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Molecular Oncology Nov 2023Targeted therapies for prostate, breast, and ovarian cancers are based on their activity against primary tumors rather than their anti-metastatic activity. Consequently,...
Targeted therapies for prostate, breast, and ovarian cancers are based on their activity against primary tumors rather than their anti-metastatic activity. Consequently, there is an urgent need for new agents targeting the metastatic process. Emerging evidence correlates in vitro and in vivo cancer invasion and metastasis with increased activity of the proteases mesotrypsin (prostate and breast cancer) and kallikrein 6 (KLK6; ovarian cancer). Thus, mesotrypsin and KLK6 are attractive putative targets for therapeutic intervention. As potential therapeutics for advanced metastatic prostate, breast, and ovarian cancers, we report novel mesotrypsin- and KLK6-based therapies, based on our previously developed mutants of the human amyloid β-protein precursor Kunitz protease inhibitor domain (APPI). These mutants, designated APPI-3M (prostate and breast cancer) and APPI-4M (ovarian cancer), demonstrated significant accumulation in tumors and therapeutic efficacy in orthotopic preclinical models, with the advantages of long retention times in vivo, high affinity and favorable pharmacokinetic properties. The applicability of the APPIs, as a novel therapy and for imaging purposes, is supported by their good safety profile and their controlled and scalable manufacturability in bioreactors.
Topics: Male; Humans; Female; Serine Proteinase Inhibitors; Amyloid beta-Peptides; Prostate; Amyloid beta-Protein Precursor; Breast Neoplasms; Ovarian Neoplasms; Kallikreins
PubMed: 37609678
DOI: 10.1002/1878-0261.13513 -
Frontiers in Immunology 2023Neutrophils have a critical role in the innate immune response to infection and the control of inflammation. A key component of this process is the release of neutrophil... (Review)
Review
Neutrophils have a critical role in the innate immune response to infection and the control of inflammation. A key component of this process is the release of neutrophil serine proteases (NSPs), primarily neutrophil elastase, proteinase 3, cathepsin G, and NSP4, which have essential functions in immune modulation and tissue repair following injury. Normally, NSP activity is controlled and modulated by endogenous antiproteases. However, disruption of this homeostatic relationship can cause diseases in which neutrophilic inflammation is central to the pathology, such as chronic obstructive pulmonary disease (COPD), alpha-1 antitrypsin deficiency, bronchiectasis, and cystic fibrosis, as well as many non-pulmonary pathologies. Although the pathobiology of these diseases varies, evidence indicates that excessive NSP activity is common and a principal mediator of tissue damage and clinical decline. NSPs are synthesized as inactive zymogens and activated primarily by the ubiquitous enzyme dipeptidyl peptidase 1, also known as cathepsin C. Preclinical data confirm that inactivation of this protease reduces activation of NSPs. Thus, pharmacological inhibition of dipeptidyl peptidase 1 potentially reduces the contribution of aberrant NSP activity to the severity and/or progression of multiple inflammatory diseases. Initial clinical data support this view. Ongoing research continues to explore the role of NSP activation by dipeptidyl peptidase 1 in different disease states and the potential clinical benefits of dipeptidyl peptidase 1 inhibition.
Topics: Humans; Neutrophils; Serine Proteases; Protease Inhibitors; Cathepsin C; Inflammation
PubMed: 38162644
DOI: 10.3389/fimmu.2023.1239151 -
Journal of Proteome Research Apr 2024Trypsin is the gold-standard protease in bottom-up proteomics, but many sequence stretches of the proteome are inaccessible to trypsin and standard LC-MS approaches....
