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The Journal of General and Applied... Mar 2017We selected a fungus secreting a neutral protease from soil and identified it as the basidiomycete fungus Cerrena albocinnamomea according to its ITS-5.8S rDNA and 28S...
We selected a fungus secreting a neutral protease from soil and identified it as the basidiomycete fungus Cerrena albocinnamomea according to its ITS-5.8S rDNA and 28S rDNA-D1/D2 sequences. A major extracellular protease isolated from C. albocinnamomea was purified approximately 44-fold through two purification steps. SDS-PAGE analyses of the purified protease revealed a single band, and its molecular mass of 39,756 Da was determined using MALDI-TOF-MS. The enzyme was optimally active at approximately pH 7.0 and 45°C. The K and V values for the hydrolysis of azocasein were 2.46 mg/mL and 989 units/min/mg protein, respectively. The enzyme was stable at pH 3.6-8.6 for 16 h and at temperatures ≤35°C for 1 h. Enzymatic activity was completely inhibited by Cu and Zn and markedly by EDTA and phosphoramidon. The N-terminal amino acid sequence ASYRVLPIT is highly similar to those of the members of the metalloprotease family M36, such as keratinase and elastinase. However, the protease did not detectably hydrolyze keratin or elastin. In contrast, the protease hydrolyzed fibrinogen, although there were no significant sequence similarities to the N-terminal amino acid sequences of other fibrinolytic enzymes. These results suggest that the purified protease represents a new neutral metalloprotease with fibrinogenolytic activity.
Topics: Caseins; Cluster Analysis; DNA, Fungal; DNA, Ribosomal; DNA, Ribosomal Spacer; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Hydrogen-Ion Concentration; Kinetics; Metalloproteases; Molecular Weight; Phylogeny; Polyporaceae; Protease Inhibitors; RNA, Ribosomal, 28S; RNA, Ribosomal, 5.8S; Sequence Analysis, DNA; Soil Microbiology; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Temperature
PubMed: 28123132
DOI: 10.2323/jgam.2016.07.006 -
Biochimica Et Biophysica Acta Jan 2010This short review highlights some recent advances in matrix metalloproteinase inhibitor (MMPi) design and development. Three distinct approaches to improved MMP... (Review)
Review
This short review highlights some recent advances in matrix metalloproteinase inhibitor (MMPi) design and development. Three distinct approaches to improved MMP inhibition are discussed: (1) the identification and investigation of novel zinc-binding groups (ZBGs), (2) the study of non-zinc-binding MMPi, and (3) mechanism-based MMPi that form covalent adducts with the protein. Each of these strategies is discussed and their respective advantages and remaining challenges are highlighted. The studies discussed here bode well for the development of ever more selective, potent, and well-tolerated MMPi for treating several important disease pathologies.
Topics: Animals; Drug Design; Humans; Metalloproteases; Models, Molecular; Protease Inhibitors; Substrate Specificity; Zinc
PubMed: 19712708
DOI: 10.1016/j.bbamcr.2009.08.006 -
The FEBS Journal Jun 2008As more data are generated from proteome and transcriptome analyses of snake venoms, we are gaining an appreciation of the complexity of the venoms and, to some degree,... (Review)
Review
As more data are generated from proteome and transcriptome analyses of snake venoms, we are gaining an appreciation of the complexity of the venoms and, to some degree, the various sources of such complexity. However, our knowledge is still far from complete. The translation of genetic information from the snake genome to the transcriptome and ultimately the proteome is only beginning to be appreciated, and will require significantly more investigation of the snake venom genomic structure prior to a complete understanding of the genesis of venom composition. Venom complexity, however, is derived not only from the venom genomic structure but also from transcriptome generation and translation and, perhaps most importantly, post-translation modification of the nascent venom proteome. In this review, we examine the snake venom metalloproteinases, some of the predominant components in viperid venoms, with regard to possible synthesis and post-translational mechanisms that contribute to venom complexity. The aim of this review is to highlight the state of our knowledge on snake venom metalloproteinase post-translational processing and to suggest testable hypotheses regarding the cellular mechanisms associated with snake venom metalloproteinase complexity in venoms.
