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Proteasome Pathway (proteasome + pathway)
Selected AbstractsCardiac ankyrin repeat protein is a marker of skeletal muscle pathological remodellingFEBS JOURNAL, Issue 3 2009Lydie Laure In an attempt to identify potential therapeutic targets for the correction of muscle wasting, the gene expression of several pivotal proteins involved in protein metabolism was investigated in experimental atrophy induced by transient or definitive denervation, as well as in four animal models of muscular dystrophies (deficient for calpain 3, dysferlin, ,-sarcoglycan and dystrophin, respectively). The results showed that: (a) the components of the ubiquitin,proteasome pathway are upregulated during the very early phases of atrophy but do not greatly increase in the muscular dystrophy models; (b) forkhead box protein O1 mRNA expression is augmented in the muscles of a limb girdle muscular dystrophy 2A murine model; and (c) the expression of cardiac ankyrin repeat protein (CARP), a regulator of transcription factors, appears to be persistently upregulated in every condition, suggesting that CARP could be a hub protein participating in common pathological molecular pathway(s). Interestingly, the mRNA level of a cell cycle inhibitor known to be upregulated by CARP in other tissues, p21WAF1/CIP1, is consistently increased whenever CARP is upregulated. CARP overexpression in muscle fibres fails to affect their calibre, indicating that CARP per se cannot initiate atrophy. However, a switch towards fast-twitch fibres is observed, suggesting that CARP plays a role in skeletal muscle plasticity. The observation that p21WAF1/CIP1 is upregulated, put in perspective with the effects of CARP on the fibre type, fits well with the idea that the mechanisms at stake might be required to oppose muscle remodelling in skeletal muscle. [source] A novel N-terminal hydrophobic motif mediates constitutive degradation of serum- and glucocorticoid-induced kinase-1 by the ubiquitin,proteasome pathwayFEBS JOURNAL, Issue 13 2006Agata M. Bogusz Serum- and glucocorticoid-induced protein kinase-1 (SGK-1) plays a critical role in regulation of the epithelial sodium channel, ENaC. SGK-1 also shares significant catalytic domain homology with protein kinase B (PKB/AKT-1) and is a downstream effector of antiapoptotic phosphoinositide 3-kinase signaling. Steady-state levels of an active SGK-1 are tightly regulated by rapid transcriptional activation and post-translational modification including phosphorylation. We show here that endogenous SGK-1 protein is polyubiquitinated and rapidly degraded by the 26S proteasome. In contrast to other rapidly degraded kinases, neither the catalytic activity of SGK-1 nor activation site phosphorylation was required for its ubiquitin modification and degradation. Instead, SGK-1 degradation required a lysine-less six-amino-acid (amino acids 19,24) hydrophobic motif (GMVAIL) within the N-terminal domain. Deletion of amino acids 19,24 significantly increased the half-life of SGK1 and prevented its ubiquitin modification. Interestingly, this minimal region was also required for the association of SGK-1 with the endoplasmic reticulum. Ubiquitin modification and degradation of SGK-1 were increasingly inhibited by the progressive mutation of six N-terminal lysine residues surrounding the GMVAIL motif. Mutation of all six lysines to arginine did not disrupt the subcellular localization of SGK-1 despite a significant decrease in ubiquitination, implying that this modification per se was not required for targeting to the endoplasmic reticulum. These results suggest that constitutive ubiquitin-mediated degradation of SGK-1 is an important mechanism regulating its biological activity. [source] Aggresome formation by anti-Ras intracellular scFv fragmentsFEBS JOURNAL, Issue 2 2001The fate of the antigen, antibody complex Diverting the antigen from its normal intracellular location to other compartments in an antibody-mediated way represents a mode of action for intracellular antibodies [Cardinale, A., Lener, M., Messina, S., Cattaneo, A. & Biocca, S. (1998) FEBS Lett.,439, 197,202; Lener, M., Horn, I.R., Cardinale, A., Messina, S., Nielsen, U.B., Rybak, S.M., Hoogenboom, H.R., Cattaneo, A. & Biocca, S. (2000) Eur