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)

We have examined the in vitro behavior of clathrin-coated vesicles that have been stripped of their surface coats such that the majority of the clathrin is removed but substantial amounts of clathrin assembly proteins (AP) remain membrane-associated. Aggregation of these stripped coated vesicles (s-CV) is observed when they are placed under conditions that approximate the pH and ionic strength of the cell interior (pH 7.2, approximately 100 mM salt). This s-CV aggregation reaction is rapid (t1/2 < or = 0.5 min), independent of temperature within a range of 4-37 degrees C, and unaffected by ATP, guanosine-5'-O-(3-thiophosphate), and in particular EGTA, distinguishing it from Ca(2+)-dependent membrane aggregation reactions. The process is driven by the action of membrane-associated AP molecules since partial proteolysis results in a full loss of activity and since aggregation is abolished by pretreatment of the s-CVs with a monoclonal antibody that reacts with the alpha subunit of AP-2. However, vesicle aggregation is not inhibited by PPPi, indicating that the previously characterized polyphosphate-sensitive AP-2 self-association is not responsible for the reaction. The vesicle aggregation reaction can be reconstituted: liposomes of phospholipid composition approximating that found on the cytoplasmic surfaces of the plasma membrane and of coated vesicles (70% L-alpha-phosphatidylethanolamine (type I-A), 15% L-alpha-phosphatidyl-L-serine, and 15% L-alpha-phosphatidylinositol) aggregated after addition of AP-2, but not of AP-1, AP-3 (AP180), or pure clathrin triskelions. Aggregation of liposomes is abolished by limited proteolysis of AP-2 with trypsin. In addition, a highly purified AP-2 alpha preparation devoid of beta causes liposome aggregation, whereas pure beta subunit does not, consistent with results obtained in the s-CV assay which also indicate the involvement of the alpha subunit. Using a fluorescence energy transfer assay we show that AP-2 does not cause fusion of liposomes under physiological solution conditions. However, since the fusion of membranes necessarily requires the close opposition of the two participating bilayers, the AP-2-dependent vesicle aggregation events that we have identified may represent an initial step in the formation and fusion of endosomes that occur subsequent to endocytosis and clathrin uncoating in vivo.
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PMID:Clathrin assembly protein AP-2 induces aggregation of membrane vesicles: a possible role for AP-2 in endosome formation. 135 96

Platelets from patients with platelet-type von Willebrand disease (vWD) were used as immunogens for the production of murine monoclonal antibodies (MoAbs). One such MoAb, C-34, inhibited ristocetin-induced aggregation of patient or normal platelets, but not aggregation induced by other aggregating agents. As demonstrated by crossed-immunoelectrophoresis, C-34 recognized an epitope within the GPIb/IX complex. In indirect immunofluorescence studies on fresh platelets, the ratio of any of four different anti-GPIb MoAbs to one another was near unity (0.88-1.14) both for normals and for patients. In contrast, the ratio of the binding of C-34 to such a MoAb (AP-1) was 0.31 +/- 0.02 (means +/- SE) for normal platelets and significantly increased to 0.54 +/- 0.01 for patient platelets (P less than 0.001). In NP-40 lysates of 3H-labelled platelets, saturating concentrations of C-34 produced much fainter bands than did AS-2 or other anti-GPIb MoAbs in the GPIb and GPIX regions. In contrast to the other anti-GPIb MoAbs, C-34 did not bind to the purified 125I-labelled glycocalicin fragment of GPIb, to the glycocalicin derivative identified by crossed-immunoelectrophoresis, or to the amino-terminal approximately 40 kDa portion of GPIb alpha cleaved from 3H-labelled platelets by trypsin. C-34 appears to be the first MoAb that is quantitatively informative in identifying the abnormal platelets in platelet-type vWD. The observed differences between the patient and normal platelets may reflect an abnormality in the primary structure of the GPIb/IX complex. Alternatively, patient platelets may have an abnormality of other structures near this region that impose less of a steric hindrance upon binding of antibody to the C-34 epitope.
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PMID:Increased platelet sensitivity to ristocetin is predicted by the binding characteristics of a GPIb/IX determinant. 169 30

