Serine protease Rv2569c facilitates transmission of Mycobacterium tuberculosis via disrupting the epithelial barrier by cleaving E-cadherin.

Epithelial cells function as the primary line of defense against invading pathogens. However, bacterial pathogens possess the ability to compromise this barrier and facilitate the transmigration of bacteria. Nonetheless, the specific molecular mechanism employed by Mycobacterium tuberculosis (M.tb) in this process is not fully understood. Here, we investigated the role of Rv2569c in M.tb translocation by assessing its ability to cleave E-cadherin, a crucial component of cell-cell adhesion junctions that are disrupted during bacterial invasion. By utilizing recombinant Rv2569c expressed in Escherichia coli and subsequently purified through affinity chromatography, we demonstrated that Rv2569c exhibited cell wall-associated serine protease activity. Furthermore, Rv2569c was capable of degrading a range of protein substrates, including casein, fibrinogen, fibronectin, and E-cadherin. We also determined that the optimal conditions for the protease activity of Rv2569c occurred at a temperature of 37°C and a pH of 9.0, in the presence of MgCl2. To investigate the function of Rv2569c in M.tb, a deletion mutant of Rv2569c and its complemented strains were generated and used to infect A549 cells and mice. The results of the A549-cell infection experiments revealed that Rv2569c had the ability to cleave E-cadherin and facilitate the transmigration of M.tb through polarized A549 epithelial cell layers. Furthermore, in vivo infection assays demonstrated that Rv2569c could disrupt E-cadherin, enhance the colonization of M.tb, and induce pathological damage in the lungs of C57BL/6 mice. Collectively, these results strongly suggest that M.tb employs the serine protease Rv2569c to disrupt epithelial defenses and facilitate its systemic dissemination by crossing the epithelial barrier.

A current view of mitochondria damage and the diversity of lipopigment inclusions in neuronal ceroid lipofuscinose type 2 from rectal biopsy.

Neuronal ceroid lipofuscinoses (NCLs) are a growing group of neurodegenerative storage diseases, in which specific features are sought to facilitate the creation of a universal diagnostic algorithm in the future. In our ultrastructural studies, the group of NCLs was represented by the CLN2 disease caused by a defect in the TPP1 gene encoding the enzyme tripeptidyl-peptidase 1. A 3.5-year-old girl was affected by this disease. Due to diagnostic difficulties, the spectrum of clinical, enzymatic, and genetic tests was extended to include analysis of the ultrastructure of cells from a rectal biopsy. The aim of our research was to search for pathognomonic features of CLN2 and to analyse the mitochondrial damage accompanying the disease. In the examined cells of the rectal mucosa, as expected, filamentous deposits of the curvilinear profile (CVP) type were found, which dominated quantitatively. Mixed deposits of the CVP/fingerprint profile (FPP) type were observed less frequently in the examined cells. A form of inclusions of unknown origin, not described so far in CLN2 disease, were wads of osmophilic material (WOMs). They occurred alone or co-formed mixed deposits. In addition, atypically damaged mitochondria were observed in muscularis mucosae. Their deformed cristae had contact with inclusions that looked like CVPs. Considering the confirmed role of the c subunit of the mitochondrial ATP synthase in the formation of filamentous lipopigment deposits in the group of NCLs, we suggest the possible significance of other mitochondrial proteins, such as mitochondrial contact site and cristae organizing system (MICOS), in the formation of these deposits. The presence of WOMs in the context of searching for ultrastructural pathognomonic features in CLN2 disease also requires further research.

Thymic Stromal Lymphopoietin (TSLP) Is Cleaved by Human Mast Cell Tryptase and Chymase.