J Biochem.267, 1196,205]. In the case of p21Ras, the sequestration of the antigen in aggregated structures in the cytoplasm of transfected cells leads to the inhibition of its biological function. We have further investigated the intracellular fate of the antigen,antibody complex by analyzing the effect of proteasome inhibitors on the formation and the intracellular localization of the aggregates. Overexpression of anti-Ras scFv fragments or inhibition of proteasomes activity leads to the formation of large perinuclear aggresomes formed of ubiquitinated-scFv fragments in which p21Ras is sequestered and degraded in an antibody-mediated way. Disruption of microtubules by nocodazole completely abrogates the accumulation of scFv fragments in a single aggresome and induces the dispersion of these structures in the periphery of the cell. Cotransfection of the GFP-scFv with a myc-tagged ubiquitin and colocalization with specific anti-proteasome antibodies indicate the recruitment of exogenous ubiquitin and proteasomes to the newly formed aggresomes. Taken together these results suggest that the intracellular antigen,antibody complex is naturally addressed to the ubiquitin,proteasome pathway and that the mechanism of ubiquitination does not inhibit the antibody binding properties and the capacity to block the antigen function. [source] Degradation of transcription factor IRF-1 by the ubiquitin,proteasome pathwayFEBS JOURNAL, Issue 6 2000The C-terminal region governs the protein stability Interferon regulatory factor-1(IRF-1) is a transcriptional activator of interferon genes and interferon-inducible genes. It has been shown that IRF-1 functions not only as a regulator of the interferon-responsive system but also as a regulator of cell growth and apoptosis. In addition, it is known that IRF-1 is a short-lived protein, but the mechanism that regulates its stability has not yet been clarified. Here, we show that IRF-1 is degraded via the ubiquitin,proteasome pathway. IRF-1 protein degradation in HeLa and NIH3T3 cells was inhibited by treatment with proteasome-specific inhibitors. Overexpression of IRF-1 protein and ubiquitin in COS7 cells revealed specific multiubiquitination of IRF-1. Although the full-length IRF-1 was unstable, IRF-1 mutants with C-terminal truncations larger than 39 amino acids were found to be almost stable, suggesting that the 39-residue C-terminal region controls the stability of IRF-1. Further analysis of the stability of a green fluorescent protein-fusion protein containing the 39-residue C-terminal region of IRF-1 showed that this C-terminal region confers instability on green fluorescent protein, a normally stable protein, suggesting that this region functions as a protein-degradation signal. Taking the results together, it can be concluded that the 39-residue C-terminal region is necessary and sufficient to control the stability of the IRF-1 protein. [source] MBSJ MCC Young Scientist Award 2009 REVIEW: Selective autophagy regulates various cellular functionsGENES TO CELLS, Issue 9 2010Masaaki Komatsu Autophagy is a self-eating system conserved among eukaryotes, in which cellular components including organelles are entrapped into a double membrane structure called the autophagosome and then degraded by lysosomal hydrolases. In addition to its role in supplying amino acids in response to nutrient starvation, autophagy is involved in quality control to maintain cell health. Thus, inactivation of autophagy causes the formation of cytoplasmic protein inclusions, which comprise misfolded proteins and the accumulation of many degenerated organelles, resulting in liver injury, diabetes, myopathy and neurodegeneration. Furthermore, although autophagy has been considered nonselective, increasing evidence points to the selectivity of autophagy in sorting vacuolar enzymes and removal of aggregate-prone proteins and unwanted organelles. Such selectivity allows diverse cellular regulation, similar to the ubiquitin proteasome pathway. In this review, we discuss the physiological roles of selective autophagy and their molecular mechanisms. [source] White muscle 20S proteasome activity is negatively correlated to growth rate at low temperature in the spotted