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)

The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli has been shown to activate members of the Rho family by deamidation of glutamine 63. This amino acid is essential for hydrolysis of GTP, and any substitution results in a constitutively active Rho. Activation of Rho induces the formation of stress fibers, filopodia, and membrane ruffles due to activation of RhoA, Cdc42, and Rac, respectively. Here we show that the level of endogenous Rac decreased in CNF1-treated HEK293 and HeLa cells. The amount of mRNA remained unaffected, leaving the possibility that Rac is subject to proteolytic degradation. Treatment of cells with lactacystin, an inhibitor of the 26S proteasome, protected Rac from degradation. We have previously shown that CNF1 activates the c-Jun N-terminal kinase (JNK) only transiently in HeLa cells (M. Lerm, J. Selzer, A. Hoffmeyer, U. R. Rapp, K. Aktories, and G. Schmidt, Infect. Immun. 67:496-503, 1998). Here we show that CNF1-induced JNK activation is stabilized in the presence of lactacystin. The data indicate that Rac is degraded by a proteasome-dependent pathway in CNF1-treated cells.
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PMID:Proteasomal degradation of cytotoxic necrotizing factor 1-activated rac. 1211 11

CapLC-ESI-MS/MS and nano-ESI-MS/MS techniques were used to identify the apoptosis associated proteins induced by inhibiting the ubiquitin-proteasome pathway in Mo7e leukaemic cells. In 2-DE, spot H was found to initiate its overexpression at 2 h after the inhibition and reached its peak at 6 h. It was identified as Rho GDI beta protein after the tandem mass spectrum and after the sequence of its tryptic peptides were obtained by the ESI-MS/MS techniques. It was not revealed by peptide mass fingerprint using MALDI-TOF-MS. Other two spots induced by the inhibition appeared close to spot H were also revealed identical to Rho GDI brg;, possibly due to unknown modifications.
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PMID:[Nano-ESI-MS/MS identification on apoptosis qssociated proteins induced by inhibiting ubiquitin-proteasome pathway]. 1219 68

CNF1 toxin is a virulence factor produced by uropathogenic Escherichia coli. Upon cell binding and introduction into the cytosol, CNF1 deamidates glutamine 63 of RhoA (or 61 of Rac and Cdc42), rendering constitutively active these GTPases. Unexpectedly, we measured in bladder cells a transient CNF1-induced activation of Rho GTPases, maximal for Rac. Deactivation of Rac correlated with the increased susceptibility of its deamidated form to ubiquitin/proteasome-mediated degradation. Sensitivity to ubiquitylation could be generalized to other permanent-activated forms of Rac and to its sustained activation by Dbl. Degradation of the toxin-activated Rac allowed both host cell motility and efficient cell invasion by uropathogenic bacteria. CNF1 toxicity thus results from a restricted activation of Rho GTPases through hijacking the host cell proteasomal machinery.
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PMID:CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion. 1247 2

Cytotoxic necrotizing factor 1 (CNF1), a virulence factor expressed by pathogenic Escherichia coli, acts on Rho-GTPases and specifically deamidates a single glutamine residue (Gln-63 in RhoA) required for GTP hydrolysis. This modification constitutively activates the effector binding function of Rho-GTPases and eventually leads to their proteasome-mediated degradation. Previous structural investigation revealed that the CNF1 active site is located in a deep and narrow pocket and that the entrance to this pocket is formed by nine loop segments. We have examined the functional importance of five of these loops (2, 6, 7, 8, and 9) by deleting them individually. We find that deletion of proximally located loops 8 and 9 in the 32 kDa catalytic domain of CNF1 (CNF1-C) nearly or completely abolishes deamidation of RhoA in vitro, identifying a potential Rho-GTPase recognition site. Deletion of loop 7 causes protein folding errors, and deletion of loop 6 has a small effect on deamidation. In contrast, deletion of loop 2 is found to increase deamidation 5-7-fold, implying that this loop rearranges in binding RhoA. None of the loop deletions or wild-type CNF1-C is able to deamidate RhoA containing Asn-63 instead of Gln-63, suggesting that the fit between the toxin and its target is highly precise. In addition, we show that the specificity constant (k(cat)/K(m)) of CNF1-C for RhoA is 825 +/- 3 M(-1) s(-1). This modest value is consistent with the confining size of the active site pocket acting to exclude nonspecific targets but also limiting reactivity toward intended targets.
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PMID:Structural elements required for deamidation of RhoA by cytotoxic necrotizing factor 1. 1459 92