Thymic stromal lymphopoietin (TSLP), mainly expressed by epithelial cells, plays a central role in asthma. In humans, TSLP exists in two variants: the long form TSLP (lfTSLP) and a shorter TSLP isoform (sfTSLP). Macrophages (HLMs) and mast cells (HLMCs) are in close proximity in the human lung and play key roles in asthma. We evaluated the early proteolytic effects of tryptase and chymase released by HLMCs on TSLP by mass spectrometry. We also investigated whether TSLP and its fragments generated by these enzymes induce angiogenic factor release from HLMs. Mass spectrometry (MS) allowed the identification of TSLP cleavage sites caused by tryptase and chymase. Recombinant human TSLP treated with recombinant tryptase showed the production of 1-97 and 98-132 fragments. Recombinant chymase treatment of TSLP generated two peptides, 1-36 and 37-132. lfTSLP induced the release of VEGF-A, the most potent angiogenic factor, from HLMs. By contrast, the four TSLP fragments generated by tryptase and chymase failed to activate HLMs. Long-term TSLP incubation with furin generated two peptides devoid of activating property on HLMs. These results unveil an intricate interplay between mast cell-derived proteases and TSLP. These findings have potential relevance in understanding novel aspects of asthma pathobiology.

Active site-directed probes targeting dipeptidyl peptidases 8 and 9.

Dipeptidyl peptidases (DPP) 8 and 9 are intracellular serine proteases that play key roles in various biological processes and recent findings highlight DPP8 and DPP9 as potential therapeutic targets for hematological and inflammasome-related diseases. Despite the substantial progress, the precise biological functions of these proteases remain elusive, and the lack of selective chemical tools hampers ongoing research. In this paper, we describe the synthesis and biochemical evaluation of the first active site-directed DPP8/9 probes which are derived from DPP8/9 inhibitors developed in-house. Specifically, we synthesized fluorescent inhibitors containing nitrobenzoxadiazole (NBD), dansyl (DNS) and cyanine-3 (Cy3) reporters to visualize intracellular DPP8/9. We demonstrate that the fluorescent inhibitors have high affinity and selectivity towards DPP8/9 over related S9 family members. The NBD-labeled DPP8/9 inhibitors were nominated as the best in class compounds to visualize DPP8/9 in human cells. Furthermore, a method has been developed for selective labeling and visualization of active DPP8/9 in vitro by fluorescence microscopy. A collection of potent and selective biotinylated DPP8/9-targeting probes was also prepared by replacing the fluorescent reporter with a biotin group. The present work provides the first DPP8/9-targeting fluorescent compounds as useful chemical tools for the study of DPP8 and DPP9's biological functions.

Isolation and Functional Characterization of Erythrofibrase: An Alfa-Fibrinogenase Enzyme from Trimeresurus erythrurus Venom of North-East India.

Green pit viper bites induce mild toxicity with painful local swelling, blistering, cellulitis, necrosis, ecchymosis and consumptive coagulopathy. Several bite cases of green pit vipers have been reported in several south-east Asian countries including the north-eastern region of India. The present study describes isolation and characterization of a haemostatically active protein from Trimeresurus erythrurus venom responsible for coagulopathy. Using a two-step chromatographic method, a snake venom serine protease erythrofibrase was purified to homogeneity. SDS-PAGE of erythrofibrase showed a single band of ~30 kDa in both reducing and non-reducing conditions. The primary structure of erythrofibrase was determined by ESI LC-MS/MS, and the partial sequence obtained showed 77% sequence similarity with other snake venom thrombin-like enzymes (SVTLEs). The partial sequence obtained had the typical 12 conserved cysteine residues, as well as the active site residues (His57, Asp102 and Ser195). Functionally, erythrofibrase showed direct fibrinogenolytic activity by degrading the Aα chain of bovine fibrinogen at a slow rate, which might be responsible for causing hypofibrinogenemia and incoagulable blood for several days in envenomated patients. Moreover, the inability of Indian polyvalent antivenom (manufactured by Premium Serum Pvt. Ltd., Maharashtra, India) to neutralize the thrombin-like and plasmin-like activity of erythrofibrase can be correlated with the clinical inefficacy of antivenom therapy. This is the first study reporting an α-fibrinogenase enzyme erythrofibrase from T. erythrurus venom, which is crucial for the pathophysiological manifestations observed in envenomated victims.

