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Ubiquitin
Terms modified by Ubiquitin Selected AbstractsCLONING AND EXPRESSION OF SPODOPTERA LITURA UBIQUITIN GENEINSECT SCIENCE, Issue 1 2003LI Zhao-fei Abstract Ubiquitin (UBI) plays a very important role in regulated non-lysosomal ATP dependent protein degradation. In the present work, the coding sequence of Spodoptera litura UBI gene was isolated (GenBank Accession No. AF436066). The length of this ORF is 228bp, encoding a protein with Mr of 8.56 kD and isoelectric point of 6.56. Multiple sequence alignment indicated that S. litura UBI is very similar to the homologous proteins of other eukaryotic species and it has 84% identity with S. litura nucleopolyhedrovirus (SpltMNPV) UBI at amino acid level. RT-PCR analysis showed that S. litura UBI gene is ubiquitously expressed in larva tissues which are susceptible to SpltMNPV infection. By constructing E. coli expression vector, S. litura UBI was highly expressed and the recombinant protein was purified using Ni-NTA resin column, and currently further study on the function of S. litura UBI in SpltMNPV infection is underway. [source] Ubiquitin at Fox Chase: Nobel LectureISRAEL JOURNAL OF CHEMISTRY, Issue 2 2006Irwin Rose First page of article [source] Damage control , a possible non-proteolytic role for ubiquitin in limiting neurodegenerationNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 2 2001D. A. Gray Ubiquitin can be detected in the neuronal and glial inclusions that are the diagnostic hallmarks of a number of human neurodegenerative diseases. It has been assumed that the presence of ubiquitin signifies the failed attempt of the cell to remove abnormal protein structures, which have been allowed to aggregate. The burden of abnormal protein arising from genetic mutations or cumulative oxidative damage might in the course of time overwhelm the ubiquitin-proteasome pathway (whose responsibility it is to eliminate misfolded or damaged proteins). However, ubiquitin may still serve a protective purpose distinct from its role in proteolysis. The physical properties of ubiquitin are such that a surface coating of ubiquitin should preclude further growth of the aggregate, prevent non-productive interactions, and conceal the contents from detection mechanisms that might ultimately kill the cell. This ,nonstick coating' hypothesis makes predictions about the nature of the conjugated ubiquitin and the consequences of removing it. [source] Der Einsatz von Relaxationserhöhungen in einer paramagnetischen Umgebung zur Proteinstrukturbestimmung mit NMR-Spektroskopie,ANGEWANDTE CHEMIE, Issue 44 2009Tobias Madl Dr. Ein Mehr an Informationen: Relaxationserhöhungen durch eine inerte paramagnetische Umgebung (PREs) wurden zusammen mit einem eingegrenzten NOE-Datensatz in einem modellfreien Strukturbestimmungsverfahren genutzt. Strukturen für zwei Proteine , Ubiquitin (8,kDa) und das Maltose-bindende Protein (42,kDa; im Komplex mit ,-Cyclodextrin) , wurden mithilfe von PREs und NOEs zwischen austauschenden Protonen ermittelt. [source] Highly Efficient and Chemoselective Peptide Ubiquitylation,ANGEWANDTE CHEMIE, Issue 43 2009Kumar Dr., S. Ajish Ein nützlicher Helfer: Mit ,-Mercaptolysin als Hilfe bei der Isopeptidbildung zwischen ,-NH2 des Lysinrests und dem aktivierten C-terminalen Glycinrest von Ubiquitin (Ub) gelang eine hoch effiziente und chemoselektive Ubiquitinylierung von Peptiden. [source] High-Resolution Solid-State NMR Studies on Uniformly [13C,15N]-Labeled UbiquitinCHEMBIOCHEM, Issue 9 2005Karsten Seidel Abstract Understanding of the effects of intermolecular interactions, molecular dynamics, and sample preparation on high-resolution magic-angle spinning NMR data is currently limited. Using the example of a uniformly [13C,15N]-labeled sample of ubiquitin, we discuss solid-state NMR methods tailored to the construction of 3D molecular structure and study the influence of solid-phase protein preparation on solid-state NMR spectra. A comparative analysis of13C,,13C,, and13C, resonance frequencies suggests that13C chemical-shift variations are most likely to occur in protein regions that exhibit an enhanced degree of molecular mobility. Our results can be refined by additional solid-state NMR techniques and serve as a reference for ongoing efforts to characterize the structure and dynamics of (membrane) proteins, protein complexes, and other biomolecules by high-resolution solid-state NMR. [source] Small-Molecule Inhibitors and Probes for Ubiquitin- and Ubiquitin-Like-Specific ProteasesCHEMBIOCHEM, Issue 2 2005Anna Borodovsky Dr. There's always a catch. The