Matrix metalloproteinases are an important group of zinc enzymes responsible for degradation of the extracellular matrix components such as collagen and proteoglycans in normal embryogenesis and remodeling and in many disease processes such as arthritis, cancer, periodontitis, and osteoporosis. A matrixin family is defined, comprising at least seven members that range in size from Mr 28,000 to 92,000 and are related in gene sequence to collagenase. All family members are secreted as zymogens that lose peptides of about 10,000 daltons upon activation. Latency is due to a conserved cysteine that binds to zinc at the active center. Latency is overcome by physical (chaotropic agents), chemical (HOCl, mercurials), and enzymatic (trypsin, plasmin) treatments that separate the cysteine residue from the zinc. Expression of the metalloproteinases is switched on by a variety of agents acting through regulatory elements of the gene, particularly the AP-1 binding site. A family of protein inhibitors of Mr 28,500 or less binds strongly and stoichiometrically in noncovalent fashion to inhibit members of the family. The serum protein alpha 2-macroglobulin and relatives are also strongly inhibitory.
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PMID:Matrix metalloproteinases and their inhibitors in connective tissue remodeling. 185 Jul 5

The core promoter for human DNA polymerase beta contains discrete binding sites for mammalian nuclear proteins, as revealed by DNasel footprinting and gel mobility shift assays. Two sites correspond to sequences identical with the Sp1 factor binding element, and a third site includes an eight residue palindromic sequence, TGACGTCA, known as the CRE element of several cAMP responsive promoters; the 5 to 10 residues flanking this palindrome on each side have no apparent sequence homology with known elements in other promoters. Nuclear extract from a variety of tissues and cells were examined; these included rat liver and testes and cultured cells of human and hamster origin. The DNasel footprint is strong over and around the palindromic element for each of the extracts and is equivalent in size (approximately 22 residues); footprinting over the Sp1 binding sites is seen also. Two potential tissue-specific binding sites, present in liver but not in testes, were found corresponding to residues -13 to -10 and +33 to +48, respectively. Protein binding to the palindromic element was confirmed by an electrophoretic mobility shift assay with the core promoter as probe. Binding specificity of the 22 residue palindromic element, as revealed by oligonucleotide competition, is different from that of AP-1 binding element. Controlled proteolysis with trypsin was used to study structural properties of proteins forming the mobility shift bands. Following digestion with trypsin, most of the palindrome binding activity of each extract corresponded to a sharp, faster migrating band, potentially representing a DNA binding domain of the palindrome binding protein.
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PMID:Protein binding elements in the human beta-polymerase promoter. 231 44

The apparent preferential expression of the elastase/cathepsin G protease inhibitor antileukoproteinase (ALP) in endometrium of species with epitheliochorial placenta suggests mechanisms of transcriptional regulation unique to these mammalian species. To begin to define the cis-acting regulatory elements involved in the endometrial transcription of the ALP gene, the porcine ALP gene was isolated and characterized. The porcine gene spans at least 13 kb and consists of 5 exons and 4 introns. This genomic structure, except for an additional exon, is similar to that of the human gene where the first three exons encode the signal peptide, trypsin/cathepsin G binding region, and elastase binding region, respectively. The positions of the 16 cysteine residues in exons 2 and 3 of the human gene are conserved in the porcine gene. The porcine gene contains a TATA box at -29 nucleotide (nt), and sequences with limited homology to those which might bind the transcription factors AP-1, AP-2, Sp-1 and Oct-1. The functional promoter activity of the ALP-5' flanking DNA was examined using chimeric ALP-chloramphenicol acetyl transferase (CAT) DNA constructs, after transient transfection in human (ECC-1, Ishikawa) and rabbit (HRE-H9) endometrial and human trophoblastic (JEG-3) cell lines. A 887 nt fragment of the ALP-5'-flanking region (-887ALP-pCAT-E) was active in these cell lines, with the highest promoter activity observed in the ECC-1. Progressive 5' deletion of the 887 nt fragment up to -243 nt had no effect on CAT gene expression in all cell lines, relative to the longest construct.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Chromosomal organization of the gene encoding porcine antileukoproteinase and functional analysis of the promoter region in endometrial and placental cells. 790 70

