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Science Advances Dec 2023Melanization and Toll pathway activation are essential innate immune mechanisms in insects, which result in the generation of reactive compounds and antimicrobial...
Melanization and Toll pathway activation are essential innate immune mechanisms in insects, which result in the generation of reactive compounds and antimicrobial peptides, respectively, to kill pathogens. These two processes are mediated by phenoloxidase (PO) and Spätzle (Spz) through an extracellular network of serine proteases. While some proteases have been identified in in genetic studies, the exact order of proteolytic activation events remains controversial. Here, we reconstituted the serine protease framework in by biochemical methods. This system comprises 10 proteases, i.e., ModSP, cSP48, Grass, Psh, Hayan-PA, Hayan-PB, Sp7, MP1, SPE and Ser7, which form cascade pathways that recognize microbial molecular patterns and virulence factors, and generate PO1, PO2, and Spz from their precursors. Furthermore, the serpin Necrotic negatively regulates the immune response progression by inhibiting ModSP and Grass. The biochemical approach, when combined with genetic analysis, is crucial for addressing problems that long stand in this important research field.
Topics: Animals; Drosophila; Drosophila melanogaster; Drosophila Proteins; Serine Endopeptidases; Serine Proteases
PubMed: 38117884
DOI: 10.1126/sciadv.adk2756 -
IUBMB Life May 2009Over one third of all known proteolytic enzymes are serine proteases. Among these, the trypsins underwent the most predominant genetic expansion yielding the enzymes... (Review)
Review
Over one third of all known proteolytic enzymes are serine proteases. Among these, the trypsins underwent the most predominant genetic expansion yielding the enzymes responsible for digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis, and immunity. The success of this expansion resides in a highly efficient fold that couples catalysis and regulatory interactions. Added complexity comes from the recent observation of a significant conformational plasticity of the trypsin fold. A new paradigm emerges where two forms of the protease, E* and E, are in allosteric equilibrium and determine biological activity and specificity.
Topics: Isoenzymes; Models, Molecular; Protein Binding; Protein Conformation; Serine Endopeptidases; Substrate Specificity
PubMed: 19180666
DOI: 10.1002/iub.186 -
The FEBS Journal May 2023Serine proteases (SPs) constitute a very important family of enzymes, both physiologically and pathologically. The effects produced by these proteins have been explained... (Review)
Review
Serine proteases (SPs) constitute a very important family of enzymes, both physiologically and pathologically. The effects produced by these proteins have been explained by their proteolytic activity. However, the discovery of pharmacologically active SP molecules that show no enzymatic activity, as the so-called pseudo SPs or SP homologs (SPHs), has exposed a profoundly neglected possibility of nonenzymatic functions of these SP molecules. In this review, the most thoroughly described SPHs are presented. The main physiological domains in which SPHs operate appear to be in reproduction, embryonic development, immune response, host defense, and hemostasis. Hitherto unexplained actions of SPs should therefore be considered also as the result of the ligand-like attributes of SPs. The gain of a novel function by an SPH is a consequence of specific amino acid replacements that have resulted in a novel interaction interface or a 'catalytic trap'. Unraveling the SP/SPH interactome will provide a description of previously unknown physiological functions of SPs/SPHs, aiding the creation of innovative medical approaches.
Topics: Serine; Serine Proteases; Serine Endopeptidases; Immunity
PubMed: 35032346
DOI: 10.1111/febs.16355 -
Insect Biochemistry and Molecular... May 2022Phenoloxidase (PO) is a crucial component of the insect immune response against microbial infection. In the tobacco hornworm Manduca sexta, PO is generated from its...
