-
Scientific Reports Nov 2023This study addresses the environmental risks associated with the accumulation of keratin waste from poultry, which is resistant to conventional protein degradation...
This study addresses the environmental risks associated with the accumulation of keratin waste from poultry, which is resistant to conventional protein degradation methods. To tackle this issue, microbial keratinases have emerged as promising tools for transforming resilient keratin materials into valuable products. We focus on the Metalloprotease (MetPr) gene isolated from novel Pichia kudriavzevii YK46, sequenced, and deposited in the NCBI GenBank database with the accession number OQ511281. The MetPr gene encodes a protein consisting of 557 amino acids and demonstrates a keratinase activity of 164.04 U/ml. The 3D structure of the protein was validated using Ramachandran's plot, revealing that 93% and 97.26% of the 557 residues were situated within the most favoured region for the MetPr proteins of template Pichia kudriavzevii strain 129 and Pichia kudriavzevii YK46, respectively. Computational analyses were employed to determine the binding affinities between the deduced protein and beta keratin. Molecular docking studies elucidated the optimal binding affinities between the metalloprotease (MetPr) and beta-keratin, yielding values of - 260.75 kcal/mol and - 257.02 kcal/mol for the template strains Pichia kudriavzevii strain 129 and Pichia kudriavzevii YK46, respectively. Subsequent molecular cloning and expression of the MetPr gene in E. coli DH5α led to a significantly higher keratinase activity of 281 ± 12.34 U/ml. These findings provide valuable insights into the potential of the MetPr gene and its encoded protein for keratin waste biotransformation, with implications for addressing environmental concerns related to keratinous waste accumulation.
Topics: Animals; Feathers; Escherichia coli; Molecular Docking Simulation; Pichia; Metalloproteases; Keratins; Cloning, Molecular
PubMed: 37968282
DOI: 10.1038/s41598-023-47179-5 -
The European Respiratory Journal Nov 1994One critical event of tumour invasion that signals the initiation of the metastatic cascade is thought to be interaction of the tumour cell with the basement membrane.... (Review)
Review
One critical event of tumour invasion that signals the initiation of the metastatic cascade is thought to be interaction of the tumour cell with the basement membrane. Basement membranes may also pose as barriers to tumour cell invasion at multiple points later in the metastatic cascade, including during the processes of vascular infiltration and extravasation. Thus, an important proteolytic event in the metastatic cascade, and also angiogenesis, appears to be degradation of basement membrane components. A specific class of extracellular matrix degrading metalloenzymes, the matrix metalloproteases, and their endogenous inhibitors, the tissue inhibitors of metalloproteases, are thought to have a role in the creation of the proteolytic defect in basement membrane type IV collagen. We will review the evidence which indicates that matrix metalloproteases and tissue inhibitors of metalloproteases are essential for tumour cell invasion and angiogenesis. The regulation of matrix metalloproteases will be discussed, including gene activation and transcription, messenger ribonucleic acid (mRNA) stability, binding of proenzymes to cell membranes and/or matrix components, proenzyme activation, and inactivation by endogenous inhibitors. We will also discuss the mechanism for tissue inhibitor of metalloproteases-mediated inhibition of tumour invasion and angiogenesis. This appears, at least in part, to be through inhibition of protease activity required for cellular invasion, although recent observations suggest that tissue inhibitors of metalloproteases affect other distinct groups of biological activities through mechanisms other than matrix metalloprotease inhibition.
Topics: Amino Acid Sequence; Animals; Glycoproteins; Humans; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Neovascularization, Pathologic; Proteins; Tissue Inhibitor of Metalloproteinase-2; Tissue Inhibitor of Metalloproteinase-3; Tissue Inhibitor of Metalloproteinases
PubMed: 7533104
DOI: No ID Found -
Scientific Reports Sep 2015Mitochondria are involved in key cellular functions including energy production, metabolic homeostasis, and apoptosis. Normal mitochondrial function is preserved by...
