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International Journal For Parasitology Apr 2010The exoskeleton or cuticle performs many key roles in the development and survival of all nematodes. This structure is predominantly collagenous in nature and requires...
The exoskeleton or cuticle performs many key roles in the development and survival of all nematodes. This structure is predominantly collagenous in nature and requires numerous enzymes to properly fold, modify, process and cross-link these essential structural proteins. The cuticle structure and its collagen components are conserved throughout the nematode phylum but differ from the collagenous matrices found in vertebrates. This structure, its formation and the enzymology of nematode cuticle collagen biogenesis have been elucidated in the free-living nematode Caenorhabditis elegans. The dpy-31 gene in C. elegans encodes a procollagen C-terminal processing enzyme of the astacin metalloprotease or bone morphogenetic protein class that, when mutated, results in a temperature-sensitive lethal phenotype associated with cuticle defects. In this study, orthologues of this essential gene have been identified in the phylogenetically diverse parasitic nematodes Haemonchus contortus and Brugia malayi. The DPY-31 protein is expressed in the gut and secretory system of C. elegans, a location also confirmed when a B. malayi transcriptional dpy-31 promoter-reporter gene fusion was expressed in C. elegans. Functional conservation between the nematode enzymes was supported by the fact that heterologous expression of the H. contortus dpy-31 orthologue in a C. elegans dpy-31 mutant resulted in the full rescue of the mutant body form. This interspecies conservation was further established when the recombinant nematode enzymes were found to have a similar range of inhibitable protease activities. In addition, the recombinant DPY-31 enzymes from both H. contortus and B. malayi were shown to efficiently process the C. elegans cuticle collagen SQT-3 at the correct C-terminal procollagen processing site.
Topics: Amino Acid Sequence; Animals; Brugia malayi; Caenorhabditis elegans Proteins; Collagen; Gastrointestinal Tract; Genes, Reporter; Haemonchus; Helminth Proteins; Metalloendopeptidases; Metalloproteases; Microscopy; Microscopy, Fluorescence; Molecular Sequence Data; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Skin
PubMed: 19883650
DOI: 10.1016/j.ijpara.2009.10.007 -
Toxins Dec 2017Snake venom metalloproteinases (SVMPs) are predominant in viperid venoms, which provoke hemorrhage and affect hemostasis and thrombosis. P-I class enzymes consist only... (Review)
Review
Snake venom metalloproteinases (SVMPs) are predominant in viperid venoms, which provoke hemorrhage and affect hemostasis and thrombosis. P-I class enzymes consist only of a single metalloproteinase domain. Despite sharing high sequence homology, only some of them induce hemorrhage. They have direct fibrin(ogen)olytic activity. Their main biological substrate is fibrin(ogen), whose Aα-chain is degraded rapidly and independently of activation of plasminogen. It is important to understand their biochemical and physiological mechanisms, as well as their applications, to study the etiology of some human diseases and to identify sites of potential intervention. As compared to all current antiplatelet therapies to treat cardiovascular events, the SVMPs have outstanding biochemical attributes: (a) they are insensitive to plasma serine proteinase inhibitors; (b) they have the potential to avoid bleeding risk; (c) mechanistically, they are inactivated/cleared by α2-macroglobulin that limits their range of action in circulation; and (d) few of them also impair platelet aggregation that represent an important target for therapeutic intervention. This review will briefly highlight the structure-function relationships of these few direct-acting fibrinolytic agents, including, barnettlysin-I, isolated from venom, that could be considered as potential agent to treat major thrombotic disorders. Some of their pharmacological advantages are compared with plasmin.