Phenoloxidase (PO) is a crucial component of the insect immune response against microbial infection. In the tobacco hornworm Manduca sexta, PO is generated from its precursor proPO by prophenoloxidase activating proteases (PAPs) in the presence of two noncatalytic serine protease homologs (SPHs). cDNA cloning and genome analysis indicate that SPH1a (formerly known as SPH1), SPH1b, SPH4, SPH101, and SPH2 contain a clip domain, a linker, and a protease-like domain (PLD). The first 22 residues of the SPH1b, SPH4, and SPH101 PLDs are identical, and differ from SPH1a only at position 4, Thr substituted with Asn in SPH1a. While the sequence from Edman degradation was used to establish PAP cofactor as a high M complex of SPH1a and SPH2, this assignment needed further validation, especially because SPH1b mRNA levels are much higher than SPH1a's and better correlate with SPH2 transcription. Thus, here we determined expression profiles of these SPH genes in different tissues from various developmental stages using highly specific primers. High levels of SPH1b and SPH2 proteins, low SPH4, and no SPH1a or SPH101 were detected in hemolymph from larvae in the feeding, wandering and bar stages, pupae, and adults by targeted LC-MS/MS analysis, based on unique peptides from the trypsin-treated SPHs. We expressed the five proSPHs in baculovirus-infected Sf9 cells for use as standards to identify and quantify their counterparts in plasma samples. Moreover, we tested their cleavage by PAP3 and efficacy of the SPH1a, 1b, 4, and 101 as SPH2 partners in PAP3-mediated proPO activation. PAP3 processed proSPH1b and 101 more readily than proSPH1a and 4; PAP3 activated proPO more efficiently in the presence of SPH2 with SPH101 or SPH1b than with SPH1a or SPH4. These results generally agree with their order of appearance or sequence similarity: SPH101 > SPH1b (98%) > SPH1a (90%) > SPH4 (83%). In other words, likely due to positive selection, products of the newly duplicated genes (SPH1b and SPH101) are more favorable substrates of PAP3 and better SPH2 partners in forming a high M cofactor than SPH1a or SPH4 is. Electrophoresis on native gel and immunoblot analysis further indicated that SPH101 or 1b form high M complexes more readily than SPH1a or 4 does. In comparison, SPH2 showed a small mobility decrease and then increase on native gel after PAP3 cleavage at the first site. Since the natural cofactor in bar-stage hemolymph is complexes of SPH1 and 2 with an average M of 790 kDa, PAP3-activated SPH2 may associate with the higher M SPH1b scaffolds to form super-complexes. Their structures and formation in relation to cleavage of SPH1b at different sites await further exploration.
Topics: Animals; Ankyrins; Catechol Oxidase; Chromatography, Liquid; Enzyme Precursors; Hemolymph; Insect Proteins; Manduca; Monophenol Monooxygenase; Serine Endopeptidases; Serine Proteases; Spherocytosis, Hereditary; Tandem Mass Spectrometry
PubMed: 35395380
DOI: 10.1016/j.ibmb.2022.103762 -
International Journal of Molecular... May 2023Hemostasis is a delicate balance between coagulation and fibrinolysis that regulates the formation and removal of fibrin, respectively. Positive and negative feedback...
Hemostasis is a delicate balance between coagulation and fibrinolysis that regulates the formation and removal of fibrin, respectively. Positive and negative feedback loops and crosstalk between coagulation and fibrinolytic serine proteases maintain the hemostatic balance to prevent both excessive bleeding and thrombosis. Here, we identify a novel role for the glycosylphosphatidylinositol (GPI)-anchored serine protease testisin in the regulation of pericellular hemostasis. Using in vitro cell-based fibrin generation assays, we found that the expression of catalytically active testisin on the cell surface accelerates thrombin-dependent fibrin polymerization, and intriguingly, that it subsequently promotes accelerated fibrinolysis. We find that the testisin-dependent fibrin formation is inhibited by rivaroxaban, a specific inhibitor of the central prothrombin-activating serine protease factor Xa (FXa), demonstrating that cell-surface testisin acts upstream of factor X (FX) to promote fibrin formation at the cell surface. Unexpectedly, testisin was also found to accelerate fibrinolysis by stimulating the plasmin-dependent degradation of fibrin and enhancing plasmin-dependent cell invasion through polymerized fibrin. Testisin was not a direct activator of plasminogen, but it is able to induce zymogen cleavage and the activation of pro-urokinase plasminogen activator (pro-uPA), which converts plasminogen to plasmin. These data identify a new proteolytic component that can regulate pericellular hemostatic cascades at the cell surface, which has implications for angiogenesis, cancer biology, and male fertility.
Topics: Male; Humans; Fibrinolysis; Fibrinolysin; Glycosylphosphatidylinositols; Serine Proteases; Serine Endopeptidases; Plasminogen; Urokinase-Type Plasminogen Activator; Fibrin; Hemostatics
PubMed: 37298257
DOI: 10.3390/ijms24119306 -
Cellular and Molecular Life Sciences :... Aug 2023HtrA2/Omi is a mitochondrial serine protease with ascribed pro-apoptotic as well as pro-necroptotic functions. Here, we establish that HtrA2/Omi also controls...