post-translational modification of proteins with ubiquitin (Ub) or ubiquitin-like (Ubl) modifiers is an important signal in the regulation of a variety of biological processes, such as degradation and regulation of gene expression. Here we report the synthesis of a panel of peptide vinyl sulfones (see scheme) harboring various portions of the Ub C terminus by using a safety-catch linker. Depending on their length, such compounds can efficiently target Ubl-specific proteases. [source] 2135: Influence of Hsp90 and HDAC inhibition and tubulin acetylation on perinuclear protein aggregation in human retinal pigment epithelial cellsACTA OPHTHALMOLOGICA, Issue 2010K KAARNIRANTA Purpose Retinal pigment epithelial (RPE) cells are continually exposed to oxidative stress that contributes to protein misfolding, aggregation and functional abnormalities during aging. The protein aggregates formed at the cell periphery are delivered along the microtubulus network by dynein dependent retrograde trafficking to a juxtanuclear location. Methods Cellular organelles were analysed by transmission electron microscopy of ARPE-19 cells exposed 5 µM MG-132, 0.25 µM geldanamycin (GA), 1 µM trichostatin A (TSA), 1 µM taxol (TAX) or 5 µM nocodazole (NOC) for 24 hours. In addition, the cells were treated simultaneously with GA or TSA or TAX or NOC and MG-132 up to 24 hours. Ubiquitin, Hsp90, Hsp70, acetylated tubulin and Hsc70 protein levels were analyzed by western blotting. Results Hsp90 inhibition by geldanamycin can effectively suppress proteasome inhibitor, MG-132 ,induced protein aggregation in a way that is an independent of HDAC inhibition, or the tubulin acetylation levels in ARPE-19 cells. However, the tubulin acetylation and polymerization state affects the localization of the proteasome-inhibitor ,induced aggregation. Conclusion Hsp90 inhibition is effectively related to regulation of protein aggregation that is independent of HDAC inhibition or tubulin acetylation levels in the RPE cells. Our findings open new perspectives for understanding the pathogenesis of protein aggregation in retinal cells and can be useful for the development of therapeutic treatments to prevent retinal cell deterioration. [source] Rnf19a, a ubiquitin protein ligase, and Psmc3, a component of the 26S proteasome, Tether to the acrosome membranes and the head,tail coupling apparatus during rat spermatid developmentDEVELOPMENTAL DYNAMICS, Issue 7 2009Eugene Rivkin Abstract We report the cDNA cloning of rat testis Rnf19a, a ubiquitin protein ligase, and show 98% and 93% protein sequence identity of testicular mouse and human Rnf19a, respectively. Rnf19a interacts with Psmc3, a protein component of the 19S regulatory cap of the 26S proteasome. During spermatid development, Rnf19a and Psmc3 are initially found in Golgi-derived proacrosomal vesicles. Later on, Rnf19a, Psmc3, and ubiquitin are seen along the cytosolic side of the acrosomal membranes and the acroplaxome, a cytoskeletal plate linking the acrosome to the spermatid nuclear envelope. Rnf19a and Psmc3 accumulate at the acroplaxome marginal ring,manchette perinuclear ring region during spermatid head shaping and in the developing sperm head,tail coupling apparatus and tail. Rnf19a and Psmc3 may interact directly or indirectly with each other, presumably pointing to the participation of the ubiquitin,proteasome system in acrosome biogenesis, spermatid head shaping, and development of the head-tail coupling apparatus and tail. Developmental Dynamics 238:1851,1861, 2009. © 2009 Wiley-Liss, Inc. [source] Laparoscopic findings in non-alcoholic steatohepatitisDIGESTIVE ENDOSCOPY, Issue 4 2003Shuichi Seki Background:, Non-alcoholic steatohepatitis (NASH) is prevalent worldwide, but little attention has been paid to the gross visual appearance of NASH. The present study was performed to address the laparoscopic features of NASH and the relationship between laparoscopic and histologicalal findings. Methods:, Eleven patients were examined by laparoscopy with liver biopsy. Histological findings were examined according to the criteria of Brunt et al. with minor modification. Mallory bodies were immunohistochemically detected by an antibody to ubiquitin in addition to hematoxylin eosin staining. Results:, Laparoscopic features of NASH were swelling of the liver, formation of many depressions, and dull edges of the liver. When steatosis was present in more than one-third of lobules, yellowish markings appeared on the liver surface. NASH progressed from a smooth liver surface with or without yellowish markings, to formation of depressions on the liver surface, to