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)

Previously, we showed that the human kappa-opioid receptor (hkor) stably expressed in Chinese hamster ovary (CHO) cells underwent down-regulation after prolonged U50,488H treatment. In the present study, we determined the mechanisms underlying this process. U50, 488H caused a significant down-regulation of the hkor, although etorphine did not. Neither U50,488H nor etorphine caused down-regulation of the rat kappa-opioid receptor. Thus, similar to internalization, there are agonist and species differences in down-regulation of kappa-opioid receptors. Expression of the dominant negative mutants arrestin-2(319-418) or dynamin I-K44A significantly reduced U50,488H-induced down-regulation of the hkor. Coexpression of GRK2 or GRK2 and arrestin-2 permitted etorphine to induce down-regulation of the hkor, although expression of arrestin-2 or dynamin I alone did not. Expression of the dominant negative mutants rab5A-N133I or rab7-N125I blunted U50,488H-induced down-regulation. Pretreatment with lysosomal enzyme inhibitors [(2S, 3S)trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester or chloroquine] or proteasome inhibitors (proteasome inhibitor I, MG-132, or lactacystin) decreased the extent of U50,488H-induced down-regulation. A combination of chloroquine and proteasome inhibitor I abolished U50,488H-induced down-regulation. These results indicate that U50,488H-induced down-regulation of the hkor involves GRK-, arrestin-2-, dynamin-, rab5-, and rab7-dependent mechanisms and receptors seem to be trafficked to lysosomes and proteasomes for degradation. Thus, U50,488H-induced internalization and down-regulation of the hkor share initial common mechanisms. To the best of our knowledge, these results represent the first report on the involvement of both rab5 and rab7 in agonist-induced down-regulation of a G protein-coupled receptor. In addition, this study is among the first to show the involvement of proteasomes in agonist-induced down-regulation of a G protein-coupled receptor.
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PMID:Mechanisms of agonist-induced down-regulation of the human kappa-opioid receptor: internalization is required for down-regulation. 1099 50

Ras is a small GTPase that is activated by upstream guanine nucleotide exchange factors, one of which is Ras-GRF2. GRF2 is a widely expressed protein with several recognizable sequence motifs, including a Ras exchanger motif (REM), a PEST region containing a destruction box (DB), and a Cdc25 domain. The Cdc25 domain possesses guanine nucleotide exchange factor activity and interacts with Ras. Herein we examine if the DB motif in GRF2 results in proteolysis via the ubiquitin pathway. Based on the solved structure of the REM and Cdc25 regions of the Son-of-sevenless (Sos) protein, the REM may stabilize the Cdc25 domain during Ras binding. The DB motif of GRF2 is situated between the REM and the Cdc25 domains, tempting speculation that it may be exposed to ubiquitination machinery upon Ras binding. GRF2 protein levels decrease dramatically upon activation of GRF2, and dominant-negative Ras induces degradation of GRF2, demonstrating that signaling downstream of Ras is not required for the destruction of GRF2 and that binding to Ras is important for degradation. GRF2 is ubiquitinated in vivo, and this can be detected using mass spectrometry. In the presence of proteasome inhibitors, Ras-GRF2 accumulates as a high-molecular-weight conjugate, suggesting that GRF2 is destroyed by the 26S proteasome. Deleting the DB reduces the ubiquitination of GRF2. GRF2 lacking the Cdc25 domain is not ubiquitinated, suggesting that a protein that cannot bind Ras cannot be properly targeted for destruction. Point mutations within the Cdc25 domain that eliminate Ras binding also eliminate ubiquitination, demonstrating that binding to Ras is necessary for ubiquitination of GRF2. We conclude that conformational changes induced by GTPase binding expose the DB and thereby target GRF2 for destruction.
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PMID:Ras binding triggers ubiquitination of the Ras exchange factor Ras-GRF2. 1123 45

Racs are involved in the regulation of important cellular processes including mitogenesis. We found that the E3 ubiquitination ligase subunit Cullin-1 interacts with constitutively active Rac3 but not with wild-type Rac3 in yeast. In mammalian cell lysates, Cullin-1 bound to V12Rac3, effector domain mutants V12L37Rac3 and V12H40Rac3, and insert domain deletion mutant V12Rac3DeltaIns(124-135). Cullin-1 also formed a clearly detectable complex with other activated Rac3-related proteins including Rac1, Rac2, Cdc42 and RhoA but not with the distantly related small GTPase Rap1. Since the proteasome is involved in cell cycle control through the programmed degradation of cell cycle proteins, the possible regulation of Rac levels during the cell cycle was examined. However, Rac was expressed at constant levels throughout the cell cycle, and a specific proteasome inhibitor had no effect on Rac protein levels. These combined results indicate that the binding of activated Rac to Cullin-1 does not affect Rac protein levels, nor does it mediate the regulation of mitogenesis by Rac. However, Rac-Cullin-1 interactions may serve to regulate other E3 ligase functions such as subcellular localization. Indeed, activated Rac3 and Cullin-1 co-localized to the perinuclear region of the cell. We also detected complex formation between Rac and the APC component CDC23. These results indicate that Rac may regulate specific proteolytic processes through directed subcellular localization of SCF or APC complexes.
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PMID:The small GTPase Rac interacts with ubiquitination complex proteins Cullin-1 and CDC23. 1144 62