HtrA2/Omi is a mitochondrial serine protease with ascribed pro-apoptotic as well as pro-necroptotic functions. Here, we establish that HtrA2/Omi also controls parthanatos, a third modality of regulated cell death. Deletion of HtrA2/Omi protects cells from parthanatos while reconstitution with the protease restores the parthanatic death response. The effects of HtrA2/Omi on parthanatos are specific and cannot be recapitulated by manipulating other mitochondrial proteases such as PARL, LONP1 or PMPCA. HtrA2/Omi controls parthanatos in a manner mechanistically distinct from its action in apoptosis or necroptosis, i.e., not by cleaving cytosolic IAP proteins but rather exerting its effects without exiting mitochondria, and downstream of PARP-1, the first component of the parthanatic signaling cascade. Also, previously identified or candidate substrates of HtrA2/Omi such as PDXDC1, VPS4B or moesin are not cleaved and dispensable for parthanatos, whereas DBC-1 and stathmin are cleaved, and thus represent potential parthanatic downstream mediators of HtrA2/Omi. Moreover, mass-spectrometric screening for novel parthanatic substrates of HtrA2/Omi revealed that the induction of parthanatos does not cause a substantial proteolytic cleavage or major alterations in the abundance of mitochondrial proteins. Resolving these findings, reconstitution of HtrA2/Omi-deficient cells with a catalytically inactive HtrA2/Omi mutant restored their sensitivity against parthanatos to the same level as the protease-active HtrA2/Omi protein. Additionally, an inhibitor of HtrA2/Omi's protease activity did not confer protection against parthanatic cell death. Our results demonstrate that HtrA2/Omi controls parthanatos in a protease-independent manner, likely via novel, unanticipated functions as a scaffolding protein and an interaction with so far unknown mitochondrial proteins.
Topics: Parthanatos; Serine Proteases; Necroptosis; Serine Endopeptidases; Mitochondrial Proteins
PubMed: 37594630
DOI: 10.1007/s00018-023-04904-7 -
The Journal of Biological Chemistry Dec 2022Transmembrane protease serine 2 (TMPRSS2) is a membrane-bound protease expressed in many human epithelial tissues, including the airway and lung. TMPRSS2-mediated...
Transmembrane protease serine 2 (TMPRSS2) is a membrane-bound protease expressed in many human epithelial tissues, including the airway and lung. TMPRSS2-mediated cleavage of viral spike protein is a key mechanism in severe acute respiratory syndrome coronavirus 2 activation and host cell entry. To date, the cellular mechanisms that regulate TMPRSS2 activity and cell surface expression are not fully characterized. In this study, we examined two major post-translational events, zymogen activation and N-glycosylation, in human TMPRSS2. In experiments with human embryonic kidney 293, bronchial epithelial 16HBE, and lung alveolar epithelial A549 cells, we found that TMPRSS2 was activated via intracellular autocatalysis and that this process was blocked in the presence of hepatocyte growth factor activator inhibitors 1 and 2. By glycosidase digestion and site-directed mutagenesis, we showed that human TMPRSS2 was N-glycosylated. N-glycosylation at an evolutionarily conserved site in the scavenger receptor cysteine-rich domain was required for calnexin-assisted protein folding in the endoplasmic reticulum and subsequent intracellular trafficking, zymogen activation, and cell surface expression. Moreover, we showed that TMPRSS2 cleaved severe acute respiratory syndrome coronavirus 2 spike protein intracellularly in human embryonic kidney 293 cells. These results provide new insights into the cellular mechanism in regulating TMPRSS2 biosynthesis and function. Our findings should help to understand the role of TMPRSS2 in major respiratory viral diseases.