A common protein C inhibitor exosite partially controls the heparin induced activation and inhibition of serine proteases.

Protein C inhibitor (PCI) maintains hemostasis by inhibiting both procoagulant and anticoagulant serine proteases, and plays important roles in coagulation, fibrinolysis, reproduction, and anti-angiogenesis. The reactive site loop of PCI traps and irreversibly inhibits the proteases like APC (activating protein C), thrombin (FIIa) and factor Xa (FXa). Previous studies on antithrombin (ATIII) had identified Tyr253 and Glu255 as functional exosites that interact and aid in the inhibition of factor IXa and FXa. Presence of exosite in PCI is not known, however a sequence comparison with the PCI from different vertebrate species and ATIII identified Glu239 to be absolutely conserved. PCI residues analogous to ATIII exosite residues were mutated to R238A and E239A. Purified variant PCI in the presence of heparin (10 µg/ml) showed a 2-4 fold decrease in the rate of inhibition of the proteases. However, the stoichiometry of inhibition of FIIa, APC, and FXa by native PCI, R238A and E239A variants were found to be close to 1.0, which also indicated the formation of stable complexes based on SDS-PAGE and western blot analysis with thrombin and APC. Our findings revealed the possible presence of an exosite in PCI that influences the protease inhibition rates.

Hexavalent chromium exposure activates the non-canonical nuclear factor kappa B pathway to promote immune checkpoint protein programmed death-ligand 1 expression and lung carcinogenesis.

Lung cancer is the leading cause of cancer-related death worldwide; however, the mechanism of lung carcinogenesis has not been clearly defined. Chronic exposure to hexavalent chromium [Cr(VI)], a common environmental and occupational pollutant, causes lung cancer, representing an important lung cancer etiology factor. The mechanism of how chronic Cr(VI) exposure causes lung cancer remains largely unknown. By using cell culture and mouse models and bioinformatics analyses of human lung cancer gene expression profiles, this study investigated the mechanism of Cr(VI)-induced lung carcinogenesis. A new mouse model of Cr(VI)-induced lung carcinogenesis was developed as evidenced by the findings showing that a 16-week Cr(VI) exposure (CaCrO4, 100 µg per mouse once per week) via oropharyngeal aspiration induced lung adenocarcinomas in male and female A/J mice, whereas none of the sham-exposed control mice had lung tumors. Mechanistic studies revealed that chronic Cr(VI) exposure activated the non-canonical NFκB pathway through the long non-coding RNA (lncRNA) ABHD11-AS1/deubiquitinase USP15-mediated tumor necrosis factor receptor-associated factor 3 (TRAF3) down-regulation. The non-canonical NFκB pathway activation increased the interleukin 6 (IL-6)/Janus kinase (Jak)/signal transducer and activator of transcription 3 (Stat3) signaling. The activation of the IL-6/Jak signaling axis by Cr(VI) exposure not only promoted inflammation but also stabilized the immune checkpoint molecule programmed death-ligand 1 (PD-L1) protein in the lungs, reducing T lymphocyte infiltration to the lungs. Given the well-recognized critical role of PD-L1 in inhibiting anti-tumor immunity, these findings suggested that the lncRNA ABHD11-AS1-mediated non-canonical NFκB pathway activation and PD-L1 up-regulation may play important roles in Cr(VI)-induced lung carcinogenesis.

Mechanism-Based Macrocyclic Inhibitors of Serine Proteases.

