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:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To test whether an observed age-related increase in the level of oxidized protein in rat liver is due to a decrease in the activity of the multicatalytic proteinase (MCP), this protease was isolated from liver of young (8-month-old) and old (24-month-old) male Fischer 344 rats. Three peptidase activities of the MCP were assayed using fluorogenic peptides: trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolase. Only peptidylglutamyl-peptide hydrolase activity declined with age, with protease from old animals exhibiting approximately 50% of the activity of that from young animals. Bidimensional gel electrophoresis and thermostability studies did not reveal age-related structural modifications of the MCP subunits. Peptidylglutamyl-peptide hydrolase activity and trypsin-like activity were sensitive to metal-catalyzed oxidation. In some preparations, a 95-kDa protein that has been identified as the heat shock protein 90 copurified with the MCP. In the presence of HSP 90, trypsin-like activity is protected from oxidative inactivation and chymotrypsin-like activity is slightly activated. Peptidylglutamyl-peptide hydrolase activity remained sensitive to oxidation in protease isolated from young rats, but that from old rats was resistant to oxidative inactivation. Furthermore, addition of rat HSP 90 to rat liver MCP (purified from 8-month-old animals and free of contaminating HSP 90) was found to protect trypsin-like activity from oxidative inactivation.
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PMID:Age-related decline of rat liver multicatalytic proteinase activity and protection from oxidative inactivation by heat-shock protein 90. 866 Jul 3

When isolated from cells grown under hormone-free conditions, the glucocorticoid receptor (GR) is known to exist as a large heteroprotein complex that contains, among its multiple components, the stress protein hsp90 (heat shock protein 90). To explore hsp90's role in mediating glucocorticoid hormone action, we have examined the effects of a selective hsp90-binding agent, geldanamycin (GA), or GR structure and function. Using a steroid-responsive reporter construct, we found that GA inhibited the dexamethasone-dependent transactivating activity of GR in transfected cells. At the molecular level, GA bound hsp90, but not GR, in a stable and specific manner in intact cells. GA treatment of cells did not inhibit coprecipitation of hsp90 or hsp70 with the GR but did result in a complete loss of the recently described p23 protein from GR immunoprecipitates. This drug-induced alteration in GR heteroprotein complex composition was associated with a rapid (15-30 min), noncompetitive loss of dexamethasone-binding activity. Longer exposures of cells to GA (2-8 h), resulted in a marked decline in the cellular level of GR protein. Pulse-chase data revealed that this decline resulted from a decrease in GR protein stability, not rate of synthesis. GA-induced declines in GR protein level were blocked by cotreatment of cells with lactacystin, a selective inhibitor of 20S proteasome activity, suggesting the possible involvement of the ubiquitin-proteasome pathway in mediating GA-induced decreases in GR protein abundance. Overall, these findings provide direct pharmacological evidence that hsp90 function is required to maintain both the hormone-binding activity and stability of the GR protein in intact cells and suggest that hsp90 function may provide a novel target for the modulation of steroid hormone signaling.
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PMID:Stable and specific binding of heat shock protein 90 by geldanamycin disrupts glucocorticoid receptor function in intact cells. 877 30