Mitochondria are involved in key cellular functions including energy production, metabolic homeostasis, and apoptosis. Normal mitochondrial function is preserved by several interrelated mechanisms. One mechanism - intramitochondrial quality control (IMQC) - is represented by conserved proteases distributed across mitochondrial compartments. Many aspects and physiological roles of IMQC components remain unclear. Here, we show that the IMQC protease Oma1 is required for the stability of the respiratory supercomplexes and thus balanced and tunable bioenergetic function. Loss of Oma1 activity leads to a specific destabilization of respiratory supercomplexes and consequently to unbalanced respiration and progressive respiratory decline in yeast. Similarly, experiments in cultured Oma1-deficient mouse embryonic fibroblasts link together impeded supercomplex stability and inability to maintain proper respiration under conditions that require maximal bioenergetic output. Finally, transient knockdown of OMA1 in zebrafish leads to impeded bioenergetics and morphological defects of the heart and eyes. Together, our biochemical and genetic studies in yeast, zebrafish and mammalian cells identify a novel and conserved physiological role for Oma1 protease in fine-tuning of respiratory function. We suggest that this unexpected physiological role is important for cellular bioenergetic plasticity and may contribute to Oma1-associated disease phenotypes in humans.
Topics: Animals; Cell Line; Embryo, Nonmammalian; Energy Metabolism; Larva; Metalloproteases; Mice; Mitochondria; Mitochondrial Proteins; Morpholinos; Phenotype; Protein Stability; RNA Interference; RNA, Small Interfering; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Zebrafish; Zebrafish Proteins
PubMed: 26365306
DOI: 10.1038/srep13989 -
Cellular & Molecular Immunology Jun 2005In an attempt to isolate and characterize peptides mimicking epitopes of metalloprotease and explore their immunological protection against Schistosoma japonicum (S....
In an attempt to isolate and characterize peptides mimicking epitopes of metalloprotease and explore their immunological protection against Schistosoma japonicum (S. japonicum), polyclonal anti-metalloprotease sera was prepared to screen a 12-mer random peptide library to isolate phages binding specially to antisera IgG. Then, phage ELISA, animal immunization, DNA sequencing, Western blotting and enzymatic activity neutralizing analysis were used to characterize the selected phage clones. All of ten randomly picked clones were shown to be positive. Five peptides of different amino acid sequences deduced from DNA sequences were obtained and two of them (peptides 2 and 3) could induce significant reduction (31.0% and 31.8%, respectively) in worm burden and high reduction (52.6% and 54.9%, respectively) in liver eggs per gram (LEPG), while, unexpectedly, others (peptides 1, 4 and 5) could not elicit enough protection against infection of S. japonicum. Peptides 2 and 3 could be recognized by S. japonicum infected mouse sera (IMS) and could elicit neutralizing Abs. The results show that peptides 2 and 3 are antigenic and immunogenic. They are true mimics of epitopes of metalloprotease and useful as novel vaccine candidates against S. japonicum.
Topics: Amino Acid Sequence; Animals; Base Sequence; Epitopes; Gelatin; Immunoblotting; Metalloproteases; Mice; Molecular Mimicry; Molecular Sequence Data; Peptides; Schistosoma japonicum; Schistosomiasis japonica; Substrate Specificity; Vaccination
PubMed: 16212890
DOI: No ID Found -
Frontiers in Cellular and Infection... 2022Extracellular proteolytic enzymes are produced by a variety of pathogenic microorganisms, and contribute to host colonization by modulating virulence. Here, we present a...
Extracellular proteolytic enzymes are produced by a variety of pathogenic microorganisms, and contribute to host colonization by modulating virulence. Here, we present a first characterization of leptolysin, a metalloprotease of the pappalysin family identified in a previous exoproteomic study. Comparative molecular analysis of leptolysin with two other pappalysins from prokaryotes, ulilysin and mirolysin, reveals similarities regarding calcium, zinc, and arginine -binding sites conservation within the catalytic domain, but also discloses peculiarities. Variations observed in the primary and tertiary structures may reflect differences in primary specificities. Purified recombinant leptolysin of was obtained as a ~50 kDa protein. The protease exhibited maximal activity at pH 8.0 and 37°C, and hydrolytic activity was observed in the presence of different salts with maximum efficiency in NaCl. Substrate specificity was assessed using a small number of FRET peptides, and showed a marked preference for arginine residues at the P1 position. leptolysin proteolytic activity on proteinaceous substrates such as proteoglycans and plasma fibronectin was also evaluated. All proteins tested were efficiently degraded over time, confirming the protease´s broad-spectrum activity . In addition, leptolysin induced morphological alterations on HK-2 cells, which may be partially attributed to extracellular matrix (ECM) degradation. Hemorrhagic foci were observed in the dorsal skin of mice intradermally injected with leptolysin, as a plausible consequence of ECM disarray and vascular endothelium glycocalyx damage. Assuming that leptospiral proteases play an important role in all stages of the infectious process, characterizing their functional properties, substrates and mechanisms of action is of great importance for therapeutic purposes.
