Tissue distribution and subcellular localizations determine in vivo functional relationship among prostasin, matriptase, HAI-1, and HAI-2 in human skin
Shiao‐Pieng LeeChen‐Yu KaoShun-Cheng ChangYi‐Lin ChiuYen‐Ju ChenMing-Hsing G. ChenChun-Chia ChangYu-Wen LinChien‐Ping ChiangJehng-Kang WangChen‐Yong LinMichael D. Johnson
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The membrane-bound serine proteases prostasin and matriptase and the Kunitz-type protease inhibitors HAI-1 and HAI-2 are all expressed in human skin and may form a tightly regulated proteolysis network, contributing to skin pathophysiology. Evidence from other systems, however, suggests that the relationship between matriptase and prostasin and between the proteases and the inhibitors can be context-dependent. In this study the in vivo zymogen activation and protease inhibition status of matriptase and prostasin were investigated in the human skin. Immunohistochemistry detected high levels of activated prostasin in the granular layer, but only low levels of activated matriptase restricted to the basal layer. Immunoblot analysis of foreskin lysates confirmed this in vivo zymogen activation status and further revealed that HAI-1 but not HAI-2 is the prominent inhibitor for prostasin and matriptase in skin. The zymogen activation status and location of the proteases does not support a close functional relation between matriptase and prostasin in the human skin. The limited role for HAI-2 in the inhibition of matriptase and prostasin is the result of its primarily intracellular localization in basal and spinous layer keratinocytes, which probably prevents the Kunitz inhibitor from interacting with active prostasin or matriptase. In contrast, the cell surface expression of HAI-1 in all viable epidermal layers renders it an effective regulator for matriptase and prostasin. Collectively, our study suggests the importance of tissue distribution and subcellular localization in the functional relationship between proteases and protease inhibitors.Keywords:
Zymogen
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Proteolysis
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With more than 6 million patients affected with them in the United States, chronic ulcers represent one of the greatest problems in wound care. High levels of corrosive proteases, particularly matrix metalloproteinases (MMPs), within the wound environment are thoughtto contribute to the persistence of these wounds through denaturation of connective tissue proteins crucial to healing progression. Therefore, there is considerable interest in protease modulation using wound dressings to promote healing in chronic wounds. Such modulation could be achieved by direct absorption of proteases, by depleting co- factors within the wound, or by release of protease inhibitors.The aim of this study is to examine protease modulation of a range of dressings with different chemistries, particularly those having demonstrated efficacy in chronic wound healing.XTRASORB® HCS (dressing A) and XTRASORB® Foam (dressing B) were able to modulate proteases by both direct absorption of MMPs and depleting metal ion co-factors, and resulted in complete elimination of protease activity in the assay used. Duoderm® (dressing C) was able to modulate proteases by direct absorption only, and not by co-factor depletion. Promogran® (dressing D) was able to reduce MMP activity, but this was shown to be pH dependant, with any protease modulation being lost at neutral pH. Neither Allevyn® (dressing E) nor Vigilon® (dressing F) were able to modulate proteases by any mechanism. None of the protease modulating dressings acted through the release of protease inhibitors.Of the dressings studied, dressing A and dressing B were the most effective protease modulators due to their acting through 2 separate mechanisms. .
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Chronic wound
Protease inhibitor (pharmacology)
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Exocellular protease production was examined in two separate strains of Pseudomonas aeruginosa, one a clinical isolate and the other a laboratory strain. Both strains produced two separate proteases (proteases 1 and 2) which were indistinguishable from one strain to the other. The two proteases were purified by a two-step procedure of gel filtration chromatography followed by ion-exchange chromatography. Proteases 1 and 2 were shown to be distinct serologically and unrelated by physiochemical parameters examined. Protease 1 was the major exocellular protein produced and contributed about 95% of the total protease activity of the culture. It was etimated to have a molecular weight of 34850 and was also shown to contain 10% glucosamine by weight. Protease 2, in contrast, had an estimated molecular weight of 52750 and contained no detectable carbohydrate. Proteases 1 and 2 were both stimulated by Ca2+, and Mg2+ and inhibited by Co2+Zn2+, and 1,10-o-phenanthroline. Protease 1 was also inhibited by EDTA. In addition to protease activity, both proteases 1 and 2 demonstrated elastase activity as well as a limited collagenase activity. Specificity of the two protease against synthetic peptides was, however, quite different. Protease 1, but not protease 2, showed a preference for peptide bonds in which the amino group was contributed by an amino acid with a hydrophovic R group.