cirrhosis with or without hepatocellular carcinoma (HCC). Conclusion:, Laparoscopy may provide useful information in the diagnosis and progression of NASH. [source] N -methyl- d -aspartate receptor- and metabotropic glutamate receptor-dependent long-term depression are differentially regulated by the ubiquitin-proteasome systemEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2009Ami Citri Abstract Long-term depression (LTD) in CA1 pyramidal neurons can be induced by activation of either N -methyl- d -aspartate receptors (NMDARs) or metabotropic glutamate receptors (mGluRs), both of which elicit changes in synaptic efficacy through ,-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) endocytosis. To address the role of the ubiquitin-proteasome system in regulating AMPAR endocytosis during these forms of LTD, we examined the effects of pharmacological inhibitors of proteasomal degradation and protein ubiquitination on endocytosis of glutamate receptor 1 (GluR1) -containing AMPARs in dissociated rat hippocampal cultures as well as LTD of excitatory synaptic responses in acute rat hippocampal slices. Our findings suggest that the contribution of the ubiquitin-proteasome system to NMDAR-induced vs. mGluR-induced AMPAR endocytosis and the consequent LTD differs significantly. NMDAR-induced AMPAR endocytosis and LTD occur independently of proteasome function but appear to depend, at least in part, on ubiquitination. In contrast, mGluR-induced AMPAR endocytosis and LTD are enhanced by inhibition of proteasomal degradation, as well as by the inhibitor of protein ubiquitination. Furthermore, the decay of mGluR-induced membrane depolarization and Erk activation is delayed following inhibition of either ubiquitination or proteasomal degradation. These results suggest that, although NMDAR-dependent LTD may utilize ubiquitin as a signal for AMPAR endocytosis, mGluR-induced signaling and LTD are limited by a feedback mechanism that involves the ubiquitin-proteasome system. [source] Pro-apoptotic protein glyceraldehyde-3-phosphate dehydrogenase promotes the formation of Lewy body-like inclusionsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2005Katsumi Tsuchiya Abstract Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has long been recognized as a classical glycolytic protein; however, previous studies by our group and others have demonstrated that GAPDH is a general mediator initiating one or more apoptotic cascades. Our most recent findings have elucidated that an expression of a pro-apoptotic protein GAPDH is critically regulated at the promoter region of the gene. Apoptotic signals for its subsequent aggregate formation and nuclear translocation are controlled by the respective functional domains harboured within its cDNA component. In this study, coexpression of GAPDH with either wild-type or mutant (A53T) ,-synuclein and less likely with ,-synuclein in transfected COS-7 cells was found to induce Lewy body-like cytoplasmic inclusions. Unlike its full-length construct, the deleted mutant GAPDH construct (C66) abolished these apoptotic signals, disfavouring the formation of inclusions. The generated inclusions were ubiquitin- and thioflavin S-positive appearing fibrils. Furthermore, GAPDH coimmunoprecipitated with wild-type ,-synuclein in this paradigm. Importantly, immunohistochemical examinations of post mortem materials from patients with sporadic Parkinson's disease revealed the colocalized profiles immunoreactive against these two proteins in the peripheral zone of Lewy bodies from the affected brain regions (i.e. locus coeruleus). Moreover, a quantitative assessment showed that about 20% of Lewy bodies displayed both antigenicities. These results suggest that pro-apoptotic protein GAPDH may be involved in the Lewy body formation in vivo, probably associated with the apoptotic death pathway. [source] Cardiac 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] Activated Rac1, but not the tumorigenic variant Rac1b, is ubiquitinated on Lys 147 through a JNK-regulated processFEBS JOURNAL, Issue 2 2008Orane Visvikis Ubiquitination and proteasomal degradation have recently emerged as an additional level of regulation of activated forms of Rho GTPases. To characterize this novel regulatory pathway and to gain insight into its biological significance, we studied the ubiquitination of two constitutively activated forms of Rac1, i.e. the mutationally activated Rac1L61, and the tumorigenic splice variant Rac1b, which is defective for several downstream signaling pathways, including JNK activation. Whereas Rac1L61 undergoes polyubiquitination and subsequent