Topics: Amino Acid Sequence; Animals; Disulfides; Metalloproteases; Molecular Sequence Data; Protein Folding; Protein Processing, Post-Translational; Protein Structure, Tertiary; Sequence Alignment; Viper Venoms
PubMed: 18479462
DOI: 10.1111/j.1742-4658.2008.06466.x -
MBio Apr 2020can colonize the human host and cause a variety of superficial and invasive infections. The success of as a pathogen derives from its ability to modulate its virulence...
can colonize the human host and cause a variety of superficial and invasive infections. The success of as a pathogen derives from its ability to modulate its virulence through the release, sensing of and response to cyclic signaling peptides. Here we provide, for the first time, evidence that processes and secretes small linear peptides through a specialized pathway that converts a lipoprotein leader into an extracellular peptide signal. We have identified and confirmed the machinery for each step and demonstrate that the putative membrane metalloprotease Eep and the EcsAB transporter are required to complete the processing and secretion of the peptides. In addition, we have identified several linear peptides, including the interspecies signaling molecule -cAM373, that are dependent on this processing and secretion pathway. These findings are particularly important because multiple Gram-positive bacteria rely on small linear peptides to control bacterial gene expression and virulence. Here, we provide evidence indicating that secretes small linear peptides into the environment via a novel processing and secretion pathway. The discovery of a specialized pathway for the production of small linear peptides and the identification of these peptides leads to several important questions regarding their role in biology, most interestingly, their potential to act as signaling molecules. The observations in this study provide a foundation for further in-depth studies into the biological activity of small linear peptides in .
Topics: Bacterial Proteins; Gene Expression Regulation, Bacterial; Humans; Membrane Transport Proteins; Metalloproteases; Peptides; Staphylococcus aureus; Virulence
PubMed: 32291297
DOI: 10.1128/mBio.00112-20 -
Scientific Reports Jan 2019In the ascidian Ciona robusta (formerly C. intestinalis type A), the mechanism underlying sperm penetration through the egg investment remains unknown. We previously...
In the ascidian Ciona robusta (formerly C. intestinalis type A), the mechanism underlying sperm penetration through the egg investment remains unknown. We previously reported that proteins containing both an astacin metalloprotease domain and thrombospondin type 1 repeats are abundant in the sperm surface protein-enriched fraction of C. robusta. Here we investigated the involvement of those proteins in fertilisation. We refined the sequences of astacin metalloproteases, confirmed that five of them are present in the sperm, and labelled them as tunicate astacin and thrombospondin type 1 repeat-containing (Tast) proteins. Fertilisation of C. robusta eggs was potently inhibited by a metalloprotease inhibitor GM6001. The eggs cleaved normally when they were vitelline coat-free or the inhibitor was added after insemination. Furthermore, vitelline coat proteins were degraded after incubation with intact sperm. These results suggest that sperm metalloproteases are indispensable for fertilisation, probably owing to direct or indirect mediation of vitelline-coat digestion during sperm penetration. TALEN-mediated knockout of Tast genes and the presence of GM6001 impaired larval development at the metamorphic stage, suggesting that Tast gene products play a key role in late development.
Topics: Animals; Ciona intestinalis; Egg Proteins; Female; Male; Metalloproteases; Sperm-Ovum Interactions; Spermatozoa; Vitelline Membrane
PubMed: 30700775
DOI: 10.1038/s41598-018-37721-1 -
Infection and Immunity Jun 1994Extracellular proteases have been suggested to be virulence factors in invasive aspergillosis. Since serine protease gene-disrupted mutants retain virulence, other...
Purification and characterization of an elastinolytic metalloprotease from Aspergillus fumigatus and immunoelectron microscopic evidence of secretion of this enzyme by the fungus invading the murine lung.
Extracellular proteases have been suggested to be virulence factors in invasive aspergillosis. Since serine protease gene-disrupted mutants retain virulence, other proteases are suspected to be also involved in the degradation of lung structural material. An elastinolytic neutral metalloprotease was purified 320-fold from the extracellular fluid of Aspergillus fumigatus grown on elastin by affinity chromatography on bacitracin-Sepharose 4B and gel filtration on Sephadex G-75. The molecular mass was determined to be 43 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No carbohydrate was attached to this metalloprotease, and its first 22 N-terminal amino acids did not show any homology with the known metalloproteases. The enzyme was completely inhibited by EDTA, 1,10-phenanthroline, and phosphoramidon but not by inhibitors specific for serine, aspartate, and cysteine proteases. Zn2+ and, to a lesser extent, Co2+ reversed the inhibition caused by 1,10-phenanthroline. The protease hydrolyzed the peptide bonds His-Leu, Ala-Leu, Tyr-Leu, Gly-Phe, and Phe-Phe in the B chain of insulin. Synthetic substrate Abz-Ala-Ala-Phe-Phe-pNA could be used for the fluorimetric assay of the A. fumigatus metalloprotease. This enzyme had maximum activity in the pH range 7.5 to 8.0 and at 60 degrees C. It retained 50% of the protease activity when held at 60 degrees C for 1 h. Zn2+ and Co2+ at 1 mM did not inhibit the protease activity. The metalloprotease was able to hydrolyze elastin, and its elastinolytic activity was comparable to that of the serine protease from this organism. The presence of Zn2+ in the culture medium stimulated the metalloprotease production. Rabbit antibodies prepared against the enzyme severely inhibited the enzyme activity. Immunogold electron microscopy revealed that A. fumigatus invading neutropenic mouse lungs secretes this metalloprotease.