Protease inhibitor drug discovery is challenged by the lack of cellular and oral permeability, selectivity, metabolic stability, and rapid clearance of peptides. Here, we describe the rational design, synthesis, and evaluation of peptidomimetic side-chain-cyclized macrocycles which we converted into covalent serine protease inhibitors with the addition of an electrophilic ketone warhead. We have identified potent and selective inhibitors of TMPRSS2, matriptase, hepsin, and HGFA and demonstrated their improved protease selectivity, metabolic stability, and pharmacokinetic (PK) properties. We obtained an X-ray crystal structure of phenyl ether-cyclized tripeptide VD4162 (8b) bound to matriptase, revealing an unexpected binding conformation. Cyclic biphenyl ether VD5123 (11) displayed the best PK properties in mice with a half-life of 4.5 h and compound exposure beyond 24 h. These new cyclic tripeptide scaffolds can be used as easily modifiable templates providing a new strategy to overcoming the obstacles presented by linear acyclic peptides in protease inhibitor drug discovery.

Insights from structure-activity relationships and the binding mode of peptidic α-ketoamide inhibitors of the malaria drug target subtilisin-like SUB1.

Plasmodium multi-resistance, including against artemisinin, seriously threatens malaria treatment and control. Hence, new drugs are urgently needed, ideally targeting different parasitic stages, which are not yet targeted by current drugs. The SUB1 protease is involved in both hepatic and blood stages due to its essential role in the egress of parasites from host cells, and, as potential new target, it would meet the above criteria. We report here the synthesis as well as the biological and structural evaluation of substrate-based α-ketoamide SUB1 pseudopeptidic inhibitors encompassing positions P4-P2'. By individually substituting each position of the reference compound 1 (MAM-117, Ac-Ile-Thr-Ala-AlaCO-Asp-Glu (Oall)-NH2), we better characterized the structural determinants for SUB1 binding. We first identified compound 8 with IC50 values of 50 and 570 nM against Pv- and PfSUB1, respectively (about 3.5-fold higher potency compared to 1). Compound 8 inhibited P. falciparum merozoite egress in culture by 37% at 100 µM. By increasing the overall hydrophobicity of the compounds, we could improve the PfSUB1 inhibition level and antiparasitic activity, as shown with compound 40 (IC50 values of 12 and 10 nM against Pv- and PfSUB1, respectively, IC50 value of 23 µM on P. falciparum merozoite egress). We also found that 8 was highly selective towards SUB1 over three mammalian serine peptidases, supporting the promising value of this compound. Finally, several crystal 3D-structures of SUB1-inhibitor complexes, including with 8, were solved at high resolution to decipher the binding mode of these compounds.

Neutrophil proteases are protective against SARS-CoV-2 by degrading the spike protein and dampening virus-mediated inflammation.

Studies on severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) have highlighted the crucial role of host proteases for viral replication and the immune response. The serine proteases furin and TMPRSS2 and lysosomal cysteine proteases facilitate viral entry by limited proteolytic processing of the spike (S) protein. While neutrophils are recruited to the lungs during COVID-19 pneumonia, little is known about the role of the neutrophil serine proteases (NSPs) cathepsin G (CatG), elastase (NE), and proteinase 3 (PR3) on SARS-CoV-2 entry and replication. Furthermore, the current paradigm is that NSPs may contribute to the pathogenesis of severe COVID-19. Here, we show that these proteases cleaved the S protein at multiple sites and abrogated viral entry and replication in vitro. In mouse models, CatG significantly inhibited viral replication in the lung. Importantly, lung inflammation and pathology were increased in mice deficient in NE and/or CatG. These results reveal that NSPs contribute to innate defenses against SARS-CoV-2 infection via proteolytic inactivation of the S protein and that NE and CatG limit lung inflammation in vivo. We conclude that therapeutic interventions aiming to reduce the activity of NSPs may interfere with viral clearance and inflammation in COVID-19 patients.

Post-ingestive stability of a mulberry Kunitz-type protease inhibitor MnKTI-1 in the digestive lumen of silkworm: dual inhibition towards α-amylase and serine protease.