wolffish Anarhichas minorJOURNAL OF FISH BIOLOGY, Issue 7 2010S. G. Lamarre The effect of temperature and mass on specific growth rate (G) was examined in spotted wolffish Anarhichas minor of different size classes (ranging from 60 to 1500 g) acclimated at different temperatures (4, 8 and 12° C). The relationship between G and 20S proteasome activity in heart ventricle, liver and white muscle tissue was then assessed in fish acclimated at 4 and 12° C to determine if protein degradation via the proteasome pathway could be imposing a limitation on somatic growth. Cardiac 20S proteasome activity was not affected by acclimation temperature nor fish mass and had no correlation with G. Hepatic 20S proteasome activity was higher at 12° C but did not show any relationship with G. Partial correlation analysis showed that white muscle 20S proteasome activity was negatively correlated to G (partial Pearson's r = ,0·609) but only at cold acclimation temperature (4° C). It is suggested that acclimation to cold temperature involves compensation of the mitochondrial oxidative capacity which would in turn lead to increased production of oxidatively damaged proteins that are degraded by the proteasome pathway and ultimately negatively affects G at cold temperature. [source] Netrin induces down-regulation of its receptor, Deleted in Colorectal Cancer, through the ubiquitin,proteasome pathway in the embryonic cortical neuronJOURNAL OF NEUROCHEMISTRY, Issue 1 2005Tae-Hong Kim Abstract The proper regulation of temporal and spatial expression of the axon guidance cues and their receptors is critical for the normal wiring of nervous system during development. Netrins, a family of secreted guidance cues, are involved in the midline crossing of spinal commissural axons and in the guidance of cortical efferents. Axons normally lose the responsiveness to their attractants when they arrive at their targets, where the attractant is produced. However the molecular mechanism is still unknown. We investigated the molecular mechanism of down-regulation of netrin-1 signaling in the embryonic cortical neurons. Netrin-1 induced the ubiquitination and proteolytic cleavage of Deleted in Colorectal Cancer (DCC), a transmembrane receptor for netrin, in dissociated cortical neurons. A dramatic decrease of DCC level particularly on the cell surface was also observed after netrin-1 stimulation. Specific ubiquitin,proteasome inhibitors prevented the netrin-induced DCC cleavage and decrease of cell surface DCC. We suggest that the ligand-mediated down-regulation of DCC might participate in the loss of netrin-responsiveness in the developing nervous system. [source] Sequestosome 1/p62 shuttles polyubiquitinated tau for proteasomal degradationJOURNAL OF NEUROCHEMISTRY, Issue 1 2005Jeganathan Ramesh Babu Abstract Inclusions isolated from several neurodegenerative diseases, including Alzheimer's disease (AD), are characterized by ubiquitin-positive proteinaceous aggregates. Employing confocal and immunoelectron microscopy, we find that the ubiquitin-associating protein sequestosome1/p62, co-localizes to aggregates isolated from AD but not control brain, along with the E3 ubiquitin ligase, TRAF6. This interaction could be recapitulated by co-transfection in HEK293 cells. Employing both in vitro and in vivo approaches, tau was found to be a substrate of the TRAF6, possessing lysine 63 polyubiquitin chains. Moreover, tau recovered from brain of TRAF6 knockout mice, compared with wild type, was not ubiquitinated. Tau degradation took place through the ubiquitin,proteasome pathway and was dependent upon either the K63-polyubiquitin chains or upon p62. In brain lysates of p62 knockout mice, tau fails to co-interact with Rpt1, a proteasomal subunit, thereby indicating a requirement for p62 shuttling of tau to the proteasome. Our results demonstrate that p62 interacts with K63-polyubiquitinated tau through its UBA domain and serves a novel role in regulating tau proteasomal degradation. We propose a model whereby either a decline in p62 expression or a decrease in proteasome activity may contribute to accumulation of insoluble/aggregated K63-polyubiquitinated tau. [source] Ubiquitin-dependent and -independent mitochondrial protein quality controls: