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)

Many of the subunit proteins (connexins) that form gap junctions are rather dynamic, with half-lives of only a few hours. Thus, alterations in connexin turnover and degradation may represent significant mechanisms for the regulation of intercellular communication. We describe a pharmacological approach to determining pathways of connexin degradation. Cell cultures are left untreated or treated with inhibitors of lysosomal or proteasomal proteolysis. Changes in connexin levels, localization, or decay curves (derived from pulse-chase experiments) are assessed by immunoblotting, immunofluorescence, and immunoprecipitation, respectively. Such experiments have provided evidence that connexin43 degradation involves both the lysosome and the proteasome.
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PMID:Connexin and gap junction degradation. 1067 11

Electrical activation of the heart requires current transfer from one cell to another via gap junctions, arrays of densely packed intercellular channels. The extent to which cardiac myocytes are coupled is determined by multiple mechanisms, including tissue-specific patterns of expression of diverse gap junction channel proteins (connexins), and regulatory pathways that control connexin synthesis, intracellular trafficking, assembly into channels, and degradation. Many connexins, including those expressed in the heart, have been found to turn over rapidly. Recent studies in the intact adult heart suggest that connexin43, the principal cardiac connexin, is surprisingly short-lived (half-life approximately 1.3 hours). Both the proteasome and the lysosome participate in connexin43 degradation. Other ion channel proteins, such as those forming selected voltage-gated K(+) channels, may also exhibit rapid turnover kinetics. Regulation of connexin degradation may be an important mechanism for adjusting intercellular coupling in the heart under normal and pathophysiological conditions.
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PMID:Connexin expression and turnover : implications for cardiac excitability. 1076 4

More than 130 different mutations in the gap junction integral plasma membrane protein connexin32 (Cx32) have been linked to the human peripheral neuropathy X-linked Charcot-Marie-Tooth disease (CMTX). How these various mutants are processed by the cell and the mechanism(s) by which they cause CMTX are unknown. To address these issues, we have studied the intracellular transport, assembly, and degradation of three CMTX-linked Cx32 mutants stably expressed in PC12 cells. Each mutant had a distinct fate: E208K Cx32 appeared to be retained in the endoplasmic reticulum (ER), whereas both the E186K and R142W mutants were transported to perinuclear compartments from which they trafficked either to lysosomes (R142W Cx32) or back to the ER (E186K Cx32). Despite these differences, each mutant was soluble in nonionic detergent but unable to assemble into homomeric connexons. Degradation of both mutant and wild-type connexins was rapid (t(1/2) < 3 h) and took place at least in part in the ER by a process sensitive to proteasome inhibitors. The mutants studied are therefore unlikely to cause disease by accumulating in degradation-resistant aggregates but instead are efficiently cleared from the cell by quality control processes that prevent abnormal connexin molecules from traversing the secretory pathway.
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PMID:Intracellular transport, assembly, and degradation of wild-type and disease-linked mutant gap junction proteins. 1084 20

Connexins, the integral membrane protein constituents of gap junctions, are degraded at a rate (t(12) = 1.5-5 h) much faster than most other cell surface proteins. Although the turnover of connexins has been shown to be sensitive to inhibitors of either the lysosome or of the proteasome, how connexins are targeted for degradation and whether this process can be regulated to affect intercellular communication is unknown. We show here that reducing connexin degradation with inhibitors of the proteasome (but not with lysosomal blockers) is associated with a striking increase in gap junction assembly and intercellular dye transfer in cells inefficient in both processes under basal conditions. The effect of proteasome inhibitors on wild-type connexin stability, assembly, and function was mimicked by treatment of assembly-inefficient cells with inhibitors of protein synthesis such as cycloheximide. Sensitivity of connexin degradation to cycloheximide, but not to proteasome inhibitors, was abolished when connexins were rendered structurally abnormal by perturbation of essential disulfide bonds or by mutation. Our findings provide the first evidence that intercellular communication can be up-regulated at the level of connexin turnover and that a short-lived protein may be required for conformationally mature connexins to become substrates of proteasomal degradation.
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PMID:Regulation of connexin degradation as a mechanism to increase gap junction assembly and function. 1094 Mar 15