Topics: Amino Acid Sequence; Animals; Catalytic Domain; Fibrinolytic Agents; Hemostasis; Humans; Metalloproteases; Snake Venoms; Snakes; Species Specificity; Structure-Activity Relationship
PubMed: 29206190
DOI: 10.3390/toxins9120392 -
Frontiers in Immunology 2022Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) with the involvement of immune cells and molecules, including cytokines,... (Review)
Review
Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) with the involvement of immune cells and molecules, including cytokines, chemokines and proteases. A previous extensive review about the molecular biology of matrix metalloproteases (MMPs) and tissue inhibitors of metalloproteases (TIMPs), related to intestinal barrier destruction and restoration functions in IBD, is here complemented with the literature from the last five years. We also compare IBD as a prototypic mucosal inflammation of an epithelial barrier against microorganisms with inflammatory retinopathy as a disease with a barrier dysfunction at the level of blood vessels. Multiple reasons are at the basis of halting clinical trials with monoclonal antibodies against MMP-9 for IBD treatment. These include (i) the absence of a causative role of MMP-9 in the pathology in animal models of IBD, (ii) the fact that endotoxins, crossing the intestinal barrier, induce massive local release of both neutrophil collagenase (MMP-8) and gelatinase B (MMP-9), (iii) insufficient recognition that MMPs modify the activities of cytokines, chemokines and their receptors, (iv) ignorance that MMPs exist as mixtures of proteoforms with different posttranslational modifications and with different specific activities and (v) the fact that MMPs and TIMPs act in an interactive network, possibly having also beneficial effects on IBD evolution. Nevertheless, inhibition of MMPs may be a useful therapeutic approach during specific IBD disease phases or in specific sub-phenotypes. This temporary "window of opportunity" for MMP-9 inhibition may be complemented by a locoregional one, provided that the pharmacological agents are targeted in time to affected tissues, as is achieved in ophthalmological inflammation. Thus, in order to discover spatial and temporal windows of opportunity for MMP inhibition as treatment of IBD, more preclinical work including well controlled animal studies will be further needed. In this respect, MMP-9/NGAL complex analysis in various body compartments is helpful for better stratification of IBD patients who may benefit from anti-MMP-9.
Topics: Animals; Antibodies, Monoclonal; Chemokines; Cytokines; Endotoxins; Inflammation; Inflammatory Bowel Diseases; Lipocalin-2; Matrix Metalloproteinase 8; Matrix Metalloproteinase 9; Tissue Inhibitor of Metalloproteinases
PubMed: 36164340
DOI: 10.3389/fimmu.2022.983964 -
The FEBS Journal Jun 2014Bacteroides fragilis causes the majority of anaerobic infections in humans. The presence of a pathogenicity island in the genome discriminates pathogenic and commensal...
Bacteroides fragilis causes the majority of anaerobic infections in humans. The presence of a pathogenicity island in the genome discriminates pathogenic and commensal B. fragilis strains. The island encodes metalloproteinase II (MPII), a potential virulence protein, and one of three homologous fragilysin isozymes (FRA; also termed B. fragilis toxin or BFT). Here, we report biochemical data on the structural-functional characteristics of the B. fragilis pathogenicity island proteases by reporting the crystal structure of MPII at 2.13 Å resolution, combined with detailed characterization of the cleavage preferences of MPII and FRA3 (as a representative of the FRA isoforms), identified using a high-throughput peptide cleavage assay with 18 583 substrate peptides. We suggest that the evolution of the MPII catalytic domain can be traced to human and archaebacterial proteinases, whereas the prodomain fold is a feature specific to MPII and FRA. We conclude that the catalytic domain of both MPII and FRA3 evolved differently relative to the prodomain, and that the prodomain evolved specifically to fit the B. fragilis pathogenicity. Overall, our data provide insights into the evolution of cleavage specificity and activation mechanisms in the virulent metalloproteinases.
Topics: Bacteroides fragilis; Genomic Islands; Metalloproteases
PubMed: 24698179
DOI: 10.1111/febs.12804 -
Identification and activity of inhibitors of the essential nematode-specific metalloprotease DPY-31.Bioorganic & Medicinal Chemistry Letters Dec 2015Infection by parasitic nematodes is widespread in the developing world causing extensive morbidity and mortality. Furthermore, infection of animals is a global problem,...
Infection by parasitic nematodes is widespread in the developing world causing extensive morbidity and mortality. Furthermore, infection of animals is a global problem, with a substantial impact on food production. Here we identify small molecule inhibitors of a nematode-specific metalloprotease, DPY-31, using both known metalloprotease inhibitors and virtual screening. This strategy successfully identified several μM inhibitors of DPY-31 from both the human filarial nematode Brugia malayi, and the parasitic gastrointestinal nematode of sheep Teladorsagia circumcincta. Further studies using both free living and parasitic nematodes show that these inhibitors elicit the severe body morphology defect 'Dumpy' (Dpy; shorter and fatter), a predominantly non-viable phenotype consistent with mutants lacking the DPY-31 gene. Taken together, these results represent a start point in developing DPY-31 inhibition as a totally novel mechanism for treating infection by parasitic nematodes in humans and animals.