Intramanchette transport (IMT) and intraflagellar transport (IFT) share similar molecular components: a raft protein complex transporting cargo proteins mobilized along microtubules by molecular motors. IFT, initially discovered in flagella of Chlamydomonas, has been also observed in cilia of the worm Caenorhabditis elegans and in mouse ciliated and flagellated cells. IFT has been defined as the mechanism by which protein raft components (also called IFT particles) are displaced between the flagellum and the plasma membrane in the anterograde direction by kinesin-II and in the retrograde direction by cytoplasmic dynein 1b. Mutation of the gene Tg737, encoding one of the components of the raft protein complex, designated Polaris in the mouse and IFT88 in both Chlamydomonas and mouse, results in defective ciliogenesis and flagellar development as well as asymmetry in left-right axis determination. Polaris/IFT88 is detected in the manchette of mouse and rat spermatids. Indications of an IMT mechanism originated from the finding that two proteins associated with the manchette (Sak57/K5 and TBP-1, the latter a component of the 26S proteasome) repositioned to the centrosome and sperm tail once the manchette disassembled. IMT has the features of the IFT machinery but, in addition, facilitates nucleocytoplasmic exchange activities during spermiogenesis. An example is Ran, a small GTPase present in the nucleus and cytoplasm of round spermatids and in the manchette of elongating spermatids. Upon disassembly of the manchette, Ran GTPase is found in the centrosome region of elongating spermatids. Because defective molecular motors and raft proteins result in defective flagella, cilia, and cilia-containing photoreceptor cells in the retina, IMT and IFT are emerging as essential mechanisms for managing critical aspects of sperm development. Details of specific role of Ran GTPase in nucleocytoplasmic transport and its relocation from the manchette to the centrosome to the sperm tail await elucidation.
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PMID:Intramanchette transport (IMT): managing the making of the spermatid head, centrosome, and tail. 1221 Oct 54

Serum response factor (SRF) is activated by contractile and hypertrophic agonists, such as endothelin-1 (ET1) to stimulate expression of cytoskeletal proteins in vascular smooth muscle cells (VSMCs). While studying the regulation of smooth muscle alpha-actin (SMA) expression at the level of protein stability, we discovered that inhibition of proteasome-dependent protein degradation by N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) or lactacystin (LC) did not enhance the levels of SMA, but, unexpectedly, attenuated SMA expression in response to ET1, without affecting the viability of VSMCs. Down-regulation of SMA protein by MG132 or LC occurred at the level of SMA transcription and via the inhibition of SRF activity. By contrast, MG132 and LC potentiated the activity of activator protein-1 transcription factor. Regulation of SRF by MG132 was not related to inhibition of nuclear factor-kappaB, an established target of proteasome inhibitors, and was not mediated by protein kinase A, a powerful regulator of SRF activity. Signaling studies indicate that inhibition of ET1-induced SRF activity by MG132 occurs at the level downstream of heterotrimeric G proteins Gq/11 and G13, of small GTPase RhoA, and of actin dynamics but at the level of SRF-DNA binding. MG132 treatment did not result in ubiquitination or accumulation of SRF. By contrast, the levels of c-Jun were rapidly increased upon incubation of cells with MG132, and ectopic overexpression of c-Jun mimicked the effect of MG132 on SRF activity. Together, these data suggest that inhibition of proteasome results in down-regulation of SMA expression via up-regulation of c-Jun and repression of SRF activity at the level of DNA binding.
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PMID:Regulation of serum response factor-dependent gene expression by proteasome inhibitors. 1555 Jun 77

Hypoxia is a crucial factor in tumor aggressiveness and resistance to treatment, particularly in glioma. Our previous results have shown that inhibiting the small GTPase RhoB increased oxygenation of U87 human glioblastoma xenografts, in part, by regulating angiogenesis. We investigated here whether RhoB might also control a signaling pathway that would permit glioma cells to adapt to hypoxia. We first showed that silencing RhoB with siRNA induced degradation and inhibition of the transcriptional activity of the hypoxia-inducible factor by the proteasome in U87 hypoxic cells. This RhoB-dependent degradation of hypoxia-inducible factor-1alpha in hypoxic conditions was mediated by the Akt/glycogen synthase kinase-3beta pathway. While investigating how hypoxia could activate this signaling pathway, using the GST-Rhotekin RBD pulldown assay, we showed the early activation of RhoB by reactive oxygen species under hypoxic conditions and, subsequently, its participation in the ensuing cellular adaptation to hypoxia. Overall, therefore, our results have not only highlighted a new signaling pathway for hypoxia controlled by the small GTPase RhoB, but they also strongly implicate RhoB as a potentially important therapeutic target for decreasing tumor hypoxia.
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PMID:Activation of RhoB by hypoxia controls hypoxia-inducible factor-1alpha stabilization through glycogen synthase kinase-3 in U87 glioblastoma cells. 1639 64