BACKGROUND: Adaptation of specialist insects to their host plants and defense responses of plants to phytophagous insects have been extensively recognized while the dynamic interaction between these two events has been largely underestimated. Here, we provide evidence for characterization of an unrevealed dynamic interaction mode of digestive enzymes of specialist insect silkworm and inhibitor of its host plant mulberry tree. RESULTS: MnKTI-1, a mulberry Kunitz-type protease inhibitor, whose messenger RNA (mRNA) transcription and protein expression in mulberry leaf were severely triggered and up-regulated by tens of times in a matter of hours in response to silkworm, Bombyx mori, and other mulberry pest insects, suggesting a quick response and broad spectrum to insect herbivory. MnKTI-1 proteins were detected in gut content and frass of specialist B. mori, and exhibited significant post-ingestive stability. Recombinant refolded MnKTI-1 (rMnKTI-1) displayed binding affinity to digestive enzymes and a dual inhibitory activity to α-amylase BmAmy and serine protease BmSP2956 in digestive juice of silkworm. Moreover, data from in vitro assays proved that the inhibition of recombinant rMnKTI-1 to BmAmy can be reverted by pre-incubation with BmSP15920, an inactivated silkworm digestive protease that lack of complete catalytic triad. CONCLUSION: These findings demonstrate that mulberry MnKTI-1 has the potential to inhibit the digestive enzyme activities of its specialist insect herbivore silkworm, whereas this insect may employ inactivated proteases to block protease inhibitors to accomplish food digestion. The current work provides an insight to better understand the interacting mode between host plant Kunitz protease inhibitors and herbivorous insect digestive enzymes. © 2024 Society of Chemical Industry.

A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

Long noncoding RNA ABHD11-AS1 interacts with SART3 and regulates CD44 RNA alternative splicing to promote lung carcinogenesis.

Hexavalent chromium [Cr(VI)] is a common environmental pollutant and chronic exposure to Cr(VI) causes lung cancer in humans, however, the mechanism of Cr(VI) carcinogenesis has not been well understood. Lung cancer is the leading cause of cancer-related death, although the mechanisms of how lung cancer develops and progresses have been poorly understood. While long non-coding RNAs (lncRNAs) are found abnormally expressed in cancer, how dysregulated lncRNAs contribute to carcinogenesis remains largely unknown. The goal of this study is to investigate the mechanism of Cr(VI)-induced lung carcinogenesis focusing on the role of the lncRNA ABHD11 antisense RNA 1 (tail to tail) (ABHD11-AS1). It was found that the lncRNA ABHD11-AS1 expression levels are up-regulated in chronic Cr(VI) exposure-transformed human bronchial epithelial cells, chronically Cr(VI)-exposed mouse lung tissues, and human lung cancer cells as well. Bioinformatics analysis revealed that ABHD11-AS1 levels are up-regulated in lung adenocarcinomas (LUADs) tissues and associated with worse overall survival of LUAD patients but not in lung squamous cell carcinomas. It was further determined that up-regulation of ABHD11-AS1 expression plays an important role in chronic Cr(VI) exposure-induced cell malignant transformation and tumorigenesis, and the stemness of human lung cancer cells. Mechanistically, it was found that ABHD11-AS1 directly binds SART3 (spliceosome associated factor 3, U4/U6 recycling protein). The interaction of ABHD11-AS1 with SART3 promotes USP15 (ubiquitin specific peptidase 15) nuclear localization. Nuclear localized USP15 interacts with pre-mRNA processing factor 19 (PRPF19) to increase CD44 RNA alternative splicing activating ß-catenin and enhancing cancer stemness. Together, these findings indicate that lncRNA ABHD11-AS1 interacts with SART3 and regulates CD44 RNA alternative splicing to promote cell malignant transformation and lung carcinogenesis.

Pseudomonas environmental strain produces a DegQ-derived and PDZ domain containing peptide with protease activity.