Trypsin is the gold-standard protease in bottom-up proteomics, but many sequence stretches of the proteome are inaccessible to trypsin and standard LC-MS approaches. Thus, multienzyme strategies are used to maximize sequence coverage in post-translational modification profiling. We present fast and robust SP3- and STRAP-based protocols for the broad-specificity proteases subtilisin, proteinase K, and thermolysin. All three enzymes are remarkably fast, producing near-complete digests in 1-5 min, and cost 200-1000× less than proteomics-grade trypsin. Using FragPipe resolved a major challenge by drastically reducing the duration of the required "unspecific" searches. In-depth analyses of proteinase K, subtilisin, and thermolysin Jurkat digests identified 7374, 8178, and 8753 unique proteins with average sequence coverages of 21, 29, and 37%, including 10,000s of amino acids not reported in PeptideAtlas' >2400 experiments. While we could not identify distinct cleavage patterns, machine learning could distinguish true protease products from random cleavages, potentially enabling the prediction of cleavage products. Finally, proteinase K, subtilisin, and thermolysin enabled label-free quantitation of 3111, 3659, and 4196 unique Jurkat proteins, which in our hands is comparable to trypsin. Our data demonstrate that broad-specificity proteases enable quantitative proteomics of uncharted areas of the proteome. Their fast kinetics may allow "on-the-fly" digestion of samples in the future.
Topics: Peptide Hydrolases; Proteomics; Trypsin; Proteome; Endopeptidase K; Thermolysin; Subtilisins
PubMed: 38457694
DOI: 10.1021/acs.jproteome.3c00852 -
Microbiology Spectrum Dec 2023Mycoparasites play important roles in the biocontrol of plant fungal diseases, during which they secret multiple hydrolases such as serine proteases to degrade their...
Mycoparasites play important roles in the biocontrol of plant fungal diseases, during which they secret multiple hydrolases such as serine proteases to degrade their fungal hosts. In this study, we demonstrated that the serine protease CrKP43 was involved in development and mycoparasitism with the regulation of Crmapk. To the best of our knowledge, it is the first report on the functions and regulatory mechanisms of serine proteases in . Our findings will provide new insight into the regulatory mechanisms of serine proteases in mycoparasites and contribute to clarifying the mechanisms underlying mycoparasitism of , which will facilitate the development of highly efficient fungal biocontrol agents as well.
Topics: Serine Proteases; Hypocreales; Serine Endopeptidases; Plant Diseases; Fungal Proteins
PubMed: 37831480
DOI: 10.1128/spectrum.02448-23 -
Die Pharmazie Dec 2023The global pandemic of COVID-19 disease is caused by the pathogenic factor called SARS-CoV-2. Meanwhile, a series of vaccines and small-molecule drugs, including the... (Review)
Review
The global pandemic of COVID-19 disease is caused by the pathogenic factor called SARS-CoV-2. Meanwhile, a series of vaccines and small-molecule drugs, including the mRNA vaccines and Paxlovid, have been approved, but their efficacy is decreased significantly due to the constant emergence of mutant viral strains. The R&D of host-directed therapeutics has great potential to overcome such limitations and provide new prevention and therapy options for patients with COVID-19 or high-risk group for SARS-CoV-2 infections. Transmembrane serine protease 2 (TMPRSS2) is belonging to a protein family with highly conserved serine protease domain whose crucial role in viral entry is to activate the spike protein of viruses to induce the fusion between host cells and viruses. In this review, we sketch the critical position of TMPRSS2 in the SARS-CoV-2 viral entry and summarize the advanced research and development of TMPRSS2 inhibitors, including repurposed drugs, as a new way to fight COVID-19.
Topics: Humans; COVID-19; Drug Repositioning; Serine Endopeptidases; Serine Proteases; Serine Proteinase Inhibitors
PubMed: 38178286
DOI: 10.1691/ph.2023.3578 -
Progress in Retinal and Eye Research Nov 2023Age-related macular degeneration (AMD) is the leading cause of severe irreversible central vision loss in individuals over 65 years old. Genome-wide association studies... (Review)
Review
Age-related macular degeneration (AMD) is the leading cause of severe irreversible central vision loss in individuals over 65 years old. Genome-wide association studies (GWASs) have shown that the region at chromosome 10q26, where the age-related maculopathy susceptibility (ARMS2/LOC387715) and HtrA serine peptidase 1 (HTRA1) genes are located, represents one of the strongest associated loci for AMD. However, the underlying biological mechanism of this genetic association has remained elusive. In this article, we extensively review the literature by us and others regarding the ARMS2/HTRA1 risk alleles and their functional significance. We also review the literature regarding the presumed function of the ARMS2 protein and the molecular processes of the HTRA1 protein in AMD pathogenesis in vitro and in vivo, including those of transgenic mice overexpressing HtrA1/HTRA1 which developed Bruch's membrane (BM) damage, choroidal neovascularization (CNV), and polypoidal choroidal vasculopathy (PCV), similar to human AMD patients. The elucidation of the molecular mechanisms of the ARMS2 and HTRA1 susceptibility loci has begun to untangle the complex biological pathways underlying AMD pathophysiology, pointing to new testable paradigms for treatment.
