Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In recent work we have shown that a serine proteinase, stratum corneum chymotryptic enzyme, with properties compatible with a role in desquamation in vitro as well as in vivo, is generally present in human stratum corneum. The enzymologic properties of the stratum corneum chymotryptic enzyme in a KCl extract of dissociated plantar corneocytes were compared with those of other known chymotryptic serine proteinases. Stratum corneum chymotryptic enzyme was found to differ significantly from bovine chymotrypsin, human cathepsin G, and human mast cell chymases in regard to inhibitor profile and substrate specificity. Stratum corneum chymotryptic enzyme was further purified from KCl extracts of dissociated plantar corneocytes by affinity chromatography on gels with covalently linked soybean trypsin inhibitor. The purified preparation contained one major component with apparent molecular weight 25 kD and one minor component with slightly higher apparent molecular weight as revealed by Coomassie staining after electrophoresis in polyacrylamide gels with sodium dodecyl sulphate of samples that had not been reduced. Both these components were associated with chymotrypsin-like activity as revealed by zymography in polyacrylamide gels with co-polymerized casein. On zymography gels, the purified preparation was also found to contain minor amounts of components with trypsin-like activity. The major purified protein had an apparent molecular weight of around 28 kD after reduction and full denaturation and was shown to contain carbohydrate.
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PMID:Purification and preliminary characterization of stratum corneum chymotryptic enzyme: a proteinase that may be involved in desquamation. 839 2

A new human 33-kDa serine protease was purified from human epidermis, and its cDNA was cloned from a keratinocyte library, from mRNA from a human keratinocyte line (HaCat) and from mRNA from human skin. Polyclonal antibodies specific for the new protein detected three groups of proteins in partially purified extracts of cornified eptihelium of human plantar skin. The three components are proposed to correspond to proenzyme, active enzyme, and proteolytically modified active enzyme. After N-deglycosylation, there was a decrease in apparent molecular mass of all detected components. Expression of the cloned cDNA in a eukaryotic virus-derived system yielded a recombinant protein that could be converted to an active protease by treatment with trypsin. Polymerase chain reaction analyses of cDNA from a number of human tissues showed high expression of the new enzyme in the skin and low expression in brain, placenta, and kidney. Homology searches yielded the highest score for porcine enamel matrix protease (55% amino acid sequence homology). High scores were also obtained for human and mouse neuropsin and for human stratum corneum chymotryptic enzyme. The function of this new protease, tentatively named stratum corneum tryptic enzyme, may be related to stratum corneum turnover and desquamation in the epidermis.
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PMID:Purification, molecular cloning, and expression of a human stratum corneum trypsin-like serine protease with possible function in desquamation. 1051 89

Exposure to a dry environment leads to depletion of water from the peripheral stratum corneum layers in a process dependent on the relative humidity (RH) and the intrinsic properties of the tissue. We hypothesized that by modulating the water content of the stratum corneum in the surface layers, RH effects the rate of desquamation by modulating the activity of the desquamatory enzymes, and specifically stratum corneum chymotryptic enzyme (SCCE). Using a novel air interface in vitro desquamatory model, we demonstrated RH-dependent corneocyte release with desquamatory rates decreasing below 80% RH. Application of 10% glycerol or a glycerol-containing moisturizing lotion further increased desquamation, even in humid conditions, demonstrating that water was the rate-limiting factor in the final stages of desquamation. Furthermore, even in humid conditions desquamation was sub-maximal. In situ stratum corneum SCCE activity showed a dependence on RH: activity was significantly higher at 100% than at 44% RH. Further increases in SCCE activity were induced by applying a 10% glycerol solution. Since SCCE, a water-requiring enzyme, must function in the water-depleted outer stratum corneum, we sought to determine whether this enzyme has a tolerance to lowered water activity. Using concentrated sucrose solutions to lower water activity, we analysed the activity of recombinant SCCE and compared it to that of trypsin and chymotrypsin. SCCE activity demonstrated a tolerance to water restriction, and this may be an adaptation to maintain enzyme activity even within the water-depleted stratum corneum intercellular space. Overall these findings support the concept that in the upper stratum corneum, RH modulates desquamation by its effect upon SCCE activity, and possibly other desquamatory hydrolases. In addition, SCCE may be adapted to function in the water-restricted stratum corneum intercellular space.
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PMID:Water modulation of stratum corneum chymotryptic enzyme activity and desquamation. 1175 90