proteasomal degradation in HEK293 cells, Rac1b is poorly ubiquitinated and appears to be much more resistant to proteasomal degradation than Rac1L61. Mutational analysis of all lysine residues in Rac1 revealed that the major target site for Rac1 ubiquitination is Lys147, a solvent-accessible residue that has a similar conformation in Rac1b. Like Rac1L61, Rac1b was found to be largely associated with plasma membrane, a known prerequisite for Rac1 ubiquitination. Interestingly, Rac1b ubiquitination could be stimulated by coexpression of Rac1L61, suggesting positive regulation of Rac1 ubiquitination by Rac1 downstream signaling. Indeed, ubiquitination of Rac1L61 is critically dependent on JNK activation. In conclusion: (a) Rac1b appears to be more stable than Rac1L61 with regard to the ubiquitin,proteasome system, and this may be of importance for the expression and tumorigenic capacity of Rac1b; and (b) ubiquitination of activated Rac1 occurs through a JNK-activated process, which may explain the defective ubiquitination of Rac1b. The JNK-dependent activation of Rac1 ubiquitination would create a regulatory loop allowing the cell to counteract excessive activation of Rac1 GTPase. [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] Preserving organelle vitality: peroxisomal quality control mechanisms in yeastFEMS YEAST RESEARCH, Issue 6 2009Eda Bener Aksam Abstract Cellular proteins and organelles such as peroxisomes are under continuous quality control. Upon synthesis in the cytosol, peroxisomal proteins are kept in an import-competent state by chaperones or specific proteins with an analogous function to prevent degradation by the ubiquitin,proteasome system. During protein translocation into the organelle, the peroxisomal targeting signal receptors (Pex5, Pex20) are also continuously undergoing quality control to enable efficient functioning of the translocon (RADAR pathway). Even upon maturation of peroxisomes, matrix enzymes and peroxisomal membranes remain subjected to quality control. As a result of their oxidative metabolism, peroxisomes are producers of reactive oxygen species (ROS), which may damage proteins and lipids. To counteract ROS-induced damage, yeast peroxisomes contain two important antioxidant enzymes: catalase and an organelle-specific peroxiredoxin. Additionally, a Lon-type protease has recently been identified in the peroxisomal matrix, which is capable of degrading nonfunctional proteins. Finally, cellular housekeeping processes keep track of the functioning of peroxisomes so that dysfunctional organelles can be quickly removed via selective autophagy (pexophagy). This review provides an overview of the major processes involved in quality control of yeast peroxisomes. [source] Decorating Liquid Crystal Surfaces with Proteins for Real-Time Detection of Specific Protein,Protein BindingADVANCED FUNCTIONAL MATERIALS, Issue 22 2009Deny Hartono Abstract Here, a novel method of immobilizing proteins with well-defined orientation directly on liquid crystal surfaces that allow subsequent real-time imaging of specific protein,protein binding events on these surfaces is reported. Self-assembly of nitrilotriacetic acid terminated amphiphiles loaded with Ni2+ ions at aqueous-liquid crystal interface creates a surface capable of immobilizing histidine-tagged ubiquitin through complex formation between Ni2+ and histidine. When these surfaces containing immobilized histidine-tagged ubiquitin are exposed to anti-ubiquitin antibody, the spatial and temporal of specific protein,protein binding events trigger orientational transitions of liquid crystals. As a result, sharp liquid crystal optical switching from dark to bright can readily be observed under polarized lighting. The protein,protein binding can be observed within seconds and only requires nanogram quantities of proteins. This work demonstrates a simple strategy to immobilize proteins with well-defined orientation on liquid crystal surfaces for real-time and label-free detection of specific protein,protein binding events, which may find use in biomedical diagnostics. [source] Molecular mechanism of ubiquitin recognition by GGA3 GAT domainGENES TO CELLS, Issue 7 2005Masato Kawasaki GGA (Golgi-localizing, ,-adaptin ear domain homology, ARF-binding) proteins, which constitute a family of clathrin coat adaptor proteins, have recently been shown to be involved in the ubiquitin-dependent sorting of receptors, through the interaction between the C-terminal three-helix-bundle of the GAT (GGA and Tom1) domain (C-GAT) and ubiquitin. We report here the crystal structure of human GGA3 C-GAT in complex