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KALCHEV, K., Y. RABADJIEV, D. GANCHEV, M. TSENOVA, I. ILIEV and I. IVANOVA, 2013. Study of proteases and protease inhibitors from Streptomyces strains. Bulg. J. Agric. Sci., Supplement 2, 19: 65–67 Proteases and protease inhibitors are important enzymes in Streptomyces physiology and differentiation. The objective of this paper was to investigate the optimal conditions for production of proteases and protease inhibitors secreted by three newly isolated Streptomyces strains. The optimal growth and inhibition activities were detected after cultivation on different media and the maximum of protease inhibition activities were specifi c for the examined strains. The extracellular proteins were purifi ed by ammonium sulfate precipitation and SDS-PAGE. One of the examined strains showed protease inhibitory activity perspective for future research.
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Protease inhibitor (pharmacology)
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Proteases constitute one of the most important groups of industrial enzymes, therefore screening of these proteases from bacteria is important. The aim of this study was to characterize proteases from plant swamp bacteria. The result of proteolytic assays revealed three isolates possesed proteolytic index >1.00 (T1P4, H1P4 and K1P4). The optimum fermentation time of each isolate was 48 hours. The optimum pH of proteases from T1P4, H1P4 and K1P4 was 8.0; 7.5; and 8.0, respectively. The optimum temperature of T1P4, H1P4 and K1P4 protease was 50 °C; 40 °C; and 50 °C, respectively. Metal ions (Fe, K, Mn, and Zn) inhibited T1P4 protease except Fe2+ (5 mM), H1P4 protease except Zn2+ (5 mM), K1P4 protease except Fe (1 and 5 mM). Effect of inhibtor specific (EDTA) shown EDTA inhibit all proteases. Study on the effect of metals ion and spesific inhibitors indicated that all protease were metaloprotease. The moleculer weights of T1P4, H1P4 and K1P4 proteases were determinited by using SDS-PAGE and zymography technique were 151; 138; and 127 kD, respectively. Key words: bacteria, characterization, protease, swamp
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Proteolytic enzymes
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Since its identification, HIV has continued to have a detrimental impact on the lives of millions of people throughout the world. The protease of HIV is a major target in antiviral treatment. The South African HIV-1 subtype C (C-SA) protease displays weaker binding affinity for some clinically approved protease inhibitors in comparison with the HIV-1 subtype B protease. The heavy HIV burden in sub-Saharan Africa, where subtype C HIV-1 predominates, makes this disparity a topic of great interest. In light of this, the enzyme activity and affinity of protease inhibitors for the subtype B and C-SA proteases were determined. The relative vitality, indicating the selective advantage of polymorphisms, of the C-SA protease relative to the subtype B protease in the presence of ritonavir and darunavir was four- and tenfold greater, respectively. Dynamic differences that contribute to the reduced drug susceptibility of the C-SA protease were investigated by performing hydrogen-deuterium exchange/mass spectrometry (HDX/MS) on unbound subtype B and C-SA proteases. The reduced propensity to form the E35-R57 salt bridge, and alterations in the hydrophobic core of the C-SA protease, are proposed to affect the anchoring of the flexible flaps, resulting in an increased proportion of the fully open flap conformation. HDX/MS data suggested that the N-terminus of both proteases is less stable than the C-terminus of the proteases, thus explaining the increased efficacy of dimerization inhibitors targeted toward the C-terminus of HIV proteases. As far as we are aware, this is the first report on assessment of HIV protease dynamics using HDX/MS.