Topics: Animals; Humans; Genetic Predisposition to Disease; Genotype; High-Temperature Requirement A Serine Peptidase 1; Macular Degeneration; Polymorphism, Single Nucleotide; Proteins; Risk Factors; Serine Endopeptidases
PubMed: 36581531
DOI: 10.1016/j.preteyeres.2022.101159 -
Frontiers in Immunology 2023Insect phenoloxidases (POs) catalyze phenol oxygenation and -diphenol oxidation to form reactive intermediates that kill invading pathogens and form melanin polymers. To...
Insect phenoloxidases (POs) catalyze phenol oxygenation and -diphenol oxidation to form reactive intermediates that kill invading pathogens and form melanin polymers. To reduce their toxicity to host cells, POs are produced as prophenoloxidases (PPOs) and activated by a serine protease cascade as required. In most insects studied so far, PPO activating proteases (PAPs) generate active POs in the presence of a high cofactor, comprising two serine protease homologs (SPHs) each with a Gly residue replacing the catalytic Ser of an S1A serine protease (SP). These SPHs have a regulatory clip domain at the N-terminus, like most of the SP cascade members including PAPs. In , PPO activation and PO-catalyzed melanization have been examined in genetic analyses but it is unclear if a cofactor is required for PPO activation. In this study, we produced the recombinant cSPH35 and cSPH242 precursors, activated them with PAP3, and confirmed their predicted role as a cofactor for PPO1 activation by MP2 (., Sp7). The cleavage sites and mechanisms for complex formation and cofactor function are highly similar to those reported in . In the presence of high complexes of the cSPHs, PO at a high specific activity of 260 U/μg was generated . To complement the analysis, we measured hemolymph PO activity levels in wild-type flies, cSPH35, and cSPH242 RNAi lines. Compared with the wild-type flies, only 4.4% and 18% of the control PO level (26 U/μl) was detected in the cSPH35 and cSPH242 knockdowns, respectively. Consistently, percentages of adults with a melanin spot at the site of septic pricking were 82% in wild-type, 30% in cSPH35 RNAi, and 53% in cSPH242 RNAi lines; the survival rate of the control (45%) was significantly higher than those (30% and 15%) of the two RNAi lines. These data suggest that cSPH35 and cSPH242 are components of a cofactor for MP2-mediated PPO1 activation, which are indispensable for early melanization in adults.
Topics: Animals; Drosophila melanogaster; Drosophila Proteins; Melanins; Monophenol Monooxygenase; Serine Endopeptidases; Serine Proteases; Catechol Oxidase; Enzyme Precursors
PubMed: 37781370
DOI: 10.3389/fimmu.2023.1244792 -
Diabetes Dec 2023There is clinical evidence that increased urinary serine proteases are associated with the disease severity in the setting of diabetic nephropathy (DN). Elevation of...
UNLABELLED
There is clinical evidence that increased urinary serine proteases are associated with the disease severity in the setting of diabetic nephropathy (DN). Elevation of serine proteases may mediate [Ca2+]i dynamics in podocytes through the protease-activated receptors (PARs) pathway, including associated activation of nonspecific cation channels. Cultured human podocytes and freshly isolated glomeruli were used for fluorescence and immunohistochemistry stainings, calcium imaging, Western blot analysis, scanning ion conductance microscopy, and patch clamp analysis. Goto-Kakizaki, Wistar, type 2 DN (T2DN), and a novel PAR1 knockout on T2DN rat background rats were used to test the importance of PAR1-mediated signaling in DN settings. We found that PAR1 activation increases [Ca2+]i via TRPC6 channels. Both human cultured podocytes exposed to high glucose and podocytes from freshly isolated glomeruli of T2DN rats had increased PAR1-mediated [Ca2+]i compared with controls. Imaging experiments revealed that PAR1 activation plays a role in podocyte morphological changes. T2DN rats exhibited a significantly higher response to thrombin and urokinase. Moreover, the plasma concentration of thrombin in T2DN rats was significantly elevated compared with Wistar rats. T2DNPar1-/- rats were embryonically lethal. T2DNPar1+/- rats had a significant decrease in glomerular damage associated with DN lesions. Overall, these data provide evidence that, during the development of DN, elevated levels of serine proteases promote an excessive [Ca2+]i influx in podocytes through PAR1-TRPC6 signaling, ultimately leading to podocyte apoptosis, the development of albuminuria, and glomeruli damage.