Topics: Animals; Binding Sites; Brugia malayi; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Helminth Proteins; Humans; Hydroxamic Acids; Inhibitory Concentration 50; Metalloendopeptidases; Metalloproteases; Molecular Docking Simulation; Nematoda; Protease Inhibitors; Protein Structure, Tertiary; Recombinant Proteins; Sheep
PubMed: 26546217
DOI: 10.1016/j.bmcl.2015.10.077 -
MBio Jul 2013The sexually transmitted infection gonorrhea is caused exclusively by the human-specific pathogen Neisseria gonorrhoeae. Type IV pili are an essential virulence factor...
UNLABELLED
The sexually transmitted infection gonorrhea is caused exclusively by the human-specific pathogen Neisseria gonorrhoeae. Type IV pili are an essential virulence factor uniformly expressed on clinical gonococcal isolates and are required for several aspects of gonococcal pathogenesis, including adherence to host tissues, autoagglutination, twitching motility, and the uptake of DNA during transformation. Symptomatic gonococcal infection is characterized by the influx of neutrophils or polymorphonuclear leukocytes (PMNs) to the site of infection. PMNs are a key component of gonococcal pathogenesis, mediating the innate immune response through the use of oxidative and nonoxidative killing mechanisms. The M23B family zinc metallopeptidase NGO1686 is required for gonococci to survive oxidative killing by H2O2- and PMN-mediated killing through unknown mechanisms, but the only known target of NGO1686 is peptidoglycan. We report that the effect of NGO1686 on survival after exposure to H2O2 and PMNs is mediated through its role in elaborating pili and that nonpiliated mutants of N. gonorrhoeae are less resistant to killing by H2O2, LL-37, and PMNs than the corresponding piliated strains. These findings add to the various virulence-associated functions attributable to gonococcal pili and may explain the selection basis for piliation in clinical isolates of N. gonorrhoeae.
IMPORTANCE
Successful infectious agents need to overcome host defense systems to establish infection. We show that the Neisseria pilus, a major virulence factor of this organism, which causes gonorrhea, helps protect the bacterium from two major killing mechanisms used by the host to combat infections. We also show that to express the pilus, an enzyme needs to partially degrade the cell wall of the bacterium.
Topics: Fimbriae, Bacterial; Host-Pathogen Interactions; Humans; Hydrogen Peroxide; Metalloproteases; Microbial Viability; Neisseria gonorrhoeae; Neutrophils; Oxidative Stress; Virulence; Virulence Factors
PubMed: 23839218
DOI: 10.1128/mBio.00399-13 -
Analytical Biochemistry Jan 2011Inductively coupled plasma-mass spectrometry (ICP-MS)-based assays lend themselves to multiplexing due to the high resolution between mass channels, the sensitivity, and...
Inductively coupled plasma-mass spectrometry (ICP-MS)-based assays lend themselves to multiplexing due to the high resolution between mass channels, the sensitivity, and the reliability of the technique. Here the potential of ICP-MS-based protease assays is demonstrated with a quadruplex assay of cysteine proteases and metalloproteases. Four orthogonal peptide substrates were synthesized for the proteases calpain-1, caspase-3, matrix metalloprotease-9 (MMP-9), and a disintegrin and metalloprotease-10 (ADAM10). Each substrate carries a biotin tag at the C terminus and a diethylenetriaminepentaacetic acid (DTPA)-based lanthanide complex at the N terminus. The results demonstrate that this is a simple and reproducible analysis technique with excellent correlation between the single and multiplex assay formats.