The X gene product of hepatitis B virus (HBV) has a trans-activation function. The AP-1, AP-2, kappa B-like, and C/EBP-like sequences, and the 26-bp element in HBV enhancer were identified as X-responsive elements. Although the X protein possesses a transcriptional activation domain, it doesn't bind to the X-responsive elements. However, CREB/ATF-2 becomes able to bind to a CRE-related sequence in the 26-bp element once it complexes with X protein. In addition, X protein was shown to have amino acid sequences homologous to the essential domain of Kunitz-type serine protease inhibitors and directly interacted with the protease, tryptase TL2. Results suggest that X protein modulates the tryptase TL2 activity, which may be involved in the proteolytic cleavage of cellular transcription factors.
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PMID:[Mechanism of hepatocarcinogenesis by hepatitis B virus]. 838 38

We have compared the abilities of mammalian ADP-ribosylation factors (ARFs) 1, 5, and 6 and Saccharomyces cerevisiae ARF2 to serve as substrates for the rat liver Golgi membrane guanine nucleotide exchange factor and to initiate the formation of clathrin- and coatomer protein (COP) I-coated vesicles on these membranes. While Golgi membranes stimulated the exchange of GTPgammaS for GDP on all of the ARFs tested, mammalian ARF1 was the best substrate, with an apparent Km of 5 microM. In all cases myristoylation of ARF was required for stimulation. Agents that inhibit the Golgi membrane guanine nucleotide exchange factor (the fungal metabolite brefeldin A and trypsin treatment) selectively inhibited the guanine nucleotide exchange on mammalian ARF1. Taken together, these data indicate that of the ARFs tested, only mammalian ARF1 is activated efficiently by the Golgi guanine nucleotide exchange factor. The other ARFs are activated mainly by another mechanism, possibly phospholipid-mediated. Once activated, all of the membrane-associated, myristoylated ARFs promoted the recruitment of coatomer to about the same extent. Mammalian ARFs 1 and 5 were the most effective in promoting the recruitment of the AP-1 adaptor complex, whereas yeast ARF2 was the least active. These data indicate that the specificity for ARF action on the Golgi membranes is primarily determined by the Golgi guanine nucleotide exchange factor, which has a strong preference for myristoylated mammalian ARF1.
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PMID:Comparative activity of ADP-ribosylation factor family members in the early steps of coated vesicle formation on rat liver Golgi membranes. 902 Jan 26

Proteinase inhibitor I (Inh I) and proteinase inhibitor II (Inh II) from potato tubers are effective proteinase inhibitors of chymotrypsin and trypsin. Inh I and Inh II were shown to suppress irradiation-induced transformation in mouse embryo fibroblasts suggesting that they possess anticarcinogenic characteristics. We have previously demonstrated that Inh I and Inh II could effectively block UV irradiation-induced activation of transcription activator protein 1 (AP-1) in mouse JB6 epidermal cells, which mechanistically may explain their anticarcinogenic actions. In the present study, we investigated the effects of Inh I and Inh II on the expression and composition pattern of the AP-1 complex following stimulation by UV B (UVB) irradiation in the JB6 model. We found that Inh I and Inh II specifically inhibited UVB-induced AP-1, but not NFkappaB, activity in JB6 cells. Both Inh I and Inh II up-regulated AP-1 constituent proteins, JunD and Fra-2, and suppressed c-Jun and c-Fos expression and composition in bound AP-1 in response to UVB stimulation. This regulation of the AP-1 protein compositional pattern in response to Inh I or Inh II may be critical for the inhibition of UVB-induced AP-1 activity by these agents found in potatoes.
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PMID:Proteinase inhibitors I and II from potatoes block UVB-induced AP-1 activity by regulating the AP-1 protein compositional patterns in JB6 cells. 1133 71