In the search of new enzymatic activities with a possible industrial application, we focused on those microorganisms and their molecular mechanisms that allow them to succeed in the environment, particularly in the proteolytic activity and its central role in the microorganisms' successful permanence. The use of highly active serine proteases for industrial applications is a modern need, especially for the formulation of detergents, protein processing, and hair removal from animal skins. This report provides the isolation and identification of a highly proteolytic fragment derived from DegQ produced by a Pseudomonas fluorescens environmental strain isolated from a frog carcass. Zymograms demonstrate that a 10 kDa protein mainly generates the total proteolytic activity of this strain, which is enhanced by the detergent SDS. Mass spectroscopy analysis revealed that the protein derived a couple of peptides, the ones showing the highest coverage belonging to DegQ. Interestingly, this small protein fragment contains a PDZ domain but no obvious residues indicating that it is a protease. Protein model analysis shows that this fragment corresponds to the main PDZ domain from DegQ, and its unique sequence and structure render a proteolytic peptide. The results presented here indicate that a novel DegQ fragment is sufficient for obtaining high protease activity highlighting that the analysis of environmental microorganisms can render new strains or enzymes with helpful biotechnological characteristics.

Campylobacter jejuni Surface-Bound Protease HtrA, but Not the Secreted Protease nor Protease in Shed Membrane Vesicles, Disrupts Epithelial Cell-to-Cell Junctions.

Fundamental functions of the intestinal epithelium include the digestion of food, absorption of nutrients, and its ability to act as the first barrier against intruding microbes. Campylobacter jejuni is a major zoonotic pathogen accounting for a substantial portion of bacterial foodborne illnesses. The germ colonizes the intestines of birds and is mainly transmitted to humans through the consumption of contaminated poultry meat. In the human gastrointestinal tract, the bacterium triggers campylobacteriosis that can progress to serious secondary disorders, including reactive arthritis, inflammatory bowel disease and Guillain-Barré syndrome. We recently discovered that C. jejuni serine protease HtrA disrupts intestinal epithelial barrier functions via cleavage of the tight and adherens junction components occludin, claudin-8 and E-cadherin. However, it is unknown whether epithelial damage is mediated by the secreted soluble enzyme, by HtrA contained in shed outer-membrane vesicles (OMVs) or by another mechanism that has yet to be identified. In the present study, we investigated whether soluble recombinant HtrA and/or purified OMVs induce junctional damage to polarized intestinal epithelial cells compared to live C. jejuni bacteria. By using electron and confocal immunofluorescence microscopy, we show that HtrA-expressing C. jejuni bacteria trigger efficient junctional cell damage, but not soluble purified HtrA or HtrA-containing OMVs, not even at high concentrations far exceeding physiological levels. Instead, we found that only bacteria with active protein biosynthesis effectively cleave junctional proteins, which is followed by paracellular transmigration of C. jejuni through the epithelial cell layer. These findings shed new light on the pathogenic activities of HtrA and virulence strategies of C. jejuni.

Extended Cleavage Specificity of two Hematopoietic Serine Proteases from a Ray-Finned Fish, the Spotted Gar (Lepisosteus oculatus).

The extended cleavage specificities of two hematopoietic serine proteases originating from the ray-finned fish, the spotted gar (Lepisosteus oculatus), have been characterized using substrate phage display. The preference for particular amino acids at and surrounding the cleavage site was further validated using a panel of recombinant substrates. For one of the enzymes, the gar granzyme G, a strict preference for the aromatic amino acid Tyr was observed at the cleavable P1 position. Using a set of recombinant substrates showed that the gar granzyme G had a high selectivity for Tyr but a lower activity for cleaving after Phe but not after Trp. Instead, the second enzyme, gar DDN1, showed a high preference for Leu in the P1 position of substrates. This latter enzyme also showed a high preference for Pro in the P2 position and Arg in both P4 and P5 positions. The selectivity for the two Arg residues in positions P4 and P5 suggests a highly specific substrate selectivity of this enzyme. The screening of the gar proteome with the consensus sequences obtained by substrate phage display for these two proteases resulted in a very diverse set of potential targets. Due to this diversity, a clear candidate for a specific immune function of these two enzymes cannot yet be identified. Antisera developed against the recombinant gar enzymes were used to study their tissue distribution. Tissue sections from juvenile fish showed the expression of both proteases in cells in Peyer's patch-like structures in the intestinal region, indicating they may be expressed in T or NK cells. However, due to the lack of antibodies to specific surface markers in the gar, it has not been possible to specify the exact cellular origin. A marked difference in abundance was observed for the two proteases where gar DDN1 was expressed at higher levels than gar granzyme G. However, both appear to be expressed in the same or similar cells, having a lymphocyte-like appearance.