Serine proteases belonging to the kallikrein group may play a central role in desquamation. We have identified human kallikreins 5, 7, and 14 (hK5, hK7, hK14) in catalytically active form in stratum corneum. All three enzymes are produced as inactive precursors. In this work, we prepared recombinant enzymes and enzyme precursors and characterized the catalytic properties of hK5 and hK14. With peptide substrates hK5 and hK14 both showed trypsin-like specificity and alkaline pH-optima. For the substrates tested, hK14 was superior to hK5 as regards maximum catalytic rate as well as catalytic efficiency. hK5, but not hK14, could activate pro-hK7 in a reaction which was optimal at pH 5-7. hK5 could activate its own precursor as well as pro-hK14. This was in contrast to hK14, which could activate pro-hK5 but not its own precursor. The activation of pro-hK5 either by auto-activation or by hK14 occurred at maximum rate at neutral or weakly alkaline pH, whereas activation of pro-hK14 by hK5 was optimal at pH 6-7. We conclude that the enzymes studied may be part of a protease cascade in the stratum corneum, and that the observed pH effects may have physiological relevance.
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PMID:A proteolytic cascade of kallikreins in the stratum corneum. 1565 74

LEKTI is a 120-kDa protein that plays an important role in skin development, as mutations affecting LEKTI synthesis underlie Netherton syndrome, an inherited skin disorder producing severe scaling. Its primary sequence indicates that the protein consists of 15 domains, all resembling a Kazal-type serine protease inhibitor. LEKTI and two serine proteases belonging to the human tissue kallikrein (hK) family (hK5 and hK7) are expressed in the granular layer of skin. In this study, we characterize the interaction of two recombinant LEKTI fragments containing three or four intact Kazal domains (domains 6-8 and 9-12) with recombinant rhK5, a trypsin-like protease, and recombinant rhK7, a chymotrypsin-like protease. Both fragments inhibited rhK5 similarly in binding and kinetic studies performed at pH 8.0, as well as pH 5.0, the pH of the stratum corneum where both LEKTI and proteases may function. Inhibition equilibrium constants (Ki) measured either directly in concentration-dependent studies or calculated from measured association (kass) and dissociation (kdis) rate constants were 1.2-5.5 nM at pH 8.0 and 10-20 nM at pH 5.0. At pH 8.0, kass and kdis values were 4.7 x 10(5) M(-1) s(-1) and 5.5 x 10(-4) s(-1), and at pH 5.0 they were 4.0 x 10(4) M(-1) s(-1) and 4.3 x 10(-4) s(-1), respectively. The low Ki and kdis values (t1/2 of 20-25 min) indicate tight and specific association. Only fragment 6-9' was a good inhibitor of rhK7, demonstrating a Ki of 11 nM at pH 8.0 in a reaction that was rapidly reversible. These results show that LEKTI, at least in fragment form, is a potent inhibitor of rhK5 and that this protease may be a target of LEKTI in human skin.
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PMID:Inhibition of human kallikreins 5 and 7 by the serine protease inhibitor lympho-epithelial Kazal-type inhibitor (LEKTI). 1630 83

Human tissue kallikreins are a family of 15 trypsin or chymotrypsin-like secreted serine proteases (hK1-hK15). hK5, hK6, hK7, hK8, and hK13 have been identified in the stratum corneum (SC), stratum granulosum, and skin appendages. It has been reported that hK5 and hK7 degrade desmosomes/corneodesmosomes, suggesting that kallikreins are responsible for desquamation. We report the quantification of hK5, hK6, hK7, hK8, hK10, hK11, hK13, and hK14 in the SC by ELISA and their variation among age groups. The total SC trypsin and chymotrypsin-like activities were also measured. The amount of hK7, hK8, and hK11 (ng per mg dry weight) were high, and varied from 6 to 14, hK5 (2.0-4.0) was present at intermediate levels, and hK10 (0.65-1.0), hK14 (0.1-0.3), hK6 (0.1-0.3), and hK13 (0.02-0.1) were present at lower levels. hK6 and hK14 were significantly lower in females between 20 and 59 y. hK5, hK7, hK10, hK11, and hK14 were not significantly different across the age groups. hK8 was lowest at extremes of age (highest at 30-39 y), hK6 was lower at >30 y, and hK13 was lower at >20 y. Overall trypsin-like activity did not differ across age groups but was higher in subjects <11 y. Overall chymotrypsin-like activity was not related to age. In conclusion, we found multiple kallikreins in the SC and suggest that these enzymes may be responsible for desquamation through an enzymatic cascade pathway.
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PMID:Quantification of human tissue kallikreins in the stratum corneum: dependence on age and gender. 1635 88

Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved (hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors.
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PMID:Specificity profiling of seven human tissue kallikreins reveals individual subsite preferences. 1674 Jun 31