Akey step in ER-associated degradation (ERAD) is dislocation of the substrate protein from the ER into the cytosol to gain access to the proteasome. Very little is known about how this process is regulated, especially in the case of polytopic proteins. Using pulse-chase analysis combined with subcellular fractionation, we show that connexins, the four transmembrane structural components of gap junctions, can be chased in an intact form from the ER membrane into the cytosol of proteasome inhibitor-treated cells. Dislocation of endogenously expressed connexin from the ER was reduced 50-80% when the cytosolic heat shock response was induced by mild oxidative or thermal stress, but not by treatments that instead upregulate the ER unfolded protein response. Cytosolic but not ER stresses slowed the normally rapid degradation of connexins, and led to a striking increase in gap junction formation and function in otherwise assembly-inefficient cell types. These treatments also inhibited the dislocation and turnover of a connexin-unrelated ERAD substrate, unassembled major histocompatibility complex class I heavy chain. Our findings demonstrate that dislocation is negatively regulated by physiologically relevant, nonlethal stress. They also reveal a previously unrecognized relationship between cytosolic stress and intercellular communication.
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PMID:Dislocation and degradation from the ER are regulated by cytosolic stress. 1198 Sep 15

The central role of the supporting cell population, or epithelial support complex (ESC), in cochlear homeostasis has gained general acceptance. That the details of this role may vary markedly with location, however, remains poorly appreciated. For example, the K+ recirculation pathway may well be dictated by position along the cochlear axis: a perilymphatic route near the apex and a transcellular one near the base. The ESC expresses very high levels of OCP1 and OCP2, now known to be components of a novel, organ of Corti (OC)-specific SCF ubiquitin ligase (SCF(OCP1)). In the SCF(OCP1) cnmplex, OCP1 presumably binds selected protein targets, positioning them for ubiquitination. The recent demonstration that recombinant OCP1 interacts non-covalently with Cx26 suggests that the connexins may be target proteins for SCF(OCP1). Although ubiquitination has classically been viewed as a signal for subsequent destruction by the 26S proteasome, the energy-limited state of the OC prompts consideration of alternative fates, e.g. reversible internalization. The ESC also expresses several components of the Wingless/Wnt signaling pathway. Significantly, two of the gap-junction proteins expressed in the OC, Cx43 and Cx30, are known targets of the Wnt pathway. On the basis of these observations, a working hypothesis is proposed wherein the Wnt pathway activates connexin expression, while OCP1 regulates its degradation.
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PMID:Toward an understanding of cochlear homeostasis: the impact of location and the role of OCP1 and OCP2. 1270 41

Connexins are membrane-spanning proteins that form gap junction channels between adjacent cells. Connexin43 (Cx43), the most widely expressed member of the connexin family in tissues and cell lines, has a rapid turnover rate and its degradation involves both the lysosomal and ubiquitin-proteasome pathway. It was previously shown that the proteasome is involved in regulating the number of functional gap junctions at the plasma membrane. However, little is known about how proteasome-dependent turnover of Cx43 is controlled. Epidermal growth factor (EGF) induces hyperphosphorylation of Cx43 and a rapid, transient decrease in gap junctional intercellular communication. In this study, we show that, along with inhibition of gap junctional intercellular communication, EGF induces disorganization, internalization and degradation of Cx43 gap junction plaques in IAR20 rat liver epithelial cells. These EGF-induced modifications of Cx43 were counteracted by the MEK1 inhibitor PD98059, indicating that the effects were mediated by the mitogen-activated protein kinase pathway. The EGF-induced destruction of Cx43 was proteasome-dependent, because the loss of Cx43 protein was counteracted by the proteasome inhibitor MG132 but not the lysosomal inhibitor leupeptin. Furthermore, EGF induced ubiquitination of Cx43, which was associated with the Cx43 hyperphosphorylation. The EGF-induced Cx43 ubiquitination was counteracted by PD98059. The EGF-induced internalization of Cx43 was blocked by hypertonic sucrose treatment, indicating that EGF mediates internalization of Cx43 via a clathrin-dependent mechanism. Our results indicate that ubiquitination of Cx43 occurs at the plasma membrane before Cx43 internalization. Taken together, these data provide the first evidence that EGF-induced phosphorylation of Cx43 induces binding of ubiquitin and targets Cx43 for internalization and degradation in a proteasome-dependent manner.
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PMID:Epidermal growth factor regulates ubiquitination, internalization and proteasome-dependent degradation of connexin43. 1497 Feb 63