Topics: Amino Acid Sequence; Animals; Aspergillus fumigatus; Elastin; Lung; Metalloendopeptidases; Mice; Microscopy, Immunoelectron; Molecular Sequence Data; Zinc
PubMed: 8188335
DOI: 10.1128/iai.62.6.2149-2157.1994 -
The FEBS Journal Jan 2023Proteases are organised in interconnected networks, together forming the protease web whose disturbance can have detrimental consequences for tissue homeostasis and...
Proteases are organised in interconnected networks, together forming the protease web whose disturbance can have detrimental consequences for tissue homeostasis and response to environmental insults. Membrane-anchored sheddases are proteases that themselves can be released into the pericellular space by ectodomain shedding. Werny et al. have uncovered unexpected promiscuity in ectodomain shedding of meprin β, a metalloprotease with critical functions in inflammation and fibrosis. These findings suggest new links within complex proteolytic networks like the epidermal protease network with potential implications for skin homeostasis, inflammation and response to injury. Comment on: https://doi.org/10.1111/febs.16586.
Topics: Peptide Hydrolases; Metalloendopeptidases; Metalloproteases; Proteolysis
PubMed: 36102354
DOI: 10.1111/febs.16621 -
Toxins Apr 2022The Australasian region is home to the most diverse elapid snake radiation on the planet (Hydrophiinae). Many of these snakes have evolved into unique ecomorphs compared...
The Australasian region is home to the most diverse elapid snake radiation on the planet (Hydrophiinae). Many of these snakes have evolved into unique ecomorphs compared to elapids on other continents; however, their venom compositions are poorly known. The Australian elapid (Stephen's banded snake) is an arboreal snake with a unique morphology. Human envenoming results in venom-induced consumption coagulopathy, without neurotoxicity. Using transcriptomics and a multi-step fractionation method involving reverse-phase high-performance liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis and bottom-up proteomics, we characterized the venom proteome of 92% of the total protein component of the venom by weight was characterized, and included all dominant protein families and 4 secondary protein families. Eighteen toxins made up 76% of the venom, four previously characterized and 14 new toxins. The four dominant protein families made up 77% of the venom, including snake venom metalloprotease (SVMP; 36.7%; three identified toxins), phospholipase A (PLA; 24.0%; five identified toxins), three-finger toxin (3FTx; 10.2%; two toxins) and snake venom serine protease (SVSP; 5.9%; one toxin; Hopsarin). Secondary protein families included L-amino acid oxidase (LAAO; 10.8%; one toxin), natriuretic peptide (NP; 0.8%; two toxins), cysteine-rich secretory protein (CRiSP; 1.7%; two toxins), c-type lectin (CTL; 1.1%; one toxin), and one minor protein family, nerve growth factor (NGF; 0.8%; one toxin). The venom composition of differs to other elapids, with a large proportion of SVMP and LAAO, and a relatively small amount of 3FTx. venom appeared to have less toxin diversity than other elapids, with only 18 toxins making up three-quarters of the venom.
Topics: Animals; Australia; Elapidae; Humans; Metalloproteases; Proteome; Snake Venoms; Toxins, Biological
PubMed: 35622563
DOI: 10.3390/toxins14050314 -
European Review For Medical and... Feb 2021The incidence of thyroid cancer is rising globally. Most patients progress slowly, but some patients develop lymph node and distant metastasis earlier, and their...
OBJECTIVE
The incidence of thyroid cancer is rising globally. Most patients progress slowly, but some patients develop lymph node and distant metastasis earlier, and their prognosis is poor. Therefore, early diagnosis and warning of malignancy are very meaningful for such patients. SAS1B gene is a newly discovered protein expressed on the surface of mature egg cells and has metalloendopeptidase activity. We aimed at exploring whether SAS1B is involved in the occurrence of thyroid cancer, and at providing evidence for early diagnosis and targeted therapy of thyroid cancer.