Rhobtb2 is a candidate tumor suppressor located on human chromosome 8p21, a region commonly deleted in cancer. Rhobtb2 is homozygously deleted in 3.5% of primary breast cancers, and gene expression is ablated in approximately 50% of breast and lung cancer cell lines. RhoBTB2 is an 83-kD, atypical Rho GTPase of unknown function, comprising an N-terminal Rho GTPase domain and two tandem BTB domains. In this report, we demonstrate that RhoBTB2 binds to the ubiquitin ligase scaffold, Cul3, via its first BTB domain and show in vitro and in vivo that RhoBTB2 is a substrate for a Cul3-based ubiquitin ligase complex. Moreover, we show that a RhoBTB2 missense mutant identified in a lung cancer cell line is neither able to bind Cul3 nor is it regulated by the ubiquitin/proteasome system, resulting in increased RhoBTB2 protein levels in vivo. We suggest a model in which RhoBTB2 functions as a tumor suppressor by recruiting proteins to a Cul3 ubiquitin ligase complex for degradation.
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PMID:RhoBTB2 is a substrate of the mammalian Cul3 ubiquitin ligase complex. 1510 2

Botulinum toxin type A (BoNT/A) produced by Clostridium botulinum inhibits Ca2+-dependent acetylcholine (ACh) release (neuroexocytosis) at peripheral neuromuscular junctions, sometimes causing neuromuscular paralysis. This inhibitory effect is attributed to its metalloprotease activity to cleave the 25-kDa synaptosomal-associated protein, which is essential for the exocytotic machinery. However, deletion of this protein does not result in a complete block of neuroexocytosis, suggesting that botulinum-mediated inhibition may occur via another mechanism. Rho GTPases, a class of small GTP-binding proteins (G proteins), control actin cytoskeletal organization, thereby regulating a variety of cellular functions in various cells, including neuronal cells. We have shown that the G protein activator lysophosphatidic acid (LPA) triggered actin reorganization followed by Ca2+-dependent ACh release in nerve growth factor-treated PC12 cells and that BoNT/A blocked both events through degradation of RhoB by the proteasome. Overexpression of wild-type RhoB caused actin reorganization and enhanced the release of ACh by LPA to overcome toxin's inhibitory effect on actin reorganization/exocytosis stimulated by LPA, whereas overexpression of a dominant negative RhoB inhibited ACh release, regardless of LPA and/or toxin treatment. Finally, a knockdown of the RhoB gene via sequence-specific, post-transcriptional gene silencing reduced RhoB expression in PC12 cells, resulting in total inhibition of both actin reorganization and ACh release induced by LPA. We conclude that the RhoB signaling pathway regulates ACh release via actin cytoskeletal reorganization and that botulinum toxin inhibits neuroexocytosis by targeting RhoB pathway.
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PMID:Botulinum toxin type A targets RhoB to inhibit lysophosphatidic acid-stimulated actin reorganization and acetylcholine release in nerve growth factor-treated PC12 cells. 1514 Sep 14

The cytotoxic necrotizing factor-1 (CNF1), a bacterial toxin of uropathogenic bacteria (UPEC), hijacks cellular Rho proteins of the Ras GTPase super-family. Recently, we have made three important findings. First, we have established that, following Rho protein activation by deamidation, these cellular proteins are ubiquitylated and degraded by the proteasome. Second, the low level of activated Rho proteins which results from the dual molecular mechanism of action of CNF1 (Rho protein activation followed by their degradation), confers high invasive properties to UPECs. Finally, we have reported that ubiquitylation and degradation of Rac is lost in HEp-2 carcinoma cells, thereby suggesting a possible link between Rho protein ubiquitylation and tumor progression.
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PMID:E. coli CNF1 toxin: a two-in-one system for host-cell invasion. 1514 26