RK13 cell lines generated to express bovine PrP(C) with a four extra octarepeat insertional mutation (Bo-10ORPrP(C)) show partially insoluble PrP(C) and lower rates of cell growth when compared to either the same cells expressing wild type Bo-6ORPrP(C) or the original RK13 cell line. The expression of Bo-10ORPrP(C) in cell cultures was also associated with changes in cell size and reorganization of the actin cytoskeleton. This last process was reversed by Clostridium difficile toxin-B, a specific inhibitor of small GTPase proteins. Further, in clones expressing Bo-10ORPrP(C), increased proportions of cells at cell cycle stage G2/M were observed. Proteasome inhibitors caused a further expansion of G2/M-stage cells that was more marked in cell lines expressing Bo-10ORPrP(C) than those expressing Bo-6ORPrP(C), while this effect was minimal or null in the original RK13 cell line. Hence, the presence of Bo-10ORPrP(C) in RK13 cells promotes cell cycle arrest at G2/M, and the effect is amplified by proteasome inhibition. These findings suggest a role for PrP(C) in cell morphology and cell cycle regulation, and open new avenues for understanding the mechanisms underlying PrP mutation-associated diseases.
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PMID:Cell expression of a four extra octarepeat mutated PrPC modifies cell structure and cell cycle regulation. 1682 87

Alzheimer's disease (AD) is characterized by deposits of aggregated proteins. Accumulation of aggregation-prone proteins activates protein quality control mechanisms, such as the unfolded protein response (UPR) in the endoplasmic reticulum (ER). We previously reported upregulation of the UPR marker BiP in AD brain. In this study, we investigated the small GTPase Rab6, which is involved in retrograde Golgi-ER trafficking and may function as a post-ER quality control system. Using immunohistochemistry and semiquantitative Western blotting, the expression of Rab6 was analysed in hippocampus, entorhinal and temporal cortex of 10 AD patients and six nondemented control subjects. Rab6 is upregulated in AD temporal cortex from Braak stage 3/4, the same stage that UPR activation is found. We observe increased neuronal Rab6 immunoreactivity in all brain areas examined. Although some neurones show colocalization of immunoreactivity for Rab6 and hyperphosphorylated tau, strong Rab6 staining does not colocalize with tangles. We find a highly significant correlation between the Rab6 and BiP levels. In vitro data show that Rab6 is not upregulated as a result of UPR activation or proteasome inhibition indicating an independent regulatory mechanism. Our data suggest that ER and post-ER protein quality control mechanisms are activated early in the pathology of AD.
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PMID:Rab6 is increased in Alzheimer's disease brain and correlates with endoplasmic reticulum stress. 1757 8

Simvastatin is a competitive inhibitor of HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway required for the biosynthesis of cholesterol and higher isoprenoids such as geranylgeranyl pyrophosphate (GGPP). Apart from its capacity to lower cholesterol plasma levels and to protect against cardiovascular disease, simvastatin induces apoptosis in various cancer cells. We have generated human Namalwa Burkitt lymphoma cells that display general apoptosis resistance and hyperproliferation due to increased expression and proteolytic activity of 26S proteasomes in response to continuous treatment of the cells with the proteasome inhibitor bortezomib. In these cells, simvastatin does not inhibit proteasome activity, but induces apoptosis, G2/M cell cycle arrest and accumulation of p21(Waf1/Cip1), and effectively inhibits hyperproliferation. These effects are reversed by the addition of GGPP. GGPP-dependent plasma membrane localization of the small GTPase RhoA that is required for RhoA-mediated oncogenic signaling is completely inhibited by simvastatin. Finally, bortezomib but not simvastatin induces accumulation and stabilization of the anti-apoptotic protein Mcl-1, which is known to confer resistance to apoptosis in cancer cells. Thus, simvastatin overcomes bortezomib-induced apoptosis resistance by inhibiting synthesis of GGPP and disrupting a GGPP-dependent survival pathway.
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PMID:HMG-CoA reductase inhibitor simvastatin overcomes bortezomib-induced apoptosis resistance by disrupting a geranylgeranyl pyrophosphate-dependent survival pathway. 1862 2

Oncogenic mutations in the small GTPase Ras are highly prevalent in cancer, but an understanding of the vulnerabilities of these cancers is lacking. We undertook a genome-wide RNAi screen to identify synthetic lethal interactions with the KRAS oncogene. We discovered a diverse set of proteins whose depletion selectively impaired the viability of Ras mutant cells. Among these we observed a strong enrichment for genes with mitotic functions. We describe a pathway involving the mitotic kinase PLK1, the anaphase-promoting complex/cyclosome, and the proteasome that, when inhibited, results in prometaphase accumulation and the subsequent death of Ras mutant cells. Gene expression analysis indicates that reduced expression of genes in this pathway correlates with increased survival of patients bearing tumors with a Ras transcriptional signature. Our results suggest a previously underappreciated role for Ras in mitotic progression and demonstrate a pharmacologically tractable pathway for the potential treatment of cancers harboring Ras mutations.
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PMID:A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. 1971 64


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