PATIENTS AND METHODS
In this study, a rabbit anti-human SAS1B polyclonal antibody was prepared by gene recombination technology. The indirect ELISA method was used to detect the SAS1B protein expression in the serum of 69 patients with thyroid cancer and 55 normal controls, and the relevant pathological factors were analyzed. Immunohistochemistry and PCR technology were used to investigate the expression levels of SAS1B protein and mRNA in 30 thyroid cancer tissues and 23 control thyroid tissues.
RESULTS
The titer of SAS1B recombinant antibody was 1:51200. The expression of SAS1B in the serum of patients with thyroid cancer was higher than that in the normal control group (p<0.01). The antibody had a good sensitivity in serum detection of cancer patients (p=0.008<0.01), the linear regression analysis result was that the expression of SAS1B gene was related to tumor envelope invasion and lymph node metastasis (p=0.003<0.01, p=0.003<0.01), and it was irrelevant to the patient's gender, age, tumor mass size, number of cancer foci, pathological stage, etc. (p>0.05). The results of immunohistochemistry showed that SAS1B protein was mainly located in the cytoplasm and membrane of thyroid cancer cells. The expression intensity in thyroid cancer tissues was higher than that in control tissues (p<0.05), but it was not expressed in normal thyroid tissues. Antibodies showed a good sensitivity that was used to detect thyroid cancer tissues (p=0.000<0.01). The results of ordinary PCR detection using thyroid cancer tissue and control thyroid tissue showed that the amplification products of the three domains (N-terminal, C-terminal and catalytic domain) of the SAS1B gene showed high expression in thyroid cancer tissue. q-PCR results showed that the expression of SAS1B gene in thyroid cancer and control thyroid tissue was higher than that in control group (p<0.05), and the genes of Aurora A and BARD1 related to centrosome replication and DNA replication forks protection during the proliferation were highly expressed in thyroid cancer tissue. The study results suggested that SAS1B was involved in the carcinogenesis of thyroid cancer. The Hum_mPLoc.2.0 software, PSORT Ⅱ software and UniProt software were used to predict that SAS1B protein had secretory protein properties.
CONCLUSIONS
The above data indicate that the SAS1B gene is closely related to the process of thyroid cancer and can serve as a good tumor marker that can be used for early diagnosis and early warning of thyroid malignancy.
Topics: Adult; Aged; Female; Humans; Male; Metalloproteases; Middle Aged; Thyroid Neoplasms
PubMed: 33629311
DOI: 10.26355/eurrev_202102_24849 -
The Journal of Biological Chemistry Jun 2003ADAMs are membrane-anchored glycoproteins with functions in fertilization, heart development, neurogenesis, and protein ectodomain shedding. Here we report an evaluation...
ADAMs are membrane-anchored glycoproteins with functions in fertilization, heart development, neurogenesis, and protein ectodomain shedding. Here we report an evaluation of the catalytic activity of recombinantly expressed soluble forms of ADAM19, a protein that is essential for cardiovascular morphogenesis. Proteolytic activity of soluble forms of ADAM19 was first demonstrated by their autocatalytic removal of a purification tag (Myc-His) and their ability to cleave myelin basic protein and the insulin B chain. The metalloprotease activity of ADAM19 is sensitive to the hydroxamic acid-type metalloprotease inhibitor BB94 (batimastat) but not to tissue inhibitors of metalloproteases (TIMPs) 1-3. Moreover, ADAM19 cleaves peptides corresponding to the known cleavage sites of tumor necrosis factor-alpha (TNF-alpha), TNF-related activation-induced cytokine (TRANCE, also referred to as osteoprotegerin ligand), and kit ligand-1 (KL-1) in vitro. Although ADAM19 is not required for shedding of TNFalpha and TRANCE in mouse embryonic fibroblasts, its overexpression in COS-7 cells results in strongly increased TRANCE shedding. This suggests a potential role for ADAM19 in shedding TRANCE in cells where both molecules are highly expressed, such as in osteoblasts. Interestingly, our results also indicate that ADAM19 can function as a negative regulator of KL-1 shedding in both COS-7 cells and mouse embryonic fibroblasts, instead of acting directly on KL-1. The identification of potential in vitro substrates offers the basis for further functional studies of ADAM19 in cells and in mice.
Topics: ADAM Proteins; Amino Acid Sequence; Animals; Binding Sites; COS Cells; Catalysis; Cell Line; Chlorocebus aethiops; Disintegrins; Fibroblasts; Kinetics; Membrane Proteins; Metalloendopeptidases; Metalloproteases; Mice; Mice, Knockout; Molecular Sequence Data; Muscle Proteins; Recombinant Proteins; Spodoptera; Transfection
PubMed: 12682046
DOI: 10.1074/jbc.M302781200