The Inhibition of Serine Proteases by Serpins Is Augmented by Negatively Charged Heparin: A Concise Review of Some Clinically Relevant Interactions.

Serine proteases are members of a large family of hydrolytic enzymes in which a particular serine residue in the active site performs an essential role as a nucleophile, which is required for their proteolytic cleavage function. The array of functions performed by serine proteases is vast and includes, among others, the following: (i) the ability to fight infections; (ii) the activation of blood coagulation or blood clot lysis systems; (iii) the activation of digestive enzymes; and (iv) reproduction. Serine protease activity is highly regulated by multiple families of protease inhibitors, known collectively as the SERine Protease INhibitor (SERPIN). The serpins use a conformational change mechanism to inhibit proteases in an irreversible way. The unusual conformational change required for serpin function provides an elegant opportunity for allosteric regulation by the binding of cofactors, of which the most well-studied is heparin. The goal of this review is to discuss some of the clinically relevant serine protease-serpin interactions that may be enhanced by heparin or other negatively charged polysaccharides. The paired serine protease-serpin in the framework of heparin that we review includes the following: thrombin-antithrombin III, plasmin-anti-plasmin, C1 esterase/kallikrein-C1 esterase inhibitor, and furin/TMPRSS2 (serine protease Transmembrane Protease 2)-alpha-1-antitrypsin, with the latter in the context of COVID-19 and prostate cancer.

Genome-wide bioinformatics analysis of human protease capacity for proteolytic cleavage of the SARS-CoV-2 spike glycoprotein.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) primarily enters the cell by binding the virus's spike (S) glycoprotein to the angiotensin-converting enzyme 2 receptor on the cell surface, followed by proteolytic cleavage by host proteases. Studies have identified furin and transmembrane protease serine 2 proteases in priming and triggering cleavages of the S glycoprotein, converting it into a fusion-competent form and initiating membrane fusion, respectively. Alternatively, SARS-CoV-2 can enter the cell through the endocytic pathway, where activation is triggered by lysosomal cathepsin L. However, other proteases are also suspected to be involved in both entry routes. In this study, we conducted a genome-wide bioinformatics analysis to explore the capacity of human proteases in hydrolyzing peptide bonds of the S glycoprotein. Predictive models of sequence specificity for 169 human proteases were constructed and applied to the S glycoprotein together with the method for predicting structural susceptibility to proteolysis of protein regions. After validating our approach on extensively studied S2' and S1/S2 cleavage sites, we applied our method to each peptide bond of the S glycoprotein across all 169 proteases. Our results indicate that various members of the proprotein convertase subtilisin/kexin type, type II transmembrane family serine protease, and kallikrein families, as well as specific coagulation factors, are capable of cleaving S2' or S1/S2 sites. We have also identified a potential cleavage site of cathepsin L at the K790 position within the S2' loop. Structural analysis suggests that cleavage of this site induces conformational changes similar to the cleavage at the R815 (S2') position, leading to the exposure of the fusion peptide and subsequent fusion with the membrane. Other potential cleavage sites and the influence of mutations in common SARS-CoV-2 variants on proteolytic efficiency are discussed.IMPORTANCEThe entry of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) into the cell, activated by host proteases, is considerably more complex in coronaviruses than in most other viruses and is not fully understood. There is evidence that other proteases beyond the known furin and transmembrane protease serine 2 can activate the spike protein. Another example of uncertainty is the cleavage site for the alternative endocytic route of SARS-CoV-2 entrance, which is still unknown. Bioinformatics methods, modeling protease specificity and estimating the structural susceptibility of protein regions to proteolysis, can aid in studying this topic by predicting the involved proteases and their cleavage sites, thereby substantially reducing the amount of experimental work. Elucidating the mechanisms of spike protein activation is crucial for preventing possible future coronavirus pandemics and developing antiviral drugs.

