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)

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

The human HSP70 gene was modified in vitro using oligonucleotide-directed mutagenesis to add sequences encoding a peptide from the testis-specific form of human lactate dehydrogenase (LDH) to the carboxy terminus of HSP70. The peptide-tagged HSP70 can be distinguished from the endogenous HSP70 protein using an LDH peptide-specific antiserum in indirect immunofluorescence assays of cells transiently transfected with an expression vector containing the tagged HSP70 gene regulated by the human HSP70 promoter. A series of deletion mutants within the HSP70 protein coding region were generated. Using double-label indirect immunofluorescence with the LDH peptide-specific antiserum and HSP70-specific mAbs, we compared the intracellular distribution of the deletion mutants to that of endogenous HSP70. We have determined that sequences in the carboxy terminus of HSP70 are necessary for proper nucleolar localization after heat shock. In contrast, sequences in the amino terminus of HSP70 are responsible for the ATP-binding ability of the protein. Mutants that were unable to bind ATP, however, still displayed nucleolar association, indicating that ATP binding is apparently not required for interaction with substrate. Additional support that HSP70 appears to be composed of at least two domains follows from the results of trypsin digestions of wild type and mutant HSP70. Protease digestion of the mutant HSP70 proteins identified a region of HSP70 that, when deleted, affected HSP70 conformation.
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PMID:Mutational analysis of the human HSP70 protein: distinct domains for nucleolar localization and adenosine triphosphate binding. 268 Dec 24

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

We previously demonstrated the stimulation of human apurinic/apyrimidinic endonuclease 1 (HAP1) by heat shock protein 70 (HSP70). In this work, we further defined the functional interaction between these proteins. Digestion of HSP70 by trypsin released 48 and 43 kDa amino terminal fragments that retained the ability to stimulate HAP1. In agreement with this result, an HSP70 N-terminal deletion mutant protein containing amino acids 1-385 was comparable to the full-length protein in its ability to enhance HAP1 activity. HSP70 mutants containing carboxy terminal amino acids 386-640 stimulated HAP1 only slightly, as did unrelated proteins. These results implicate the amino terminal portion of HSP70 in stimulating the activity of HAP1.
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PMID:Specific stimulation of human apurinic/apyrimidinic endonuclease by heat shock protein 70. 1254 89

Mature Epstein-Barr virus (EBV) was purified from the culture medium of infected lymphocytes made functionally conditional for Zta activation of lytic replication by an in-frame fusion with a mutant estrogen receptor. Proteins in purified virus preparations were separated by gradient gel electrophoresis and trypsin-digested; peptides were then analyzed by tandem hydrophobic chromatography, tandem MS sequencing, and MS scans. Potential peptides were matched with EBV and human gene ORFs. Mature EBV was mostly composed of homologues of proteins previously found in a herpes virion. However, EBV homologues to herpes simplex virus capsid-associated or tegument components UL7 (BBRF2), UL14 (BGLF3), and EBV BFRF1 were not significantly detected. Instead, probable tegument components included the EBV and gamma-herpesvirus-encoded BLRF2, BRRF2, BDLF2 and BKRF4 proteins. Actin was also a major tegument protein, and cofilin, tubulin, heat shock protein 90, and heat shock protein 70 were substantial components. EBV envelope glycoprotein gp350 was highly abundant, followed by glycoprotein gH, intact and furin-cleaved gB, gM, gp42, gL, gp78, gp150, and gN. BILF1 (gp64) and proteins associated with latent EBV infection were not detected in virions.
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PMID:Proteins of purified Epstein-Barr virus. 1553 16

Metallothionein (MT)-3, originally called growth inhibitory factor (GIF), was initially identified through its ability to inhibit the growth of neuronal cells in the presence of brain extract. MT-3 is the brain specific isoform of the MT family whose specific biological activity associates it with neurological disorders. Indeed, studies report that MT-3 is decreased by ~30% in brains of patients with Alzheimer disease (AD). Furthermore, many lines of evidence suggest that MT-3 engages in specific protein interactions. To address this, we conducted immunoaffinity chromatography experiments using an immobilized anti-mouse MT-3 antibody. We identified five associated proteins from the pool of sixteen recovered using mass spectrometry and tandem mass spectrometry after in-gel trypsin digestion of bands from the affinity chromatography. The proteins identified were: heat shock protein 84 (HSP84), heat shock protein 70 (HSP70), dihydropyrimidinase-like protein-2 (DRP-2), creatine kinase (CK) and beta-actin. Coimmunoprecipitation experiments, also conducted on whole mouse brain extract using the anti-mouse MT-3 antibody along with commercially available antibodies against HSP84 and CK, confirmed that these three proteins were in a single protein complex. Immunohistochemical experiments were then conducted on the perfused mouse brain that confirmed the in situ colocalization of CK and MT-3 in the hippocampus region. These data provide new insights into the involvement of MT-3 in a multiprotein complex, which will be used to understand the biological activity of MT-3 and its role in neurological disease.
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PMID:Metallothionein-3 is a component of a multiprotein complex in the mouse brain. 1701 72

