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 obtained highly purified preparations of the heme-controlled eukaryotic initiation factor 2 alpha-subunit (eIF-2 alpha) kinase (HCI) from rabbit reticulocyte lysates containing five different polypeptides. One of these is a 87-kDa (p87) phosphopeptide which appears to show an autokinase activity. The controlled digestion with trypsin of HCI preparations leads to the suggestion that phosphorylation of p87 is not needed for kinase activity and, furthermore, that another 89-kDa polypeptide could be the kinase catalytic subunit. In agreement with this, monoclonal antibodies directed against p87 do not interfere with eIF-2 alpha kinase activity. Moreover, the anti-p87 antibodies and those directed against the mammalian 90-kDa heat shock protein recognize the same p87 polypeptide from rabbit reticulocyte lysates. Upon incubation of the HCI preparation with hemin (5-10 microM), the eIF-2 alpha kinase is converted into an inactive form and appears to become associated with related peptides forming high molecular weight complexes which can be reversibly activated by 2-mercaptoethanol. The maintenance of the integrity of the porphyrin ring is absolutely required for kinase inactivation and although the presence of metal ion is not essential, the iron and cobalt metalloporphyrins are more effective than protoporphyrin IX. The formation of the inactive form of HCI by hemin is prevented by either N-ethylmaleimide, monoclonal antibodies directed against p87, or phosphorylation of p87. The data strongly suggest that hemin regulates eIF-2 alpha kinase activity by promoting formation of the inactive dimer HCI.p87 via disulfide bonds and direct binding of hemin. A model of HCI regulation is discussed.
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PMID:Regulation of heme-controlled eukaryotic polypeptide chain initiation factor 2 alpha-subunit kinase of reticulocyte lysates. 135 Jul 84

The intracellular concentration of the 27-kDa mammalian heat shock protein, HSP27, increases several-fold after heat and other metabolic stresses and is closely associated with the acquisition of thermotolerance. Posttranslational modifications may also affect the function of HSP27. Heat shock of HeLa cell cultures, or treatment with arsenite, phorbol ester, or tumor necrosis factor, caused a rapid phosphorylation of preexisting HSP27 and the appearance of three phosphorylated isoforms, HSP27 B, C, and D. Digestion with trypsin and fractionation of the peptides by reverse phase high performance liquid chromatography revealed three 32P-labeled phosphopeptides. Microsequence analysis identified peak I as Ala76-Leu77-Ser78-Arg79 and peak II as Gln80-Leu81-Ser82-Ser83-Gly84-Val85- Ser86-Glu87-Ile88-Arg89; peak III contained the undigested peptide pair Ala76-Arg89. Ser82 was the major site and Ser78 the minor site of phosphorylation. Mutant proteins with Ser78 or Ser82 altered to glycine or Ser78-Ser82 double mutants were phosphorylated to reduced extents in vivo after heat or arsenite treatment. Ser78 and Ser82 (and Ser15) occur in the sequence motif RXXS, which is recognized by ribosomal protein S6 kinase II. Mitogenic stimulation of serum-deprived, Go-arrested Chinese hamster cells with serum, thrombin, or fibroblast growth factor also stimulated phosphorylation of HSP27 Ser78 and Ser82, and mitogenic stimulation and heat shock activated protein kinase activities that phosphorylated HSP27 and protein S6 in vitro. These results suggest that HSP27 may exert phosphorylation-activated functions linked with growth signaling pathways in unstressed cells. A homeostatic function at this level could protect cells from adverse effects of signal transduction systems which may be activated inappropriately during stress.
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PMID:Human HSP27 is phosphorylated at serines 78 and 82 by heat shock and mitogen-activated kinases that recognize the same amino acid motif as S6 kinase II. 173 Jun 70