We previously reported three families with type A insulin-resistant syndrome who had mutations, either Asp1179 or Leu1193, in the kinase domain of the insulin receptor. The extreme insulin resistance of these patients was found to be caused by the decreased number of insulin receptors on the cell surface, due to the intracellular rapid degradation (Imamura, T., Takata, Y., Sasaoka, T., Takada, Y., Morioka, H., Haruta, T., Sawa, T., Iwanishi, M., Yang, G. H., Suzuki, Y., Hamada, J., and Kobayashi, M. (1994) J. Biol. Chem. 269, 31019-31027). In the present study, we first examined whether these mutations caused rapid degradation of unprocessed proreceptors, using the exon 13 deleted mutant insulin receptors (DeltaEx13-IR), which were accumulated in the endoplasmic reticulum as unprocessed proreceptors. The addition of Asp1179 or Leu1193 mutation to DeltaEx13-IR caused accelerated degradation of the unprocessed DeltaEx13-IR in the transfected COS-7 cells. Next, we tested whether these mutant receptors were degraded by the proteasome. Treatment with proteasome inhibitors Z-Leu-Leu-Nva-H (MG-115) or Z-Leu-Leu-Leu-H (MG-132) prevented the accelerated degradation of these mutant receptors, resulting in increased amounts of the mutant receptors in the COS-7 cells. Essentially the same results were obtained in the patient's transformed lymphocytes. Finally, we found that these mutant receptors bound to heat shock protein 90 (Hsp90). To determine whether Hsp90 played an important role in the accelerated receptor degradation, we examined the effect of anti-Hsp90 antibody on the mutant receptor degradation. The microinjection of anti-Hsp90 antibody into cells prevented the accelerated degradation of both Asp1179 and Leu1193 mutant insulin receptors. Taken together, these results suggest that Hsp90 is involved in dislocation of the mutant insulin receptors out of the endoplasmic reticulum into the cytosol, where the mutant receptors are degraded by the proteasome.
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PMID:Involvement of heat shock protein 90 in the degradation of mutant insulin receptors by the proteasome. 955 7

The proteasome is a large protease complex that is thought to be responsible for proteolytic removal of damaged proteins. We have previously shown that the level of proteolytic activity due to the proteasome is lower in lens epithelium from human cataractous lenses compared to the activity in epithelium from clear donor lenses. This study aimed to characterize the three main peptidase activities of the proteasome in human lens epithelium with respect to kinetic properties and sensitivity to heat and oxidation. Human lens epithelia were obtained from cataract surgery and analysis performed on pools of epithelial cell cytoplasm. Using the fluorogenic peptide substrates Suc-Leu-Leu-Val-Tyr-AMC (LLVY), Boc-Val-Gly-Arg-AMC (VGR) and Z-Leu-Leu-Glu-betaNA (LLE), Km-values of 56, 678 and 108 micrometers were obtained. All peptidase activities were inhibited by lactacystin, a specific proteasome inhibitor, but at very different rates; with LLVY-hydrolysing activity being the most sensitive (Ki50%=0.15 micrometers). Thermostability was investigated by performing the proteolytic assay at 20 degrees, 37 degrees and 53 degrees C. The trypsin-like activity, as measured by VGR, was completely stable at 53 degrees C for at least 24 hr whereas hydrolysis of LLVY and LLE declined after a few hours at 37 degrees C. Oxidative inhibition was induced by incubation of the samples in 0.5 m m H2O2for 1 or 24 hr. One hour exposure to H2O2caused moderate inhibition of all peptidase activities. The activity could be partially restored by adding 1 m m dithiotreitol, indicating the dependency on intact SH-groups. After 24 hr, peptidase activities were decreased to 25% (LLVY), 73% (VGR) and 44% (LLE) of corresponding control. This inhibition was irreversible for VGR and LLE, but could be partly prevented by the presence of heat shock protein 90 (LLVY and VGR) or alpha-crystallin (LLVY). These data show that the peptidase activities of the human lens proteasome can be modulated by metabolites, such as reactive oxygen species, and by endogenous proteins such as alpha-crystallin and heat shock protein 90.
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PMID:Differential inhibition of three peptidase activities of the proteasome in human lens epithelium by heat and oxidation. 1037 57

The DeltaF508 mutation of cystic fibrosis transmembrane conductance regulator (CFTR) is a trafficking mutant, which is retained and degraded in the endoplasmic reticulum by the ubiquitin-proteasome pathway. The mutant protein fails to reach a completely folded conformation that is no longer a substrate for ubiquitination ("stable B"). Wild type protein reaches this state with 25% efficiency. In this study the rabbit reticulocyte lysate with added microsomal membranes has been used to reproduce the post-translational events in the folding of wild type and DeltaF508 CFTR. In this system wild type CFTR does not reach the stable B form if the post-translational temperature is 37 degrees C, whereas at 30 degrees C the behavior of both wild type and mutant proteins mimics that observed in the cell. Geldanamycin stabilizes DeltaF508 CFTR with respect to ubiquitination only when added post-translationally. The interaction of wild type and mutant CFTR with the molecular chaperones heat shock cognate 70 (hsc70) and heat shock protein 90 (hsp90) has been assessed. Release of wild type protein from hsc70 coincides with the cessation of ubiquitination and formation of stable B. Geldanamycin immediately prevents the binding of hsp90 to DeltaF508 CFTR, and after a delay releases it from hsc70. Release of mutant protein from hsc70 also coincides with the formation of stable B DeltaF508 CFTR.
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PMID:Post-translational disruption of the delta F508 cystic fibrosis transmembrane conductance regulator (CFTR)-molecular chaperone complex with geldanamycin stabilizes delta F508 CFTR in the rabbit reticulocyte lysate. 1098 7