Gap junctions are specialized plasma membrane domains enriched in connexin proteins that form channels between adjacent cells. Gap junctions are highly dynamic, and modulation of the connexin turnover rate is considered to play an important role in the regulation of gap junctional intercellular communication. In the present study, we show that the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induces ubiquitination of connexin-43 (Cx43) in IAR20 rat liver epithelial cells. The accelerated ubiquitination of Cx43 in response to TPA occurred concomitantly with Cx43 hyperphosphorylation and inhibition of cell-cell communication via gap junctions. The TPA-induced ubiquitination of Cx43 was mediated via protein kinase C and partly involved the mitogen-activated protein kinase pathway. Following ubiquitination, Cx43 was internalized and degraded. The loss of Cx43 protein was counteracted by ammonium chloride, indicating that acidification of internalized Cx43 gap junctions is a prerequisite for its degradation. Furthermore, the Cx43 degradation was partly counteracted by leupeptin, an inhibitor of cathepsin B, H, and L. Cx43 internalization and subsequent degradation were blocked by inhibitors of the proteasome. Evidence is provided that Cx43 is modified by multiple monoubiquitins rather than a polyubiquitin chain in response to TPA. Moreover, the TPA-induced ubiquitination of Cx43 was blocked by proteasomal inhibitors. Taken together, the data indicate that Cx43 ubiquitination is a highly regulated process. Moreover, the results suggest that the proteasome might play an indirect role in Cx43 degradation by affecting the level of monoubiquitin conjugation and trafficking of Cx43 to endosomal compartments.
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PMID:Ubiquitination and down-regulation of gap junction protein connexin-43 in response to 12-O-tetradecanoylphorbol 13-acetate treatment. 1537 42

The protein constituents of gap junctions, connexins, have a rapid basal rate of degradation even after transport to the cell surface. We have used cell surface biotinylation to label gap junction-unassembled plasma membrane pools of connexin43 (Cx43) and show that their degradation is inhibited by mild hyperthermia, oxidative stress, and proteasome inhibitors. Cytosolic stress does not perturb endocytosis of biotinylated Cx43, but instead it seems to interfere with its targeting and/or transport to the lysosome, possibly by increasing the level of unfolded protein in the cytosol. This allows more Cx43 molecules to recycle to the cell surface, where they are assembled into long-lived, functional gap junctions in otherwise gap junction assembly-inefficient cells. Cytosolic stress also slowed degradation of biotinylated Cx43 in gap junction assembly-efficient normal rat kidney fibroblasts, and reduced the rate at which gap junctions disappeared from cell interfaces under conditions that blocked transport of nascent connexin molecules to the plasma membrane. These data demonstrate that degradation from the cell surface can be down-regulated by physiologically relevant forms of stress. For connexins, this may serve to enhance or preserve gap junction-mediated intercellular communication even under conditions in which protein synthesis and/or intracellular transport are compromised.
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PMID:Cytosolic stress reduces degradation of connexin43 internalized from the cell surface and enhances gap junction formation and function. 1613 29

Bowman-Birk protease inhibitor (BBI) from soybean acts as a potential chemopreventive agent in several types of tumors. However, the mechanism is still unclear. The present study was undertaken to estimate a mechanism of BBI-dependent negative growth control of human osteosarcoma cell (U2OS cell). BBI had negative growth control of the cells via induction of connexin (Cx) 43, a tumor suppressor gene in U2OS cells. This negative growth control by BBI was abrogated under down-regulation of Cx43 induced by a Cx43 antisense nucleotide treatment. It was also found that the BBI-dependent induction of Cx43 was due to elevation of Cx43 mRNA and stabilization of Cx43 protein. Especially, BBI-dependent inhibition of chymotrypsin-like activity in proteasome contributed to stabilization of Cx43 protein. These results suggest that a major negative growth effect of BBI is based on the restoration of Cx43 expression in U2OS cells.
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PMID:Negative growth control of osteosarcoma cell by Bowman-Birk protease inhibitor from soybean; involvement of connexin 43. 1734 82


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