The Src homology phosphotyrosyl phosphatase, SHP2, is a positive effector of EGFR signaling. However, the molecular mechanism and biological functions of SHP2 regulation are still not completely known. To better understand the cellular processes in which SHP2 participates, we carried out mass spectrometry to find SHP2 binding proteins. FLAG-SHP2 complexes were isolated by affinity purification, and associated proteins were identified by in-gel trypsin digestion followed by LC/MS/MS mass spectrometry. Among the identified proteins, we focus in this report on the heat shock protein 70 (HSP70). Physical interactions of SHP2 with HSP70 were confirmed in vivo. Further experiments demonstrate that EGF does not activate binding of SHP2 with HSP70 rather the binding appears to be constitutive. However, the formation of an HSP70/SHP2 complex affected the binding of SHP2 with EGFR and (or) GAB1. These data suggest that binding of HSP70 with SHP2 regulates to some extent the EGF signaling pathway. In addition, immunostaining experiments indicated that SHP2 and HSP70 co-localized in the cell membrane region after EGF treatment. Our findings propose a possible involvement of HSP70 in the regulation of EGF signaling pathway by SHP2.
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PMID:HSP70 binds to SHP2 and has effects on the SHP2-related EGFR/GAB1 signaling pathway. 1709 51

Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.
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PMID:Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members. 1714 52

Cystic fibrosis (CF) is caused by mutations to the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common of these mutations is deletion of a phenylalanine residue at position 508 (Delta F508), which accounts for approximately 70% of all CF alleles. This mutation interferes with the biogenesis and maturation of Delta F508-CFTR to the plasma membrane. However, Delta F508-CFTR can partially function upon proper localization. Thus, pharmacological correction of Delta F508-CFTR maturation holds promise in CF therapy. Our previous studies indicate that a single non-cytotoxic dose of the anthracycline doxorubicin (Dox) significantly increase Delta F508-CFTR-associated chloride secretion in MDCK cells by increasing the expression of this protein at the apical plasma membrane. We report here that Dox alters the biogenesis of Delta F508-CFTR. Treatment with Dox increases the resistance of Delta F508-CFTR to trypsin digestion, possibly by expediting protein folding. Further, treatment with Dox reduces the amount of polyubiquitinated Delta F508-CFTR in cells and prolongs the half-life of this protein. Concomitantly, treatment with Dox decreases the association of Delta F508-CFTR with HSP70 but does not alter the expression of major HSP70 family members. Based on these results, we propose that Dox expedites the folding and maturation of Delta F508-CFTR by acting as a pharmacological chaperone, which consequently promotes the functional expression of this protein in MDCK cells.
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PMID:Altered biogenesis of deltaF508-CFTR following treatment with doxorubicin. 1776 73

A clonal human embryonic kidney (HEK) 293 cell line was established that stably expressed the rat kappa-opioid receptor (rKOR) with a FLAG epitope at the amino terminus. The Kd for [3H]diprenorphine was 1.1+/-0.2 nM, and the Bmax was 2.6+/-0.4 pmol/mg. Dynorphin A (1-13), U69,593 and naloxone competitively inhibited [3H]diprenorphine binding with Ki values of 2.0, 18 and 18 nM, respectively, in good agreement with previously reported affinities for the unmodified receptor. U69,593 stimulated [35S]GTPgammaS binding in a concentration-dependent manner and caused phosphorylation of mitogen-activated protein (MAP) kinase, indicating that the activated epitope-tagged receptor triggered appropriate signaling pathways. Immunoblot analysis demonstrated that two immunoreactive receptor species with apparent molecular masses of 42 and 52 kDa were expressed. Previous studies indicated that the 42 kDa protein was localized intracellularly and was a precursor of the 52 kDa receptor, which was present at the cell surface. rKOR was extracted from transfected HEK 293 cell membranes with n-dodecyl-beta-D-maltopyranoside. Sequential use of wheat germ agglutinin chromatography, Sephacryl S300 gel filtration chromatography, anti-FLAG immunoaffinity chromatography and SDS/PAGE permitted purification of the 52 kDa receptor. MALDI-TOF mass spectrometry was used to identify peptides derived from rKOR following sequential in-gel digestion with trypsin and cyanogen bromide. Eighteen rKOR peptides were detected, corresponding to 27.1% coverage of the receptor. Precursor-selective MS/MS confirmed the identity of most of these peptides. In addition, we have identified heat shock protein 70 (HSP70) as a rKOR-interacting protein.
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PMID:Purification and mass spectrometric analysis of the kappa opioid receptor. 1865 60


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