The effect of heat shock protein 90 (Hsp-90) and several other proteins on the catalytic activities of the 20 S proteasome (MPC) was examined. The chymotrypsin-like (ChT-L) and peptidylglutamyl-peptide hydrolyzing (PGPH) activities of the pituitary MPC were inhibited by Hsp-90 with IC50 values of 8 and 28 nM, respectively. Bovine serum albumin and two other proteins tested inhibited the same activities with much higher IC50 values. The trypsin-like and branched-chain amino-acid-preferring activities were not affected by any of the proteins. None of the activities of the bovine spleen MPC, an enzyme form in which the X, Y, and Z subunits are virtually completely replaced by the LMP2, LMP7, and LMP10 subunits, was affected by either Hsp-90 or the other proteins tested. Hsp-90 inhibited the degradation of the oxidized B-chain of insulin by the pituitary MPC but not by its spleen counterpart. The PA28 activator (11 S regulator; REG) of the proteasome abolished the inhibitory effect of Hsp-90 and other proteins on the ChT-L and PGPH activities of the pituitary MPC. It is suggested that Hsp-90 induces conformational changes that affect the ChT-L and PGPH activities expressed by the X and Y subunits, respectively, but does not affect the activities expressed by LMP subunits.
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PMID:Heat shock protein-90 and the catalytic activities of the 20 S proteasome (multicatalytic proteinase complex). 1136 78

Cisplatin is an antineoplastic drug that binds to DNA, thereby inhibiting cell division and tumor growth. Cisplatin may also disrupt the function of some proteins, including heat shock protein 90 (Hsp90). We report that cisplatin dose-dependently inhibited transcriptional activity of the androgen receptor and the glucocorticoid receptor (GR) in transient reporter assays. A truncated, hormone-independent GR was only partially inhibited at significantly higher doses of cisplatin. Cisplatin treatment of neuroblastoma cells led to an immediate inhibition of hormone binding by GR, followed by proteasome-dependent degradation of the receptor. Other Hsp90-regulated proteins, i.e. the phosphokinases raf-1, lck, and c-src, were not affected, indicating a specific functional interference of cisplatin with the steroid receptors GR and androgen receptor. Cisplatin did not elicit a stress response, in contrast to geldanamycin. Immunoprecipitation revealed that cisplatin disrupts binding of GR to Hsp90. Moreover, cisplatin-treated Hsp90 was unable to associate with untreated ligand binding domain of GR. Reticulocyte lysate was able to restore hormone binding of GR in vitro, but not when the lysate was pretreated with geldanamycin. Our data reveal that cisplatin influences steroid receptors also independently of its DNA-mediated effects and, thus, suggest a novel modes of action for this cytostatic drug.
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PMID:The heat shock protein 90-targeting drug cisplatin selectively inhibits steroid receptor activation. 1286 91