We have previously presented evidence that two human kallikrein-related peptidases, KLK5 (hK5, stratum corneum tryptic enzyme, SCTE) and KLK7 (hK7, stratum corneum chymotryptic enzyme, SCCE), which are abundant in the stratum corneum, may be involved in desquamation. Since we had noted that not all trypsin-like activity in the plantar stratum corneum could be ascribed to KLK5, we set out to identify other skin proteases with similar primary substrate specificity. Here we describe purification of a protease identified as KLK14 from plantar stratum corneum, and show that this enzyme may be responsible for as much as 50% of the total trypsin-like activity in this tissue, measured as activity towards a chromogenic substrate cleaved by a wide variety of enzymes with trypsin-like specificity. This was in spite of very low levels of KLK14 protein compared to KLK5 and KLK7. KLK14 could be detected by immunoblotting in normal superficial stratum corneum of all individuals examined. The majority of KLK14 in the plantar stratum corneum is present in its catalytically active form. KLK14 could be immunohistochemically detected in sweat ducts, preferentially in the intraepidermal parts (the acrosyringium), and in sweat glands. The role played by this very efficient protease under normal and disease conditions in the skin remains to be elucidated.
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PMID:Kallikrein-related peptidase 14 may be a major contributor to trypsin-like proteolytic activity in human stratum corneum. 1680 Jul 37

Itch is a common symptom in dry skin related to inflammatory skin diseases, normal aging, and systemic diseases such as chronic renal failure, and HIV. However, correlations between itch and objective measures of barrier function and skin dryness such as skin hydration and transepidermal water loss have been rarely found. Recent experimental evidence indicates that damage to the stratum corneum with acetone/ether and water elicits a scratching response in mice and rats. These responses correlate to the number of PGP 9.5 immunoreactive fibers in the epidermis and to FOS-like immunoreactivity in the spinal cord. Other neuromediators involved in the pathogenesis of itch in dry skin are nerve growth factor (NGF), muscarinic acetylcholine receptors, and opiates. Serine proteases such as tryptase and their respective proteinase-activating receptor 2 (PAR2), recently found in both skin and nerves of patients with atopic eczema, suggest that these molecules may have a role in itch in dry skin. This has also been exemplified in the itchy and hyperkeratotic phenotype of the stratum corneum chymotryptic enzyme (SCCE) transgenic mouse model, which is over-expressing a serine protease. Developing inhibitors to these neuropeptides and mediators may be an attractive strategy for anti-itch treatment. The significant progress made in development of moisturizers may have an additional benefit in reducing the itch associated with dry skin. Formulating topical combination therapies containing moisturizers and anti-pruritics can significantly reduce the itch associated with dry skin. This paper will review the current clinical knowledge on the association between dry skin and itch and the recent advances in understanding the pathophysiology of this problem.
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PMID:Dry skin and impairment of barrier function associated with itch - new insights. 1849 19

Eczema is widely considered to be an exacerbation of alkaline stress to the skin. Epidermal barrier dysfunction is a feature of eczema pathology, which predisposes affected individuals to distressing morbid symptoms. At least two serine proteases, stratum corneum chymotryptic enzyme (kallikrein 7 [KLK7]) and stratum corneum tryptic enzyme (kallikrien 5 [KLK5]), have increased activity levels in eczematous lesions and both have been implicated in the destruction of corneodesomosomes, which are crucial to epidermal integrity. The present in vitro study investigated whether transcriptional gene silencing after siRNA transfection could influence the activity of these signature enzymes in an in vitro model of eczema induced by alkaline shock. HaCaT epithelial cells were subjected to alkaline stress by the addition of 1,1,3,3-tetramethyl guanidine "superbase" (TMG) to the culture media. The culture media were subsequently tested for chymotryspin, trypsin, plasmin, and urokinase activity using colorimetric peptide assays and for reactive oxygen species using WST1 cell viability reagent. Cells that had been transfected with small interfering ribonucleic acid (siRNA) against KLK5 and KLK7 for 24 h before alkaline shock did not exhibit the increase in serine protease levels observed in untreated controls. Moreover, an endpoint MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) confirmed that detachment of cells from the culture substrate observed in alkaline-stressed cells did not occur in siRNA-treated cells. This in vitro study has established the proof-of-principle that siRNA therapy appears to mitigate the consequences of alkaline shock to the serine protease-associated fragility of epithelial cells that is characteristic of eczema.
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PMID:Transcriptional gene silencing of kallikrein 5 and kallikrein 7 using siRNA prevents epithelial cell detachment induced by alkaline shock in an in vitro model of eczema. 2209 88


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