Clofibrate and many of its structural analogues induce proliferation of peroxisomes in the hepatic parenchymal cells of rodents and certain nonrodent species including primates. This induction is tissue specific, occurring mainly in the liver parenchymal cells and to a lesser extent in the kidney cortical epithelium. The induction of peroxisomes is associated with a predictable pleiotropic response, characterized by hepatomegaly, and increased activities and mRNA levels of certain peroxisomal enzymes. Using affinity chromatography, we had previously isolated a protein that binds to clofibric acid. We now show that this protein is homologous with the heat shock protein HSP70 family by analysis of amino acid sequences of isolated peptides from trypsin-treated clofibric acid binding protein and by cross-reactivity with a monoclonal antibody raised against the conserved region of the 70-kDa heat shock proteins. The clofibric acid-Sepharose column could bind HSP70 proteins isolated from various species, which could then be eluted with either clofibric acid or ATP. Conversely, when a rat liver cytosol containing multiple members of the HSP70 family was passed through an ATP-agarose column, and eluted with clofibric acid, only P72 (HSC70) was eluted. These results suggest that clofibric acid, a peroxisome proliferator, preferentially interacts with P72 at or near the ATP binding site.
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PMID:Identification of cytosolic peroxisome proliferator binding protein as a member of the heat shock protein HSP70 family. 237 Dec 72

Glucocorticoid hormone receptors are present in the soluble fraction of target cell homogenates as large entities (Mr approximately 300,000) that are unable to interact with DNA. These large complexes contain an Mr approximately 94,000 steroid- and DNA-binding polypeptide, in association with an Mr approximately 90,000 non-ligand-binding entity, which has been identified as a heat shock protein, hsp90. This protein has been purified to near homogeneity as a component of the non-activated receptor complex. Characterization of the purified protein revealed its presence as a dimer in the large receptor form. Dissociation of the receptor-hsp90 complex can be induced by heat treatment only when ligand is bound to the receptor, as demonstrated by specific DNA-binding assay and sucrose gradient ultracentrifugation, hsp90 represents ca 1% of total proteins in rat liver cytosol, and milligram amounts were purified using a combination of high performance ion exchange and gel permeation chromatography. Monospecific antibodies were raised in rabbits. They were found to precipitate the intact non-activated glucocorticoid receptor, as well as the Mr approximately 27,000 steroid-binding fragment of the receptor generated by trypsin treatment, indicating that hsp90 interacts with the steroid-binding domain of the glucocorticoid receptor. Finally, translation of glucocorticoid receptor mRNA in reticulocyte lysate yields a protein which also interacts with hsp90 and binds to DNA only after ligand-binding and heat treatment. Thus, the glucocorticoid receptor is synthesized in a non-activated form also in vitro.
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PMID:The non-activated glucocorticoid receptor: structure and activation. 262 47

This brief report deals with some recent observations relating to the assoclation of the Mr approximately 90,000 heat shock protein (hsp90) with the glucocorticoid receptor. In its nonactivated state, stabilized by sodium molybdate, the glucocorticoid receptor exists as a 9S heteromeric complex containing a single Mr approximately 94,000 steroid-binding unit and a dimer of hsp90. Monospecific antibodies raised against the purified rat glucocorticoid receptor-associated hsp90 interact with the molybdate-stabilized receptor. They also immunoprecipitate the Mr approximately 27,000 steroid-binding fragment of the receptor generated by trypsin treatment. Thus, hsp90 interacts with the ligand-binding domain of the glucocorticoid receptor. Furthermore, dissociation of the glucocorticoid receptor-hsp90 complex results in a major reduction of the affinity of the Mr approximately 94,000 receptor entity for its ligand. The heteromeric 9S complex does not bind to DNA. When it is activated to a DNA-binding state, the hsp90 dissociates from the ligand-binding protein. In vitro, activation of the cytosolic rat glucocorticoid receptor to a DNA-binding state is inducible by the binding of ligand. Taken together, our observations indicate the existence of important connections between the association of hsp90 and the functions of ligand- and DNA-binding of the glucocorticoid receptor.
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PMID:The Mr approximately 90,000 heat shock protein: an important modulator of ligand and DNA-binding properties of the glucocorticoid receptor. 264 38