Topics: Humans; Serine Proteases; Glycosylation; COVID-19; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Enzyme Precursors; Virus Internalization; Serine Endopeptidases
PubMed: 36309092
DOI: 10.1016/j.jbc.2022.102643 -
Journal of Thrombosis and Haemostasis :... Oct 2016Thrombin is a multifunctional serine protease produced from prothrombin, and is a key regulator in hemostatic and non-hemostatic processes. It is the main effector... (Review)
Review
Thrombin is a multifunctional serine protease produced from prothrombin, and is a key regulator in hemostatic and non-hemostatic processes. It is the main effector protease in primary hemostasis by activating platelets, and plays a key role in secondary hemostasis. Besides its well-known functions in hemostasis, thrombin also plays a role in various non-hemostatic biological and pathophysiologic processes, predominantly mediated through activation of protease-activated receptors (PARs). Depending on several factors, such as the concentration of thrombin, the duration of activation, the location of PARs, the presence of coreceptors, and the formation of PAR heterodimers, activation of the receptor by thrombin can induce different cellular responses. Moreover, thrombin can have opposing effects in the same cell; it can induce both inflammatory and anti-inflammatory signals. Owing to the complexity of thrombin's signal transduction pathways, the exact mechanism behind the dichotomy of thrombin is yet still unknown. In this review, we highlight the hemostatic and non-hemostatic functions of thrombin, and specifically focus on the non-hemostatic dual role of thrombin under various conditions and in relation to cardiovascular disease.
Topics: Animals; Blood Coagulation; Blood Platelets; Cardiovascular Diseases; Hemostasis; Humans; Inflammation; Platelet Activation; Protein Multimerization; Receptors, Proteinase-Activated; Receptors, Thrombin; Serine Endopeptidases; Serine Proteases; Signal Transduction; Thrombin; Transcriptional Activation
PubMed: 27513692
DOI: 10.1111/jth.13441 -
Frontiers in Immunology 2022Shiga toxin-producing O157:H7 is a virulent strain causing severe gastrointestinal infection, hemolytic uremic syndrome and death. To date there are no specific...
Shiga toxin-producing O157:H7 is a virulent strain causing severe gastrointestinal infection, hemolytic uremic syndrome and death. To date there are no specific therapies to reduce progression of disease. Here we investigated the effect of pooled immunoglobulins (IgG) on the course of disease in a mouse model of intragastric O157:H7 inoculation. Intraperitoneal administration of murine IgG on day 3, or both on day 3 and 6, post-inoculation improved survival and decreased intestinal and renal pathology. When given on both day 3 and 6 post-inoculation IgG treatment also improved kidney function in infected mice. Murine and human commercially available IgG preparations bound to proteins in culture filtrates from O157:H7. Bound proteins were extracted from membranes and peptide sequences were identified by mass spectrometry. The findings showed that murine and human IgG bound to extracellular serine protease P (EspP) in the culture filtrate, the IgG Fc domain. These results were confirmed using purified recombinant EspP and comparing culture filtrates from the wild-type O157:H7 strain to a deletion mutant lacking Culture filtrates from wild-type O157:H7 exhibited enzymatic activity, specifically associated with the presence of EspP and demonstrated as pepsin cleavage, which was reduced in the presence of murine and human IgG. EspP is a virulence factor previously shown to promote colonic cell injury and the uptake of Shiga toxin by intestinal cells. The results presented here suggest that IgG binds to EspP, blocks its enzymatic activity, and protects the host from O157:H7 infection, even when given post-inoculation.
Topics: Animals; Escherichia coli Infections; Escherichia coli O157; Escherichia coli Proteins; Immunoglobulin G; Mice; Serine; Serine Endopeptidases; Serine Proteases
PubMed: 35250980
DOI: 10.3389/fimmu.2022.807959 -
Frontiers in Immunology 2022During tumor development, invasion and metastasis, the intimate interaction between tumor and stroma shapes the tumor microenvironment and dictates the fate of tumor... (Review)
Review
During tumor development, invasion and metastasis, the intimate interaction between tumor and stroma shapes the tumor microenvironment and dictates the fate of tumor cells. Stromal cells can also influence anti-tumor immunity and response to immunotherapy. Understanding the molecular mechanisms that govern this complex and dynamic interplay, thus is important for cancer diagnosis and therapy. Proteolytic enzymes that are expressed and secreted by both cancer and stromal cells play important roles in modulating tumor-stromal interaction. Among, several serine proteases such as fibroblast activation protein, urokinase-type plasminogen activator, kallikrein-related peptidases, and granzymes have attracted great attention owing to their elevated expression and dysregulated activity in the tumor microenvironment. This review highlights the role of serine proteases that are mainly derived from stromal cells in tumor progression and associated theranostic applications.
Topics: Animals; Carcinogenesis; Endopeptidases; Extracellular Matrix; Granzymes; Humans; Kallikreins; Membrane Proteins; Neoplasms; Precision Medicine; Serine Proteases; Stromal Cells; Tumor Microenvironment; Urokinase-Type Plasminogen Activator
PubMed: 35222418
DOI: 10.3389/fimmu.2022.832418