bioactivity assignments and downstream structure elucidation. To enable bioactivity-focused compound identification from complex mixtures, we develop a scalable native metabolomics approach that integrates non-targeted liquid chromatography tandem mass spectrometry and detection of protein binding via native mass spectrometry. A native metabolomics screen for protease inhibitors from an environmental cyanobacteria community reveals 30 chymotrypsin-binding cyclodepsipeptides. Guided by the native metabolomics results, we select and purify five of these compounds for full structure elucidation via tandem mass spectrometry, chemical derivatization, and nuclear magnetic resonance spectroscopy as well as evaluation of their biological activities. These results identify rivulariapeptolides as a family of serine protease inhibitors with nanomolar potency, highlighting native metabolomics as a promising approach for drug discovery, chemical ecology, and chemical biology studies.

A TIL-Type Serine Protease Inhibitor Involved in Humoral Immune Response of Asian Corn Borer Ostrinia furnaculis.

To elucidate the application value of insect endogenous protease and its inhibitor genes in pest control, we analyzed in detail the transcriptome sequence of the Asian corn borer, Ostrinia furnacalis. We obtained 12 protease genes and 11 protease inhibitor genes, and comprehensively analyzed of their spatiotemporal expression by qRT-PCR. In which, a previous unstudied serine protease inhibitor gene attracted our attention. It belongs to the canonical serine proteinase inhibitor family, a trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitor, but its TIL domain lacks two cysteine residues, and it was named as ACB-TIL. Its expression level is relatively very low in the absence of pathogen stimulation, and can be up-regulated expression induced by Gram-negative bacteria (Escherichia coli), virus (BmNPV), and dsRNA (dsEGFP), but cannot be induced by fungus spores (Metarrhizium anisopliae). Prokaryotic expressed ACB-TIL protein can significantly inhibit the melanization in vitro. Injecting this protein into insect body can inhibit the production of antimicrobial peptides of attacin, lebocin and gloverin. Inhibition of ACB-TIL by RNAi can cause the responses of other immune-, protease- and inhibitor-related genes. ACB-TIL is primarily involved in Asian corn borer humoral immunity in responses to Gram-negative bacteria and viruses. This gene can be a potential target for pest control since this will mainly affect insect immune response.

Anticoagulant SERPINs: Endogenous Regulators of Hemostasis and Thrombosis.

Appropriate activation of coagulation requires a balance between procoagulant and anticoagulant proteins in blood. Loss in this balance leads to hemorrhage and thrombosis. A number of endogenous anticoagulant proteins, such as antithrombin and heparin cofactor II, are members of the serine protease inhibitor (SERPIN) family. These SERPIN anticoagulants function by forming irreversible inhibitory complexes with target coagulation proteases. Mutations in SERPIN family members, such as antithrombin, can cause hereditary thrombophilias. In addition, low plasma levels of SERPINs have been associated with an increased risk of thrombosis. Here, we review the biological activities of the different anticoagulant SERPINs. We further consider the clinical consequences of SERPIN deficiencies and insights gained from preclinical disease models. Finally, we discuss the potential utility of engineered SERPINs as novel therapies for the treatment of thrombotic pathologies.

BAP1-Related ceRNA (NEAT1/miR-10a-5p/SERPINE1) Promotes Proliferation and Migration of Kidney Cancer Cells.

Background BAP1 is an important tumor suppressor involved in various biological processes and is commonly lost or inactivated in clear-cell renal cell carcinoma (ccRCC). However, the role of the BAP1-deficient tumor competing endogenous RNA (ceRNA) network involved in ccRCC remains unclear. Thus, this study aims to investigate the prognostic BAP1-related ceRNA in ccRCC.MethodsRaw data was obtained from the TCGA and the differentially expressed genes were screened to establish a BAP1-related ceRNA network. Subsequently, the role of the ceRNA axis was validated using phenotypic experiments. Dual-luciferase reporter assays and fluorescence in situ hybridization (FISH) assays were used to confirm the ceRNA network.ResultsNuclear enriched abundant transcript 1 (NEAT1) expression was significantly increased in kidney cancer cell lines. NEAT1 knockdown significantly inhibited cell proliferation and migration, which could be reversed by miR-10a-5p inhibitor. Dual-luciferase reporter assay confirmed miR-10a-5p as a common target of NEAT1 and Serine protease inhibitor family E member 1 (SERPINE1). FISH assays revealed the co-localization of NEAT1 and miR-10a-5p in the cytoplasm. Additionally, the methylation level of SERPINE1 in ccRCC was significantly lower than that in normal tissues. Furthermore, SERPINE1 expression was positively correlated with multiple immune cell infiltration levels.ConclusionsIn BAP1-deficient ccRCC, NEAT1 competitively binds to miR-10a-5p, indirectly upregulating SERPINE1 expression to promote kidney cancer cell proliferation. Furthermore, NEAT1/miR-10a-5p/SERPINE1 were found to be independent prognostic factors of ccRCC.