Darunavir
Hydrogen–deuterium exchange
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The loss of chlorophyll during the senescence of leaves is preceded by a decrease in protein content. Proteases responsible for the degradation of the proteins have been implicated in the regulation of the senescence process. The first leaf of the seedling of oats ( Avena sativa L.) demonstrates the typical pattern of leaf senescence in cereals and was chosen to study the properties and subcellular localization of proteases throughout leaf development.In Chapter 1, a general introduction is given that indicates the significance of the breakdown of proteins in leaves for seed yield and quality in cereals. Also in this chapter, protease classification is documented, and the possible role of changing protease activities in the regulation of protein degradation is discussed.Oat leaves contain two major proteases with pH optima at pH 4.5 ("acidic protease") and 7.5 ("neutral protease"). During natural development of greenhouse-grown plants both types of enzymes showed highest activities in young and fully-grown leaves, and decreased throughout the course of senescence. Also in detached leaves incubated in the dark and, therefore, subject to accelerated ageing, loss of protein was not accompanied by increases in protease activities. In the light, protease activities increased, but the rate of protein loss was greatly reduced. Thus, protein breakdown appears to be independent of the amount of protease present and additional synthesis of the major proteases is not required for protein loss during senescence (Chapter 1).This apparent paradox could be resolved if proteases and their substrates were spatially separated and brought into contact by a controlled decompartmentalization. Therefore, the distribution and subcellular localization of the major proteases were determined. Protoplasts were prepared as a first step in the isolation of vacuoles. However, the cell wall-degrading enzyme mixtures used for protoplast isolation were seriously contaminated by proteases interfering with the determination of the endogenous protease activity in the isolated protoplasts. These extra proteases could be inactivated by heating the cell well-degrading enzyme mixture at 50°C for 10 min at pH 6.5. This treatment did not impair the cell walldegrading activity. Protoplasts isolated with heated enzymes showed similar protease activities as washed protoplasts, isolated with untreated enzymes. This proved that contaminating proteases were effectively removed during protoplast washing, and that the protease activity measured in isolated protoplasts was derived from the protoplasts themselves. (Chapter 3).Vacuoles were isolated by osmotically lysing the protoplasts in the presence of K 2 HPO 4 . The maximum concentration of phosphate by which lysis occurred decreased progressively with increasing leaf age. By using the appropriate phosphate concentrations, it became possible to isolate clean vacuoles from leaves up to an advanced stage of senescence, when the leaves had lost more than 50% of their protein. From leaves older than 17 days, only vacuoplasts (vacuoles with adhering cytoplasm, within a resealed plasma membrane) could be obtained. The integrity of both the plasmalemma and the tonoplast decreased in these older leaves and this phenomenon might be linked with increased decompartmentalization at a late stage of senescence (Chapter 4).When the distribution of the proteases was determined in different subcellular fractions, on an average 16% of the acidic protease activity was washed out of the intercellular space of the leaves. The major part of the acidic activity was located within the vacuole. The neutral protease was absent from both these compartments and must, therefore, be cytoplasmic. During the course of leaf development, all of the acidic protease activity present in protoplasts was recovered in the vacuoles, as long as clean vacuoles could be isolated (i.e. up to 17 days). It seems most likely that protein degradation is controlled by import of protein substrates into the vacuole (Chapter 5).The acidic and neutral proteases were partly purified by gel filtration and anion-exchange chromatography (Chapter 6). The enzymes hardly separated, indicating that they have similar molecular weights and charges. The neutral activity was stabilized by merceptoethanol and inhibited by inhibitors of metallopeptidases, whereas the acidic one was not. Both activities were inhibited to varying extents by sulphydryl- and serinetype inhibitors. Both enzymes were endopeptideses. The instability of, in particular, the neutral protease seriously hampers its further purification and characterization.Furthermore, by activity staining after electrophoretic separation and using aminoacyl-2-naphthyl am ides as substrates, five aminopeptidases and one trypsin-like endopeptidase were identified (Chapter 7). The main aminopeptidase was largely unspecific. Two aminopeptidases showed preference for arginine and lysine. An iminopeptidase acted on proline. The fifth aminopeptidase was most active with methionine. All enzymes were active at pH 4.5, 6.0 and 7.5, but showed highest activities at low rather then at neutral pH. The trypsine-like endopeptidase was active at pH 4.5 and 7.5, was inhibited by o -phenanthroline and was not identical with either the acidic or the neutral protease described previously. Its activity decreased during leaf development. During storage of protein extracts in the cold this arginine-specific endopeptidase associated with ribulosebisphophate carboxylase, concomitant with a loss of this protein band. As to how far this enzyme has a function in protein breakdown invivo has to be further elucidated.The results of this study show that the levels of protease activities are not correlated with the rate of over all protein degradation. The acidic protease has no entry to the substrates to be degraded. In contrast, the neutral protease appears to be located together with protein substrates and organelles in the cytoplasm, but seems to selectively degrade only a few proteins. Whereas the mechanism of normal protein turnover remains to be elucidated, the slow, steady decline of protein during natural development may be controlled by, on the one hand, compartmentalization of the acidic protease in the vacuole and, on the other hand, by maintaining a pH removed from the optimum for exopeptidese action in the cytoplasm.
Avena
Senescence
Protein Degradation
Proteolysis
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Proteolytic enzymes are key signaling molecules in both normal physiological processes and various diseases. After synthesis, protease activity is tightly controlled. Consequently, levels of protease messenger RNA and protein often are not good indicators of total protease activity. To more accurately assign function to new proteases, investigators require methods that can be used to detect and quantify proteolysis. In this review, we describe basic principles, recent advances, and applications of biochemical methods to track protease activity, with an emphasis on the use of activity-based probes (ABPs) to detect protease activity. We describe ABP design principles and use case studies to illustrate the application of ABPs to protease enzymology, discovery and development of protease-targeted drugs, and detection and validation of proteases as biomarkers.
Proteolysis
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Pseudomonas fluorescens
Bacillus (shape)
Proteolysis
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Cleavage (geology)
MASP1
NS2-3 protease
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