implications in ageing and neurodegenerative diseasesMOLECULAR MICROBIOLOGY, Issue 6 2008Doris Germain Summary The ubiquitin-independent protein quality control of matrix proteins of the mitochondrion is well characterized and until recently the mitochondrion was considered a ,ubiquitination-free' organelle. However, a number of studies now indicate multiple roles of the ubiquitin,proteasome pathway in the regulation and maintenance of mitochondrial integrity. Of particular interest to this review is the finding of a mitochondrial ubiquitin-dependent protein quality control and that this pathway may share similarity to the endoplasmic reticulum- associated degradation (ERAD) pathway that acts to eliminate misfolded proteins from the lumen of the endoplasmic reticulum. The potential cross-talk between the ubiquitin-dependent and -independent protein quality controls and their implications in ageing and neurodegenerative diseases, notably in Parkinson's disease, are discussed. [source] Proteomics of human umbilical vein endothelial cells applied to etoposide-induced apoptosisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2005Arnaud Bruneel Dr. Abstract We have undertaken to continue the proteomic study of human umbilical vein endothelial cells (HUVECs) using the combination of 2-DE, automated trypsin digestion, and PMF analysis after MALDI-TOF MS and peptide sequencing using nano LC-ESI-MS/MS. The overall functional characterization of the 162 identified proteins from primary cultures of HUVECs confirms the metabolic capabilities of endothelium and illustrates various cellular functions more related to cell motility and angiogenesis, protein folding, anti-oxidant defenses, signal transduction, proteasome pathway and resistance to apoptosis. In comparison with controls cells, the differential proteomic analysis of HUVECs treated by the pro-apoptotic topoisomerase inhibitor etoposide further revealed the variation of eight proteins, namely, GRP78, GRP94, valosin-containing protein, proteinase inhibitor 9, cofilin, 37-kDa laminin receptor protein, bovine apolipoprotein, and tropomyosin. These data suggest that etoposide-induced apoptosis of human vascular endothelial cells results from the intricate involvement of multiple apoptosis processes including at least the mitochondrial and the ER stress pathways. The presented 2-D pattern and protein database, as well as the data related to apoptosis of HUVECs, are available at http://www.huvec.com. [source] Crystallization and preliminary X-ray crystallographic analysis of human FAF1 UBX domainACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010Wonchull Kang Fas-associated factor 1 (FAF1) is a multifunctional pro-apoptotic protein that is involved in Fas-mediated apoptosis, NF-,B signalling and the ubiquitin,proteasome pathway. In the ubiquitin,proteasome pathway, FAF1 binds to the N domain of p97/VCP, a molecular chaperone that acts in complex with the proteasome, through its C-terminal UBX domain and inhibits the proteasomal protein-degradation process. In an effort to elucidate the structural basis of the function of FAF1 in modulating p97/VCP activity related to proteasomal protein degradation, crystallographic analysis of the FAF1 UBX domain and the p97/VCP N domain was initiated. Following the recently reported crystallization of the FAF1 UBX domain bound to the p97/VCP N domain, the unbound FAF1 UBX domain was also crystallized for purposes of structural comparison. X-ray data were collected to 3.00,Å resolution and the crystals belonged to space group F4132, with unit-cell parameters a = b = c = 176.40,Å. The Matthews coefficient and solvent content were estimated to be 3.04,Å3,Da,1 and 59.5%, respectively, assuming that the asymmetric unit contained two molecules of the UBX domain, which was subsequently confirmed by molecular-replacement calculations. [source] Crystallization and preliminary X-ray crystallographic analysis of the N domain of p97/VCP in complex with the UBX domain of FAF1ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010Hwa Young Shin p97/VCP is a multifunctional AAA+ -family ATPase that is involved in diverse cellular processes. p97/VCP directly interacts with various adaptors for activity in different