We have investigated the physiochemical characteristics of trypsin-treated, molybdate-stabilized glucocorticoid-receptor complexes from rat liver in the presence of 10 mM sodium molybdate by high performance ion-exchange chromatography, high performance size-exclusion chromatography, and sedimentation analysis. Trypsin treatment was performed under conditions previously reported to degrade the monomeric Mr approximately 94,000 steroid-binding protein to an Mr approximately 27,000 ligand-binding entity (Wrange, O., and Gustafsson, J.-A. (1978) J. Biol. Chem. 253, 856-865). Also in the presence of molybdate, an Mr approximately 27,000 steroid-binding fragment was obtained by limited trypsinization. However, no major differences in the tested physicochemical parameters were seen when trypsin-treated glucocorticoid-receptor complexes were compared with crude cytosolic complexes. Furthermore, the Mr approximately 27,000 steroid-binding fragment generated in the presence of molybdate could be immunoprecipitated by antibodies specific for the glucocorticoid receptor-associated Mr approximately 90,000 heat shock protein. These results provide direct evidence for an interaction of the Mr approximately 90,000 heat shock protein with the steroid-binding domain of the glucocorticoid receptor, known to correspond to the C-terminal third of the receptor protein.
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PMID:Interaction of the Mr = 90,000 heat shock protein with the steroid-binding domain of the glucocorticoid receptor. 319 46

Reactivity to the mycobacterial 65 kDa heat shock protein (HSP 65) has been implicated in the pathogenesis of adjuvant arthritis in the rat, and may be involved in the pathogenesis of rheumatoid arthritis or other autoimmune diseases in humans. Accordingly this study sought quantitative or qualitative differences in the antibody reactivity to HSP 65 between normal controls, patients with the multisystem autoimmune diseases, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and patients with the mycobacterial infections, tuberculosis (TB) and leprosy. Levels of antibodies to recombinant HSP 65 in serum were measured by ELISA in normal subjects and in patients with RA, SLE, TB or leprosy. Antibody reactivity was examined by Western blotting using polypeptide fragments of HSP 65 derived by recombinant DNA techniques, or by digestion with trypsin or cyanogen bromide (CNBr). Reactivity to a synthetic peptide, the adjuvant arthritis T-cell epitope of HSP 65 (180-188), was tested by ELISA. High levels of antibodies to full length recombinant HSP 65 from Mycobacterium bovis were present in all the groups tested. By Western blot analysis, most reactivity with intact HSP 65 was retained in a 32 kDa tryptic fragment, judged by sequencing and size estimations to represent amino acid residues 118- approximately 388. This sequence included a major T-cell epitope for adjuvant arthritis (180-188), but these nine amino acids were not essential for B-cell reactivity since most sera also reacted with residues 188-540 which lack the T-cell epitope. Moreover, the 180-188 synthetic peptide was unreactive by ELISA, and did not inhibit reactivity with the intact recombinant HSP 65. In conclusion, most individuals had antibodies to mycobacterial HSP 65, presumably resulting from previous bacterial infections. The magnitude of the response was unrelated to the occurrence of systemic autoimmune disease, and the pattern of antibody reactivity with recombinant and proteolytic fragments of HSP 65 suggests that the major B-cell epitope is conformational and consists of discontinuous regions of the molecule.
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PMID:Antibody reactivity to mycobacterial 65 kDa heat shock protein: relevance to autoimmunity. 754 3

The peptides recognized by an H-2Db-restricted CD8 cytotoxic T lymphocyte (CTL) clone which is specific for the 60-kDa mycobacterial heat shock protein (hsp) and cross-reacts with stressed host cells were characterized. None of the nonapeptides from hsp60 conforming to the H-2Db binding motif were able to sensitize target cells for lysis by this CTL clone. Sequence analysis of the stimulatory fraction from a trypsin digest of hsp60, together with synthetic peptide studies, defined a cluster of overlapping epitopes. Carboxy-terminal extension by at least one amino acid of the nonamer predicted to bind best to H-2Db was essential for CTL recognition. Two such elongated peptides, a 10-mer and a 12-mer stimulated the clone at similarly low concentrations in the 100 pM range. We assume that these two peptides comply best with the natural epitope. In contrast, the 11-mer was inactive. The stimulatory 10-mer bound to H-2Db with an efficacy similar to that of the nonapeptide corresponding to the H-2Db motif, as revealed by peptide induced major histocompatibility complex (MHC) surface expression on RMA-S cells and competitive blocking of epitope recognition by the nonamer. Binding of these carboxy-terminally extended peptides to the MHC groove can be explained by anchoring through the amino acid residue Asn in position 5 of the peptide and by intrusion of the hydrophobic carboxy-terminal Ala (10-mer) or Leu (12-mer), but not Gly (11-mer), into the hydrophobic pocket of the H-2Db cleft. Because the carboxy-terminal part is thus larger than predicted, this region of the peptide may arch up from the binding groove. We assume that recognition of steric components of the MHC/peptide complex broaden the range of epitope specificity for a single T cell receptor. This flexibility not only promotes recognition of several overlapping peptides from a single antigen, but may also increase the chance of cross-reaction with similar peptides from unrelated proteins, including autoantigens. Consistent with this latter assumption, the T cell clone cross-recognizes mycobacterial hsp60 and stressed host cells.
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PMID:Elongated peptides, not the predicted nonapeptide stimulate a major histocompatibility complex class I-restricted cytotoxic T lymphocyte clone with specificity for a bacterial heat shock protein. 780 44