Topics: Animals; Arginine; Leptospira; Leptospirosis; Metalloproteases; Mice; Peptide Hydrolases
PubMed: 36081769
DOI: 10.3389/fcimb.2022.966370 -
Journal of Dairy Science Feb 2018This work evaluated the expression and activity of a metalloprotease released by Pseudomonas fluorescens 07A in milk. Low relative expression of the protease by the...
This work evaluated the expression and activity of a metalloprotease released by Pseudomonas fluorescens 07A in milk. Low relative expression of the protease by the strain was observed after incubation for 12 h at 25°C while the strain was in the logarithmic growth phase. After 24 h, protease production significantly increased and remained constant for up to 48 h, a time range during which the strain remained in the stationary phase. Conversely, at refrigeration temperatures, at 12 h the strain was still in the lag phase and expressed the protease at higher levels than when the logarithmic phase was reached. Casein fractions were highly degraded by P. fluorescens 07A, the purified protease, and the bacterial pellet on d 7 of incubation at 25°C and to a lesser extent at 10°C for the sample incubated with the bacterium. Heat treatment at 90°C for 5 min completely inactivated the proteolytic activity of the purified protease and the bacterial pellet. This work contributes to the knowledge about the conditions of milk storage that influence the production and activity of this extracellular metalloprotease. The results demonstrate the need to find alternative strategies to control the synthesis and activity of proteolytic enzymes in the dairy industry to ensure the quality of processed products.
Topics: Animals; Bacterial Proteins; Metalloproteases; Milk; Pseudomonas fluorescens; Temperature
PubMed: 29248219
DOI: 10.3168/jds.2017-13238 -
Cardiovascular Therapeutics 2008Metalloproteinases (MMPs, also called matrixins) are extracellular proteolytic enzymes involved in the degradation of both matrix and nonmatrix proteins. Currently, 25... (Review)
Review
Metalloproteinases (MMPs, also called matrixins) are extracellular proteolytic enzymes involved in the degradation of both matrix and nonmatrix proteins. Currently, 25 MMPs have been identified in humans, and the overexpression of one or more MMPs has been implicated in several pathologies, spanning from cancer to rheumathoid arthritis to cardiovascular disease. While research over the past 20 years has focused on understanding MMP biology and selectively inhibiting MMP activity, key issues that remain to be addressed include MMP roles in the context of normal versus pathological conditions and whether globally inhibiting MMPs improves or deteriorates overall organ function. In terms of cardiovascular disease, increased MMP expression has been demonstrated in the setting of myocardial ischemia, reperfusion injury, and during the progression to congestive heart failure. MMPs are also major contributors to the progression of atherosclerotic lesions. In this review, we focus on cardiovascular effects produced by PD 166793, a wide-broad spectrum MMP inhibitor, originally developed by Parke-Davis (now Pfizer). We will briefly review its structure, mechanism of action, and inhibitory capacity. Finally, we will illustrate the cardiac contexts, both in vivo and in vitro, in which PD166793 administration has proven beneficial.
Topics: Animals; Enzyme Activation; Genetic Therapy; Heart Failure; Humans; Hydroxamic Acids; Metalloproteases; Oligopeptides; Protease Inhibitors; Ventricular Remodeling
PubMed: 18466418
DOI: 10.1111/j.1527-3466.2007.00034.x -
Toxins Dec 2019Bothropic venoms contain enzymes such as metalloproteases, serine-proteases, and phospholipases, which acting by themselves, or in synergism, are the cause of the...