with ubiquitin. A hydrophobic patch on C-GAT helices ,1 and ,2 forms a binding site for the hydrophobic Ile44 surface of ubiquitin. Two distinct orientations of ubiquitin Arg42 determine the shape and the charge distribution of ubiquitin Ile44 surface, leading to two different binding modes. Biochemical and NMR data strongly suggest another hydrophobic binding site on C-GAT helices ,2 and ,3, opposite to the first binding site, also binds ubiquitin although weakly. The double-sided ubiquitin binding provides the GAT domain with higher efficiency in recognizing ubiquitinated receptors for lysosomal receptor degradation. [source] The crystal structure of microtubule-associated protein light chain 3, a mammalian homologue of Saccharomyces cerevisiae Atg8GENES TO CELLS, Issue 7 2004Kenji Sugawara Microtubule-associated protein light chain 3 (LC3), a mammalian homologue of yeast Atg8, plays an essential role in autophagy, which is involved in the bulk degradation of cytoplasmic components by the lysosomal system. Here, we report the crystal structure of LC3 at 2.05 Å resolution with an R-factor of 21.8% and a free R-factor of 24.9%. The structure of LC3, which is similar to those of Golgi-associated ATPase enhancer of 16 kDa (GATE-16) and GABAA receptor-associated protein (GABARAP), contains a ubiquitin core with two , helices, ,1 and ,2, attached at its N-terminus. Some common and distinct features are observed among these proteins, including the conservation of residues required to form an interaction among ,1, ,2 and the ubiquitin core. However, the electrostatic potential surfaces of these helices differ, implicating particular roles to select specific binding partners. Hydrophobic patches on the ubiquitin core of LC3, GABARAP and GATE-16 are well conserved and are similar to the E1 binding surface of ubiquitin and NEDD8. Therefore, we propose that the hydrophobic patch is a binding surface for mammalian Atg7 similar to a ubiquitin-like conjugation system. We also propose the functional implications of the ubiquitin fold as a recognition module of target proteins. [source] Autophagy activation by rapamycin eliminates mouse Mallory-Denk bodies and blocks their proteasome inhibitor-mediated formation,HEPATOLOGY, Issue 6 2008Masaru Harada The proteasomal and lysosomal/autophagy pathways in the liver and other tissues are involved in several biological processes including the degradation of misfolded proteins. Exposure of hepatocyte cell lines to proteasome inhibitors (PIs) results in the formation of inclusions that resemble Mallory-Denk bodies (MDBs). Keratins are essential for MDB formation and keratin 8 (K8)-overexpressing transgenic mice are predisposed to MDB formation. We tested the hypothesis that PIs induce MDBs in vivo and that autophagy participates in MDB turnover. The effect of the PI bortezomib (which is used to treat some malignancies) on MDB formation was tested in K8-overexpressing mice and in cultured cells. Inclusion formation was examined using immune and conventional electron microscopy (EM). Bortezomib induced MDB-like inclusions composed of keratins, ubiquitin, and p62 in cultured cells. Short-term exposure to bortezomib induced similar inclusions in K8-overexpressing but not in nontransgenic mice, without causing liver injury. In bortezomib-treated mice, autophagy was activated in hepatocytes as determined by EM and biochemical analysis. Further activation of autophagy by rapamycin (Rap) decreased the number of inclusions in bortezomib-treated K8 transgenic mice significantly. Rap also led to resorption of spontaneously formed MDBs in aging K8-overexpressing mice. Immune EM demonstrated K8-positive and ubiquitin-positive structures in autophagic vacuoles in the mouse liver. Conclusion: PIs alone are sufficient to induce MDBs in susceptible animals, while Rap-mediated activation of autophagy prevents MDB formation and causes MDB resorption. These findings suggest that some patients treated with PIs may become predisposed to MDB formation. Autophagy provides a potential cellular mechanism for the resorption of cytoplasmic inclusions. (HEPATOLOGY 2008.) [source] Ubiquitin protein modification and signal transduction: Implications for inflammatory bowel diseasesINFLAMMATORY BOWEL DISEASES, Issue 12 2005Cormac Taylor PhD Abstract A dysregulated immune response to luminal antigen(s) is associated with the development of inflammatory bowel diseases (IBDs). A complex network of inflammatory and immune mediators released by immune and nonimmune cells participate in the physiopathology of IBD. At the molecular level, events leading to the improper