Topics: ADAM Proteins; Biotin; Calpain; Caspase 3; Cysteine Proteases; Disintegrins; Mass Spectrometry; Matrix Metalloproteinase 9; Metalloproteases; Streptavidin; Substrate Specificity
PubMed: 20849809
DOI: 10.1016/j.ab.2010.09.008 -
The International Journal of... 2008Wound healing is a dynamic process that involves a coordinated response of many cell types representing distinct tissue compartments and is fundamentally similar among... (Review)
Review
Wound healing is a dynamic process that involves a coordinated response of many cell types representing distinct tissue compartments and is fundamentally similar among tissue types. Among the many gene products that are essential for restoration of normal tissue architecture, several members of the matrix metalloproteinase (MMP) family function as positive and, at times, negative regulators of repair processes. MMPs were initially thought to only function in the resolution phase of wound healing, particularly during scar resorption; however, recent evidence suggests that they also influence other wound-healing responses, such as inflammation and re-epithelialization. In this review, we discuss what is currently known about the function of MMPs in wound healing and will provide suggestions for future research directions.
Topics: Animals; Gene Expression Regulation, Enzymologic; Humans; Metalloproteases; Models, Biological; Tissue Inhibitor of Metalloproteinases; Wound Healing
PubMed: 18083622
DOI: 10.1016/j.biocel.2007.10.024 -
Journal of Medicinal Chemistry Jan 2023The mitochondrial rhomboid protease PARL regulates mitophagy by balancing intramembrane proteolysis of PINK1 and PGAM5. It has been implicated in the pathogenesis of...
The mitochondrial rhomboid protease PARL regulates mitophagy by balancing intramembrane proteolysis of PINK1 and PGAM5. It has been implicated in the pathogenesis of Parkinson's disease, but its investigation as a possible therapeutic target is challenging in this context because genetic deficiency of PARL may result in compensatory mechanisms. To address this problem, we undertook a hitherto unavailable chemical biology strategy. We developed potent PARL-targeting ketoamide inhibitors and investigated the effects of acute PARL suppression on the processing status of PINK1 intermediates and on Parkin activation. This approach revealed that PARL inhibition leads to a robust activation of the PINK1/Parkin pathway without major secondary effects on mitochondrial properties, which demonstrates that the pharmacological blockage of PARL to boost PINK1/Parkin-dependent mitophagy is a feasible approach to examine novel therapeutic strategies for Parkinson's disease. More generally, this study showcases the power of ketoamide inhibitors for cell biological studies of rhomboid proteases.
Topics: Humans; Peptide Hydrolases; Metalloproteases; Mitophagy; Parkinson Disease; Protein Kinases; Mitochondrial Proteins; Endopeptidases; Ubiquitin-Protein Ligases
PubMed: 36540942
DOI: 10.1021/acs.jmedchem.2c01092 -
International Journal of Molecular... Mar 2021Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that have been associated not only with various cellular processes, such as...
Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that have been associated not only with various cellular processes, such as embryonic development and adult wound healing but also enhanced tumor survival, angiogenesis, and metastatic spread. Proteolytic cleavage of these single-pass transmembrane receptors has been suggested to regulate biological activities of their ligands during growth and development, yet little is known about the proteases responsible for this process. In this study, we monitored the release of membrane-anchored FGFRs 1, 2, 3, and 4 in cell-based assays. We demonstrate here that metalloprotease-dependent metalloprotease family, ADAM10 and ADAM17. Loss- and gain-of-function studies in murine embryonic fibroblasts showed that constitutive shedding as well as phorbol-ester-induced processing of FGFRs 1, 3, and 4 is mediated by ADAM17. In contrast, treatment with the calcium ionophore ionomycin stimulated ADAM10-mediated FGFR2 shedding. Cell migration assays with keratinocytes in the presence or absence of soluble FGFRs suggest that ectodomain shedding can modulate the function of ligand-induced FGFR signaling during cell movement. Our data identify ADAM10 and ADAM17 as differentially regulated FGFR membrane sheddases and may therefore provide new insight into the regulation of FGFR functions.
Topics: ADAM10 Protein; ADAM17 Protein; Animals; Cell Line; Cell Movement; Enzyme Activation; Epithelial Cells; Membrane Proteins; Metalloproteases; Multigene Family; Protein Binding; Protein Interaction Domains and Motifs; Protein Isoforms; Protein Kinase C; Proteolysis; Receptors, Fibroblast Growth Factor
PubMed: 33804608
DOI: 10.3390/ijms22063165