G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine-specific protein kinase that mediates agonist-dependent phosphorylation of numerous G protein-coupled receptors. In an effort to identify proteins that regulate GRK2 function, we searched for interacting proteins by immunoprecipitation of endogenous GRK2 from HL60 cells. Subsequent analysis by gel electrophoresis and mass spectrometry revealed that GRK2 associates with heat shock protein 90 (Hsp90). GRK2 interaction with Hsp90 was confirmed by co-immunoprecipitation and was effectively disrupted by geldanamycin, an Hsp90-specific inhibitor. Interestingly, geldanamycin treatment of HL60 cells decreased the expression of endogenous GRK2 in a dose- and time-dependent manner, and metabolic labeling demonstrated that geldanamycin rapidly accelerated the degradation of newly synthesized GRK2. The use of various protease inhibitors suggested that GRK2 degradation induced by geldanamycin was predominantly through the proteasome pathway. To test whether Hsp90 plays a general role in regulating GRK maturation, additional GRKs were studied by transient expression in COS-1 cells and subsequent treatment with geldanamycin. These studies demonstrate that GRK3, GRK5, and GRK6 are also stabilized by interaction with Hsp90. Taken together, our work revealed that GRK interaction with heat shock proteins plays an important role in regulating GRK maturation.
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PMID:G protein-coupled receptor kinase interaction with Hsp90 mediates kinase maturation. 1455 68

Bisphenol A (BpA), an endocrine-disrupting chemical, is known to be a xenoestrogen and to affect the reproductive functions of animals. Recent reports have documented BpA-induced developmental abnormalities in the neuronal systems of humans and animals, and these effects appear to be non-estrogenic. In this study, we found that BpA inhibited the hypoxic response of human hepatoma cells. The expression of hypoxic response genes such as the erythropoietin (EPO) gene is done via a hypoxia inducible factor 1 (HIF-1)-dependent signaling pathway. To investigate possible structural requirements for this inhibitory effect, several BpA analogs were synthesized and added to this system. The blocking of two phenol groups in BpA did not change the effect, but the inhibition completely disappeared by the removal of two central methyl groups in BpA (the resulting compound is designated BpF). BpA, but not BpF, promoted degradation of the HIF-1alpha protein, which is a component of HIF-1, followed by inhibition of EPO induction. An immunoprecipitation assay indicated that BpA dissociated heat shock protein 90 (Hsp90) from HIF-1alpha and destabilized HIF-1alpha protein. HIF-1alpha is usually degraded first by ubiquitination and then by the proteasome pathway. Cobalt ion inhibits ubiquitination of HIF-1alpha and stabilizes it. In the present study, BpA promoted HIF-1alpha degradation in the presence of cobalt and in the presence of proteasome inhibitor. These results suggest that BpA degraded HIF-1alpha via a currently unknown pathway, and that this phenomenon required two methyl groups in BpA.
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PMID:Bisphenol A, an environmental endocrine-disrupting chemical, inhibits hypoxic response via degradation of hypoxia-inducible factor 1alpha (HIF-1alpha): structural requirement of bisphenol A for degradation of HIF-1alpha. 1514 73

It is established that neuronal NO synthase (nNOS) is ubiquitinated and proteasomally degraded. The metabolism-based inactivation of nNOS and the inhibition of heat shock protein 90 (hsp90)-based chaperones, which are known to regulate nNOS, both lead to enhanced proteasomal degradation of nNOS. The mechanism of this selective proteolytic degradation, or in essence how the nNOS becomes labilized and recognized for ubiquitination and subsequent degradation, has not been determined. In the current study, we used a crude preparation of reticulocyte proteins, which contains ubiquitin-conjugating enzymes and the proteasome, to determine how nNOS is labilized. We found that the inactive monomeric heme-deficient nNOS (apo-nNOS) is rapidly degraded in vitro, consistent with the finding that both metabolism-based inactivation and inhibition of hsp90-based chaperones cause the formation of apo-nNOS and enhance its degradation in vivo. In the current study, we discovered that destabilization of the dimeric nNOS, as determined by measuring the SDS-resistant dimer, is sufficient to trigger ubiquitin-proteasomal degradation. Treatment of nNOS with NG-nitro-L-arginine or 7-nitroindazole led to stabilization of the dimeric nNOS and decreased proteasomal degradation of the enzyme, consistent with that observed in cells. Thus, it seems that the dimeric structure is a major determinant of nNOS stability and proteolysis.
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PMID:Ubiquitination and degradation of neuronal nitric-oxide synthase in vitro: dimer stabilization protects the enzyme from proteolysis. 1523 1


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