We observed evolutionary conservation of canonical nuclear localization signal sequences (K(K/R)X(K/R)) in the C-terminal polybasic regions (PBRs) of some Rac and Rho isoforms. Canonical D-box sequences (RXXL), which target proteins for proteasome-mediated degradation, are also evolutionarily conserved near the PBRs of these small GTPases. We show that the Rac1 PBR (PVKKRKRK) promotes Rac1 nuclear accumulation, whereas the RhoA PBR (RRGKKKSG) keeps RhoA in the cytoplasm. A mutant Rac1 protein named Rac1 (pbrRhoA), in which the RhoA PBR replaces the Rac1 PBR, has greater cytoplasmic localization, enhanced resistance to proteasome-mediated degradation, and higher protein levels than Rac1. Mutating the D-box by substituting alanines at amino acids 174 and 177 significantly increases the protein levels of Rac1 but not Rac1(pbrRhoA). These results suggest that Rac1 (pbrRhoA) is more resistant than Rac1 to proteasome-mediated degradative pathways involving the D-box. The cytoplasmic localization of Rac1(pbrRhoA) provides the most obvious reason for its resistance to proteasome-mediated degradation, because we show that Rac1(pbrRhoA) does not greatly differ from Rac1 in its ability to stimulate membrane ruffling or to interact with SmgGDS and IQGAP1-calmodulin complexes. These findings support the model that nuclear localization signal sequences in the PBR direct Rac1 to the nucleus, where Rac1 participates in signaling pathways that ultimately target it for degradation.
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PMID:The Rac1 C-terminal polybasic region regulates the nuclear localization and protein degradation of Rac1. 1530 4

Insulin/IGF-I-dependent signals play important roles for the regulation of proliferation, differentiation, metabolism, and autophagy in various cells, including hematopoietic cells. Although the early protein kinase activation cascade has been intensively studied, the whole picture of intracellular signaling events has not yet been clarified. To identify novel downstream effectors of insulin-dependent signals in relatively early phases, we performed high-resolution two-dimensional electrophoresis (2-DE)-based proteomic analysis using human hematopoietic cells 1 h after insulin stimulation. We identified SRp20, a splicing factor, and CLIC1, an intracellular chloride ion channel, as novel downstream effectors besides previously reported effectors of Rho-guanine nucleotide dissociation inhibitor 2 and glutathione S-transferase-pi. Reduction in SRp20 was confirmed by one-dimensional Western blotting. Moreover, MG-132, a proteasome inhibitor, prevented this reduction. By contrast, upregulation of CLIC1 was not observed in one-dimensional Western blotting, unlike the 2-DE results. As hydrophilic proteins were predominantly recovered in 2-DE, the discrepancy between the 1-DE and 2-DE results may indicate a certain qualitative change of the protein. Indeed, the nuclear localization pattern of CLIC1 was remarkably changed by insulin stimulation. Thus insulin induces the proteasome-dependent degradation of SRp20 as well as the subnuclear relocalization of CLIC1.
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PMID:Proteomic analysis on insulin signaling in human hematopoietic cells: identification of CLIC1 and SRp20 as novel downstream effectors of insulin. 1582 65

cAMP and cyclic GMP-dependent kinases (PKA and PKG) phosphorylate the small G protein RhoA on Ser188. We have previously demonstrated that phosphorylation of Ser188 inhibits RhoA-dependent functions and positively regulates RhoA expression, and that the nitric oxide (NO)/cGMP-dependent protein kinase pathway plays an essential role, both in vitro and in vivo, in the regulation of RhoA protein expression and functions in vascular smooth muscle cells. Here we analyze the consequences of Ser188 phosphorylation on RhoA protein degradation. By expressing Ser188 phosphomimetic wild-type (WT-RhoA-S188E) and active RhoA proteins (Q63L-RhoA-S188E), we show that phosphorylation of Ser188 of RhoA protects RhoA, particularly its active form, from ubiquitin-mediated proteasomal degradation. Coimmunoprecipitation experiments indicate that the resistance of the phosphorylated active form of RhoA to proteasome-mediated degradation is because of its cytoplasmic sequestration through enhanced RhoGDI interaction. In rat aortic smooth muscle cells, stimulation of PKG and inhibition of proteasome by lactacystin, induce nonadditive increases in RhoA protein expression. In addition, stimulation of PKG leads to the accumulation of GTP-bound RhoA in the cytoplasm. In vivo stimulation of the NO/PKG signaling by treating rats with sildenafil increased RhoA level and RhoA phosphorylation, and enhanced its association to RhoGDI in the pulmonary artery, whereas opposite effects are induced by chronic inhibition of NO synthesis in N-omega-nitro-L-arginine-treated rats. Our results thus suggest that Ser188 phosphorylation-mediated protection against degradation is a physiological process regulating the level of endogenous RhoA and define a novel function for RhoGDI, as an inhibitor of Rho protein degradation.
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PMID:Phosphorylation of serine 188 protects RhoA from ubiquitin/proteasome-mediated degradation in vascular smooth muscle cells. 1589 Sep 75


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