biochemical contexts. Among these adaptors are p47 and Fas-associated factor 1 (FAF1), which contain a common UBX domain through which they bind to the N domain of p97/VCP. In the ubiquitin,proteasome pathway, p97/VCP acts as a chaperone that presents client proteins to the proteasome for degradation, while FAF1 modulates the process by interacting with ubiquitinated client proteins and also with p97/VCP. In an effort to elucidate the structural details of the interaction between p97/VCP and FAF1, the p97/VCP N domain was crystallized in complex with the FAF1 UBX domain. X-ray data were collected to 2.60,Å resolution and the crystals belonged to space group C2221, with unit-cell parameters a = 58.24, b = 72.81, c = 132.93,Å. The Matthews coefficient and solvent content were estimated to be 2.39,Å3,Da,1 and 48.4%, respectively, assuming that the asymmetric unit contained p97/VCP N domain and FAF1 molecules in a 1:1 ratio, which was subsequently confirmed by molecular-replacement calculations. [source] Wnt signaling stabilizes the DIXDC1 protein through decreased ubiquitin-dependent degradationCANCER SCIENCE, Issue 3 2010Lei Wang (Cancer Sci 2010; 101: 700,706) Wnt signaling plays key roles in development, cell growth, differentiation, polarity formation, neural development, and carcinogenesis. DIX Domain Containing 1 (DIXDC1), a novel component of the Wnt pathway, was recently cloned. DIXDC1 is the human homolog of Ccd1, a positive regulator of the Wnt signaling pathway during zebrafish neural patterning. Little has been known about DIXDC1 gene expression regulation. In the present study, we showed that the DIXDC1 protein was induced upon Wnt-3a stimulation, whereas the DIXDC1 mRNA level was not significantly increased after Wnt-3a treatment. Positive DIXDC1 staining was detected in colon cancer cells and was colocalized with ,-catenin staining. However, the DIXDC1 mRNA expression decreased in human colon cancer cells compared to the matched normal colon epithelial cells. Our further investigation showed that the DIXDC1 protein was degraded through the proteasome pathway, and the activation of canonical Wnt signaling decreased the ubiquitin-dependent degradation of both the ectopic and endogenous DIXDC1 protein. In order to explore the possible mechanism of the ubiquitination of DIXDC1, we found that the phosphorylation of DIXDC1 was inhibited by Wnt-3a. Collectively, these results indicate that canonical Wnt/,-catenin pathway activation might upregulate DIXDC1 through a post-translational mechanism by inhibiting the ubiquitin-mediated degradation of the DIXDC1 protein. [source] Regulation of TGF-, family signaling by E3 ubiquitin ligasesCANCER SCIENCE, Issue 11 2008Yasumichi Inoue Members of the transforming growth factor-, (TGF-,) family, including TGF-,, activin and bone morphogenetic proteins (BMPs), are multifunctional proteins that regulate a wide variety of cellular responses, such as proliferation, differentiation, migration and apoptosis. Alterations in their downstream signaling pathways are associated with a range of human diseases like cancer. TGF-, family members transduce signals through membrane serine/threonine kinase receptors and intracellular Smad proteins. The ubiquitin,proteasome pathway, an evolutionarily conserved cascade, tightly regulates TGF-, family signaling. In this pathway, E3 ubiquitin ligases play a crucial role in the recognition and degradation of target proteins by the 26S proteasomes. Smad degradation regulates TGF-, family signaling; HECT (homologous to the E6-accessory protein C-terminus)-type E3 ubiquitin ligases, Smad ubiquitin regulatory factor 1 (Smurf1), Smurf2, and a RING-type E3 ubiquitin ligase, ROC1-SCFFbw1a have been implicated in Smad degradation. Smurf1 and Smurf2 bind to TGF-, family receptors via the inhibitory Smads, Smad6 and Smad7, to induce their ubiquitin-dependent degradation. Arkadia, a RING-type E3 ubiquitin ligase, induces the ubiquitination and degradation of Smad7 and corepressors, c-Ski and SnoN, to enhance TGF-, family signaling. Abnormalities in E3 ubiquitin ligases that control components of TGF-, family signaling may lead to the development and progression of various cancers. (Cancer Sci 2008; 99: 2107,2112) [source] | |||||||||||||