Bothropic venoms contain enzymes such as metalloproteases, serine-proteases, and phospholipases, which acting by themselves, or in synergism, are the cause of the envenomation symptoms and death. Here, two mRNA transcripts, one that codes for a metalloprotease and another for a serine-protease, were isolated from a venom gland. The metalloprotease and serine-protease transcripts were cloned on a pCR2.1-TOPO vector and consequently expressed in a recombinant way in E. coli (strains Origami and M15, respectively), using pQE30 vectors. The recombinant proteins were named rBamSP_1 and rBamMP_1, and they were formed by an N-terminal fusion protein of 16 amino acid residues, followed by the sequence of the mature proteins. After bacterial expression, each recombinant enzyme was recovered from inclusion bodies and treated with chaotropic agents. The experimental molecular masses for rBamSP_1 and rBamMP_1 agreed with their expected theoretical ones, and their secondary structure spectra obtained by circular dichroism were comparable to that of similar proteins. Additionally, equivalent mixtures of rBamSP_1, rBamMP_1 together with a previous reported recombinant phospholipase, rBamPLA2_1, were used to immunize rabbits to produce serum antibodies, which in turn recognized serine-proteases, metalloproteases and PLA2s from and other regional viper venoms. Finally, rabbit antibodies neutralized the 3LD50 of venom.
Topics: Animals; Antibodies, Neutralizing; Bothrops; Crotalid Venoms; Metalloproteases; Phospholipases; Rabbits; Recombinant Proteins; Reptilian Proteins; Serine Proteases
PubMed: 31810356
DOI: 10.3390/toxins11120702 -
Nature Communications Oct 2022The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small...
The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal tissue homeostasis meprin α performs key roles in inflammation, immunity, and extracellular matrix remodelling. Dysregulated meprin α is associated with acute kidney injury, sepsis, urinary tract infection, metastatic colorectal carcinoma, and inflammatory bowel disease. Accordingly, meprin α is the target of drug discovery programs. In contrast to meprin β, meprin α is secreted into the extracellular space, whereupon it oligomerises to form giant assemblies and is the largest extracellular protease identified to date (~6 MDa). Here, using cryo-electron microscopy, we determine the high-resolution structure of the zymogen and mature form of meprin α, as well as the structure of the active form in complex with a prototype small molecule inhibitor and human fetuin-B. Our data reveal that meprin α forms a giant, flexible, left-handed helical assembly of roughly 22 nm in diameter. We find that oligomerisation improves proteolytic and thermal stability but does not impact substrate specificity or enzymatic activity. Furthermore, structural comparison with meprin β reveal unique features of the active site of meprin α, and helical assembly more broadly.
Topics: Humans; Fetuin-B; Cryoelectron Microscopy; Metalloendopeptidases; Metalloproteases; Enzyme Precursors; Zinc
PubMed: 36261433
DOI: 10.1038/s41467-022-33893-7 -
Mediators of Inflammation 2014The chemokine fractalkine is considered as unique since it exists both as membrane-bound adhesion molecule and as shed soluble chemoattractant. Here the hypothesis was...
The chemokine fractalkine is considered as unique since it exists both as membrane-bound adhesion molecule and as shed soluble chemoattractant. Here the hypothesis was tested whether placental fractalkine can be shed and released into the maternal circulation. Immunohistochemical staining of human first trimester and term placenta sections localized fractalkine at the apical microvillous plasma membrane of the syncytiotrophoblast. Gene expression analysis revealed abundant upregulation in placental fractalkine at term, compared to first trimester. Fractalkine expression and release were detected in the trophoblast cell line BeWo, in primary term trophoblasts and placental explants. Incubation of BeWo cells and placental explants with metalloprotease inhibitor Batimastat inhibited the release of soluble fractalkine and at the same time increased the membrane-bound form. These results demonstrate that human placenta is a source for fractalkine, which is expressed in the syncytiotrophoblast and can be released into the maternal circulation by constitutive metalloprotease dependent shedding. Increased expression and release of placental fractalkine may contribute to low grade systemic inflammatory responses in third trimester of normal pregnancy. Aberrant placental metalloprotease activity may not only affect the release of placenta derived fractalkine but may at the same time affect the abundance of the membrane-bound form of the chemokine.
Topics: Adult; Cell Line; Cell Membrane; Chemokine CX3CL1; Female; Gene Expression Regulation, Enzymologic; Humans; Immunohistochemistry; Inflammation; Metalloproteases; Microvilli; Phenylalanine; Placenta; Pregnancy; Pregnancy Trimester, First; Thiophenes; Trophoblasts
PubMed: 24771984
DOI: 10.1155/2014/839290