Stabilizing the Proteomes of Acute Myeloid Leukemia Cells: Implications for Cancer Proteomics.

Previous work has shown that inhibition of abundant myeloid azurophil granule-associated serine proteases (ELANE [neutrophil elastase], PRTN3 [protease 3], and CTSG [Cathepsin G]) is required to stabilize some proteins in myeloid cells. We therefore hypothesized that effective inhibition of these proteases may be necessary for quantitative proteomic analysis of samples containing myeloid cells. To test this hypothesis, we thawed viably preserved acute myeloid leukemia cells from cryovials in the presence or the absence of diisopropyl fluorophosphate (DFP), a cell-permeable and irreversible serine protease inhibitor. Global proteomic analysis was performed, using label-free and isobaric peptide-labeling quantitation. The presence of DFP resulted in an increase of tryptic peptides (14-57%) and proteins (9-31%). In the absence of DFP, 11 to 31% of peptide intensity came from nontryptic peptides; 52 to 75% had cleavage specificity consistent with activities of ELANE-PRTN3. Treatment with DFP reduced the intensity of nontryptic peptides to 4-8% of the total. ELANE inhibition was 95%, based on diisopropyl phosphate modification of active site serine residue. Overall, the relative abundance of 20% of proteins was significantly altered by DFP treatment. These results suggest that active myeloid serine proteases, released during sample processing, can skew quantitative proteomic measurements. Finally, significant ELANE activity was also detected in Clinical Proteomics Tumor Analysis Consortium datasets of solid tumors (many of which have known myeloid infiltration). In the pancreatic cancer dataset, the median percentage of nontryptic intensity detected across patient samples was 34%, with many patient samples having more than half of their detected peptide intensity from nontryptic cleavage events consistent with ELANE-PRTN3 cleavage specificity. Our study suggests that in vitro cleavage of proteins by myeloid serine proteases may be relevant for proteomic studies of any tumor that contains infiltrating myeloid cells.

The serine protease 2 gene regulates lipid metabolism through the LEP/ampkα1/SREBP1 pathway in bovine mammary epithelial cells.

Molecular breeding has brought about significant transformations in the milk market and production system during the twenty-first century. The primary economic characteristic of dairy production pertains to milk fat content. Our previous transcriptome analyses revealed that serine protease 2 (PRSS2) is a candidate gene that could impact milk fat synthesis in bovine mammary epithelial cells (BMECs) of Chinese Holstein dairy cows. To elucidate the function of the PRSS2 gene in milk fat synthesis, we constructed vectors for PRSS2 overexpression and interference and assessed intracellular triglycerides (TGs), cholesterol (CHOL), and nonesterified fatty acid (NEFA) contents in BMECs. Fatty acid varieties and components were also quantified using gas chromatography‒mass spectrometry (GC‒MS) technology. The regulatory pathway mediated by PRSS2 was validated through qPCR, ELISA, and WB techniques. Based on our research findings, PRSS2 emerges as a pivotal gene that regulates the expression of associated genes, thereby making a substantial contribution to lipid metabolism via the leptin (LEP)/Adenylate-activated protein kinase, alpha 1 catalytic subunit (AMPKα1)/sterol regulatory element binding protein 1(SREBP1) pathway by inhibiting TGs and CHOL accumulation while potentially promoting NEFA synthesis in BMECs. Furthermore, the PRSS2 gene enhances intracellular medium- and long-chain fatty acid metabolism by modulating genes related to the LEP/AMPKα1/SREBP1 pathway, leading to increased contents of unsaturated fatty acids C17:1N7 and C22:4N6. This study provides a robust theoretical framework for further investigation into the underlying molecular mechanisms through which PRSS2 influences lipid metabolism in dairy cows.