Cell death within atherosclerotic plaques leads to necrosis and rupture, resulting in vascular occlusion. We have previously demonstrated that addition of exogenous 70 kDa heat shock protein (HSP70) to arterial smooth muscle cells (aSMCs) in vitro can protect against toxins that may initiate necrosis. To determine whether exogenous HSP70 enters aSMCs or acts from outside cells to preserve viability, cultured rabbit aSMCs were stressed by serum deprivation and treated with fluorescently labeled (7-aminomethyl-4-coumarin-3-acetate) or 125I-radiolabeled HSP70. Cell-associated HSP70 was analyzed using Western blotting, fluorescence spectroscopy, and gamma counting/autoradiography. Surface binding of HSP70 to aSMCs was differentiated from uptake by using trypsin treatment to degrade non-internalized HSP70. Specificity of HSP70 binding was tested by inhibiting uptake of 125I-HSP70 with excess unlabeled HSP70 or bovine serum albumin (BSA). The effect of unlabeled exogenous HSP70 on endogenous HSP synthesis was also tested. Exogenous HSP70 increased total cell-associated HSP70 2.9- to 3.6-fold over levels present in unstressed aSMCs. However, < 5% of the exogenous HSP70 was trypsin-insensitive, indicating that bound HSP70 was not internalized. Binding of 125I-HSP70 was inhibited by both unlabeled HSP70 and BSA, implying a non-specific interaction with the plasmalemma. Exogenous HSP70 significantly lowered overall protein synthesis by serum-deprived aSMCs, but it did not specifically inhibit synthesis of endogenous HSPs after heat shock. The results indicate that exogenous HSP70 protects viability of stressed aSMCs through interactions with the cell surface rather than via internalization.
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PMID:Exogenous HSP70 becomes cell associated, but not internalized, by stressed arterial smooth muscle cells. 811 16

Patients with insulin-dependent diabetes (IDDM) possess antibodies to islet proteins of M(r)-64,000. Potential autoantigens of this M(r) include glutamate decarboxylase (GAD) and 65 kD heat shock protein. We have detected two distinct antibody specificities in IDDM that bind 50,000 M(r) or 37,000/40,000 M(r) proteolytic fragments of 64,000 M(r) proteins. In this study, we investigated relationships of these proteolytic fragments to GAD and heat shock proteins. Polyclonal antibodies to GAD bound 50,000 M(r) fragments of islet antigen. Recombinant GAD65, but not GAD67, blocked binding to this antigen, suggesting that 50,000 M(r) fragments are derived from islet GAD65. In contrast, GAD antibodies did not recognize 37,000/40,000 M(r) fragments, and neither GAD isoforms blocked autoantibody binding to precursors of these fragments. The 37,000/40,000 M(r) fragments, but not the 50,000 M(r) fragments, were detected after trypsin treatment of immunoprecipitates from insulinoma cells that lacked expression of major GAD isoforms. Antibodies in IDDM did not bind native or trypsinized islet heat shock proteins. Thus, IDDM patients possess antibodies to GAD, but also distinct antibodies to a 64,000 M(r) protein that is not related to known GAD isoforms or heat shock proteins.
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PMID:Detection of pancreatic islet 64,000 M(r) autoantigens in insulin-dependent diabetes distinct from glutamate decarboxylase. 832 89


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