We have previously shown that mast cells enhance eosinophil survival and activation. In this study we further characterized mast cell activity toward eosinophils. Sonicate of both rat peritoneal mast cells and the human mast cell line 1 (HMC-1) induced a concentration-dependent IL-6 and IL-8 release from human peripheral blood eosinophils (ELISA). HMC-1-induced IL-8 release was significantly reduced by the tryptase inhibitors GW-45 and GW-58 (90 and 87%, respectively, at an optimal concentration) but not by anti-stem cell factor, anti-TNF-alpha, or anti-IFN-gamma neutralizing Abs or by the antihistamine drugs pyrilamine and cimetidine. In a manner similar to HMC-1, human recombinant tryptase induced the expression of mRNA for IL-8 (RT-PCR) and caused IL-8 release from the eosinophils. Addition of cycloheximide, actinomycin D, dexamethasone, PD 98059, curcumin, or SB 202190 completely inhibited the tryptase-induced IL-6 and IL-8 release. In contrast, cyclosporin A had no effect on tryptase-induced IL-8 release. Tryptase caused phosphorylation of extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinases 1 and 2, and p38 (Western blot). Tryptase also induced the translocation of c-Jun from the cytosol to the nucleus (confocal microscopy) and enhanced AP-1 binding activity to the DNA (EMSA). Eosinophils were found to express proteinase-activated receptor 2 (FACS). When eosinophils were incubated with tryptase in the presence of anti-proteinase-activated receptor 2 antagonist Abs a significant decrease in the IL-6 and IL-8 release occurred. In summary, we have demonstrated that the preformed mast cell mediator tryptase induces cytokine production and release in human peripheral blood eosinophils by the mitogen-activated protein kinase/AP-1 pathway.
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PMID:Tryptase activates the mitogen-activated protein kinase/activator protein-1 pathway in human peripheral blood eosinophils, causing cytokine production and release. 1219 39

There is increasing evidence that the cutaneous nervous system modulates physiological and pathophysiological effects including cell growth and differentiation, immunity and inflammation as well as tissue repair. Both cutaneous nervous fibers and inflammatory cells are able to release neuromediators and thereby activate specific receptors on target cells in the skin or transient immunocompetent cells. Cutaneous neuromediators include classical neurotransmitters such as catecholamines and acetylcholine being released from the automatic nervous system or cutaneous cells. On the other hand neuropeptides including substance P, calcitonin gene related peptide (CRGP), vasointestinal peptide (VIP) or proopiomelanocortin (POMC) derived peptides such as alpha melanocyte stimulating hormone (alphaMSH) may be released from sensory or autonomic nerve fibers and several epidermal as well as dermal cells. Neuropeptides are known to activate a variety of cutaneous cells through high affinity neuropeptide receptors or by direct activation of intracellular G-protein signalling cascades. Via the modulation of transcription factor activation (NF-kappaB, AP-1, STAT-3) they regulate the expression of adhesion molecules and proinflammatory cytokines in different cells and thereby function as modulators of immune and inflammatory reactions. Accordingly, neuropeptides such as CGRP or alphaMSH in vitro were found to downregulate costimulatory molecule expression on dendritic cells and in vivo via the generation of suppressor T-lymphocytes to induce hapten specific tolerance. Proteinases such as tryptase or neural endopeptidase inactivate neuropeptides in the extracellular space or at the cell surface thereby terminating neuropeptide induced inflammatory or immune responses. Proteinase-activated receptors (PAR) are recently described receptors that may have high impact in regulating cutaneous neurogenic inflammation. In the skin PAR-2 being expressed on sensory neurons and endothelial cells is self activated by tethered peptide ligands that are exposed after extracellular amino-terminal cleavage by trypsin or mast cell tryptase. PAR-2 agonists were found to induce the release of CGRP and SP which mediate vasodilation, plasma extravasation as well as the expression of adhesion molecules on vascular endothelial cells and thus elicit neurogenic inflammation. These findings indicate that the neuromediator network including neuropeptide receptors as well as proteinases play an important role in the maintenance of tissue integrity and the regulation of inflammatory and immune responses in the skin.
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PMID:Neuromediators--a crucial component of the skin immune system. 1241 63


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