use of the signaling grid are likely responsible for the dysregulated activation of various transcription factors and subsequent induction of inflammatory genes. The posttranslational modification of signaling proteins by the ubiquitin system is a critical event in activation or repression of transcription factors. Two important transcriptional pathways in which ubiquitin is central are the nuclear factor-,B and hypoxia inducible factor-1 (HIF-1) pathways, both of which are important components of intestinal homeostasis. In this review, we discuss the role of ubiquitin modification in relation to nuclear factor-,B and HIF-1 signaling and consider its impact on intestinal inflammation. A greater understanding of posttranslational ubiquitin modification may lead to the identification of new therapeutic opportunities for the treatment of IBD. [source] Combined R-,,lipoic acid and acetyl-L-carnitine exerts efficient preventative effects in a cellular model of Parkinson's diseaseJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 1-2 2010Hongyu Zhang Abstract Mitochondrial dysfunction and oxidative damage are highly involved in the pathogenesis of Parkinson's disease (PD). Some mitochondrial antioxidants/nutrients that can improve mitochondrial function and/or attenuate oxidative damage have been implicated in PD therapy. However, few studies have evaluated the preventative effects of a combination of mitochondrial antioxidants/nutrients against PD, and even fewer have sought to optimize the doses of the combined agents. The present study examined the preventative effects of two mitochondrial antioxidant/nutrients, R-,,lipoic acid (LA) and acetyl-L-carnitine (ALC), in a chronic rotenone-induced cellular model of PD. We demonstrated that 4-week pretreatment with LA and/or ALC effectively protected SK-N-MC human neuroblastoma cells against rotenone-induced mitochondrial dysfunction, oxidative damage and accumulation of ,-synuclein and ubiquitin. Most notably, we found that when combined, LA and ALC worked at 100,1000-fold lower concentrations than they did individually. We also found that pretreatment with combined LA and ALC increased mitochondrial biogenesis and decreased production of reactive oxygen species through the up-regulation of the peroxisome proliferator-activated receptor-, coactivator 1, as a possible underlying mechanism. This study provides important evidence that combining mitochondrial antioxidant/nutrients at optimal doses might be an effective and safe prevention strategy for PD. [source] Protein identification via ion-trap collision-induced dissociation and examination of low-mass product ionsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2008Jeremiah J. Bowers Abstract A whole-protein tandem mass spectrometry approach for protein identification based on precursor ion charge state concentration via ion/ion reactions, ion-trap collisional activation, ion/ion proton-transfer reactions involving the product ions, and mass analysis over a narrow m/z range (up to m/z 2000) is described and evaluated. The experiments were carried out with a commercially available electrospray ion-trap instrument that has been modified to allow for ion/ion reactions. Reaction conditions and the approach to searching protein databases were developed with the assumption that the resolving power of the mass analyzer is insufficient to distinguish charge states on the basis of the isotope spacings. Ions derived from several charge states of cytochrome c, myoglobin, ribonuclease A, and ubiquitin were used to evaluate the approach for protein identification and to develop a two-step procedure to database searching to optimize specificity. The approach developed with the model proteins was then applied to whole cell lysate fractions of Saccharomyces cerevisiae. The results are illustrated with examples of assignments made for three a priori unknown proteins, each selected randomly from a lysate fraction. Two of the three proteins were assigned to species present in the database, whereas one did not match well any database entry. The combination of the mass measurement and the product ion masses suggested the possibility for the oxidation of two methionine residues of a protein in the database. The examples show that this limited whole-protein characterization approach can provide insights that might otherwise be lacking with approaches based on complete enzymatic digestion. Copyright © 2007 John Wiley & Sons, Ltd. [source] Effect of buffer cations and of H3O+ on the charge states of native proteins.JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 6 2003Significance