ARTICLE HIGHLIGHTS
Increased urinary serine proteases are associated with diabetic nephropathy. During the development of diabetic nephropathy in type 2 diabetes, the elevation of serine proteases could overstimulate protease-activated receptor 1 (PAR1). PAR1 signaling is involved in the development of DN via TRPC6-mediated intracellular calcium signaling. This study provides fundamental knowledge that can be used to develop efficient therapeutic approaches targeting serine proteases or corresponding PAR pathways to prevent or slow the progression of diabetes-associated kidney diseases.
Topics: Rats; Humans; Animals; Diabetic Nephropathies; Podocytes; Receptor, PAR-1; TRPC6 Cation Channel; Calcium; Diabetes Mellitus, Type 2; Thrombin; Rats, Wistar
PubMed: 37722138
DOI: 10.2337/db23-0032 -
The New England Journal of Medicine Nov 2023Two siblings presented with cardiomyopathy, hypertension, arrhythmia, and fibrosis of the left atrium. Each had a homozygous null variant in , the gene encoding atrial...
Two siblings presented with cardiomyopathy, hypertension, arrhythmia, and fibrosis of the left atrium. Each had a homozygous null variant in , the gene encoding atrial natriuretic peptide (ANP)-converting enzyme. A plasma sample obtained from one of the siblings had no detectable levels of corin or N-terminal pro-ANP but had elevated levels of B-type natriuretic peptide (BNP) and one of the two protein markers of fibrosis that we tested. These and other findings support the hypothesis that BNP cannot fully compensate for a lack of activation of the ANP pathway and that corin is critical to normal ANP activity, left atrial function, and cardiovascular homeostasis.
Topics: Humans; Arrhythmias, Cardiac; Atrial Fibrillation; Atrial Natriuretic Factor; Cardiomyopathies; Fibrosis; Heart Atria; Hypertension; Natriuretic Peptide, Brain; Serine Endopeptidases; Siblings
PubMed: 37913506
DOI: 10.1056/NEJMoa2301908 -
Matriptase drives dissemination of ovarian cancer spheroids by a PAR-2/PI3K/Akt/MMP9 signaling axis.The Journal of Cell Biology Nov 2023The transmembrane serine protease matriptase is a key regulator of both barrier-disruptive and protective epithelial cell-cell interactions. Elevated matriptase is a...
The transmembrane serine protease matriptase is a key regulator of both barrier-disruptive and protective epithelial cell-cell interactions. Elevated matriptase is a consistent feature of epithelial ovarian cancers (OvCa), where multicellular spheroids shed from the primary tumor into the peritoneal cavity are critical drivers of metastasis. Dynamic cell-to-cell adhesive contacts are required for spheroid formation and maintenance. Here, we show that overactive matriptase, reflected in an increased ratio of matriptase to its inhibitor hepatocyte growth factor activator inhibitor 1 (HAI-1), disrupts cell-cell contacts to produce loose prometastatic spheroids that display increased mesothelial cell adhesion and submesothelial invasion. We show that these activities are dependent on the matriptase activation of a protease-activated receptor-2 (PAR-2) signaling pathway involving PI3K/Akt and MMP9-induced disruption of cell-cell adhesion by the release of the soluble E-cadherin ectodomain. These data reveal a novel pathological connection between matriptase activation of PAR-2 and disruption of cell-cell adhesion, and support the clinical investigation of this signaling axis as a therapeutic strategy for aggressive metastatic OvCa.
Topics: Female; Humans; Matrix Metalloproteinase 9; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Spheroids, Cellular; Serine Endopeptidases
PubMed: 37737895
DOI: 10.1083/jcb.202209114