to determinations of stability constants of protein complexes Abstract The progressive reduction of charge in charge states of non-denatured proteins (lysozyme, ubiquitin, and cytochrome c), observed with nanospray in the positive ion mode, when the buffer salt ammonium acetate is replaced by ethylammonium acetates (EtNH3Ac, Et2NH2Ac and Et3NHAc) is rationalized on the basis of the charge residue model (CRM). The charge states of the multiply protonated protein are shown to be controlled by the increasing gas-phase basicities, GB(B), of the bases(B) NH3, EtNH2, Et2NH and Et3N. Charge states derived from evaluated apparent gas-phase basicities GBapp of the basic side-chains of the protein and the known GB(B) of the above bases are found to be in agreement with the experimentally observed charge states. This is a requirement of the CRM, because in this model the small positive ions (the buffer cations in the present case) at the surface of the electrospray droplets are the excess ions that provide the charge of the final small droplet that contains the protein molecule and on evaporation of the solvent transfer the charge to the protein. The observed charge states in the absence of buffer salts, i.e. pure water, are attributed to excess H3O+ ions produced by the electrolysis process that attends electrospray. A proposed extended mechanism provides predictions of factors that determine the sensitivity for detection of the multiply protonated proteins. Consideration of restraints imposed by the CRM lead to some simple predictions for conditions that should be present to obtain accurate determinations by electrospray and nanospray of stability constants for the protein,complex equilibrium in aqueous solution. Copyright © 2003 John Wiley & Sons, Ltd. [source] Cerebral ischemia/stroke and small ubiquitin-like modifier (SUMO) conjugation , a new target for therapeutic intervention?JOURNAL OF NEUROCHEMISTRY, Issue 3 2008Wei Yang Abstract Transient cerebral ischemia/stroke activates various post-translational protein modifications such as phosphorylation and ubiquitin conjugation that are believed to play a major role in the pathological process triggered by an interruption of blood supply and culminating in cell death. A new system of post-translational protein modification has been identified, termed as small ubiquitin-like modifier (SUMO) conjugation. Like ubiquitin, SUMO is conjugated to the lysine residue of target proteins in a complex process. This review summarizes observations from recent experiments focusing on the effect of cerebral ischemia on SUMO conjugation. Transient global and focal cerebral ischemia both induced a rapid, dramatic and long-lasting rise in levels of SUMO2/3 conjugation. After transient focal cerebral ischemia, SUMO conjugation was particularly prominent in neurons located at the border of the ischemic territory where SUMO-conjugated proteins translocated to the nucleus. Many SUMO conjugation target proteins are transcription factors and sumoylation has been shown to have a major impact on the activity, stability, and cellular localization of target proteins. The rise in levels of SUMO-conjugated proteins is therefore likely to have a major effect on the fate of post-ischemic neurons. The sumoylation process could provide an exciting new target for therapeutic intervention. [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] Dysfunction of the ubiquitin,proteasome system in Parkinson's diseaseJOURNAL OF NEUROCHEMISTRY, Issue 2003P. Jenner The cause of nigral cell degeneration and Lewy body formation in Parkinson's disease (PD) remains unknown but may involve impaired proteolysis. Evidence from both sporadic and familial forms of PD suggest the involvement of alterations in the ubiquitin,proteasomal system. In postmortem tissues from PD cases, there is a loss of 26S proteasomal enzyme activity coupled to a decrease in the expression of ,-subunits in substantia nigra while ,-subunit expression remains unchanged. The expression of PA700 is up-regulated in a number of brain regions in PD but not in substantia nigra. Interestingly, there was little or no expression of PA28 in the nigra in both aged control tissue or in PD. These data suggest that alterations in protein handling may be key to the formation of Lewy bodies in PD. Indeed, in vitro and in vivo inhibition of proteasomal activity causes the death of dopaminergic neurones. Recent evidence suggests that the formation of Lewy bodies may be linked to impaired proteasomal function in centrosomes leading to aggresome formation. [source] | |||||||||||||||||||||||||||||||||||||