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Nicole M Hjortland, Andrew D Mesecar
USP17 is a deubiquitinating enzyme that is upregulated in numerous cancers and therefore a drug target. We developed a robust expression, purification, and assay system for USP17 enabling its enzymatic and structural characterization. USP17 was expressed in E. coli as inclusion bodies and then solubilized, refolded, and purified using affinity and size-exclusion chromatography. Milligram quantities of pure USP17 can be produced that is catalytically more efficient (kcat/Km = 1500 (x10(3)) M(-1) sec(-1)) than other human USPs studied to date...
October 15, 2016: Archives of Biochemistry and Biophysics
Chrisovalantis Papadopoulos, Philipp Kirchner, Monika Bug, Daniel Grum, Lisa Koerver, Nina Schulze, Robert Poehler, Alina Dressler, Sven Fengler, Khalid Arhzaouy, Vanda Lux, Michael Ehrmann, Conrad C Weihl, Hemmo Meyer
Rupture of endosomes and lysosomes is a major cellular stress condition leading to cell death and degeneration. Here, we identified an essential role for the ubiquitin-directed AAA-ATPase, p97, in the clearance of damaged lysosomes by autophagy. Upon damage, p97 translocates to lysosomes and there cooperates with a distinct set of cofactors including UBXD1, PLAA, and the deubiquitinating enzyme YOD1, which we term ELDR components for Endo-Lysosomal Damage Response. Together, they act downstream of K63-linked ubiquitination and p62 recruitment, and selectively remove K48-linked ubiquitin conjugates from a subpopulation of damaged lysosomes to promote autophagosome formation...
October 17, 2016: EMBO Journal
Elsa Perrody, Laurence Abrami, Michal Feldman, Beatrice Kunz, Sylvie Urbé, Gisou van der Goot
Many membrane proteins fold inefficiently and require the help of enzymes and chaperones. Here we reveal a novel folding assistance system that operates on membrane proteins from the cytosolic side of the endoplasmic reticulum (ER). We show that folding of the Wnt signaling coreceptor LRP6 is promoted by ubiquitination of a specific lysine, retaining it in the ER while avoiding degradation. Subsequent ER exit requires removal of ubiquitin from this lysine by the deubiquitinating enzyme USP19. This ubiquitination-deubiquitination is conceptually reminiscent of the glucosylation-deglucosylation occurring in the ER lumen during the calnexin/calreticulin folding cycle...
October 18, 2016: ELife
Ye Cao, Kai Guan, Xiang He, Congwen Wei, Zirui Zheng, Yanhong Zhang, Shengli Ma, Hui Zhong, Wei Shi
The Yersinia outer protein J (YopJ) plays a pivotal role in evading the host immune response and establishes a persistent infection in host cells after bacterial infection. YopJ is a cysteine protease and can act as a deubiquitinating enzyme that deubiquitinates several targets in multiple signaling pathways. Stimulator of interferon genes (STING) is a critical adapter for the induction of interferon regulatory factor 3 (IRF3) phosphorylation and subsequent production of the cytokines in response to nucleic acids in the cytoplasm...
October 11, 2016: Biochimica et Biophysica Acta
Xing Lin, Qianshun Chen, Chen Huang, Xunyu Xu
Lung cancer is one of the most common cancers in the world. Cylindromatosis (CYLD) is a deubiquitination enzyme and contributes to the degradation of ubiquitin chains on RIP1. The aim of the present study is to investigate the levels of CYLD in lung cancer patients and explore the molecular mechanism of CYLD in the lung cancer pathogenesis. The levels of CYLD were detected in human lung cancer tissues and the paired paracarcinoma tissues by real-time PCR and western blotting analysis. The proliferation of human lung cancer cells was determined by MTT assay...
2016: Mediators of Inflammation
Tycho E T Mevissen, Yogesh Kulathu, Monique P C Mulder, Paul P Geurink, Sarah L Maslen, Malte Gersch, Paul R Elliott, John E Burke, Bianca D M van Tol, Masato Akutsu, Farid El Oualid, Masato Kawasaki, Stefan M V Freund, Huib Ovaa, David Komander
The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types co-exist in polyubiquitin and are independently regulated in cells. This 'ubiquitin code' determines the fate of the modified protein. Deubiquitinating enzymes of the ovarian tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system...
October 12, 2016: Nature
Lodewijk J A Toonen, Iris Schmidt, Martijn S Luijsterburg, Haico van Attikum, Willeke M C van Roon-Mom
Spinocerebellar ataxia type-3 (SCA3) is a neurodegenerative disorder caused by a polyglutamine repeat expansion in the ataxin-3 protein. Cleavage of mutant ataxin-3 by proteolytic enzymes yields ataxin-3 fragments containing the polyglutamine stretch. These shorter ataxin-3 fragments are thought to be involved in SCA3 pathogenesis due to their increased cellular toxicity and their involvement in formation of the characteristic neuronal aggregates. As a strategy to prevent formation of toxic cleavage fragments, we investigated an antisense oligonucleotide-mediated modification of the ataxin-3 pre-mRNA through exon skipping of exon 8 and 9, resulting in the removal of a central 88 amino acid region of the ataxin-3 protein...
October 12, 2016: Scientific Reports
Maggy Hologne, François-Xavier Cantrelle, Gwladys Riviere, Florence Guillière, Xavier Trivelli, Olivier Walker
AMSH is one of the deubiquitinating enzymes associated in the regulation of endocytic cargo trafficking. It shows an exquisite selectivity for Lys63-linked polyubiquitin chains that are the main chains involved in cargo sorting. The first step requires the ESCRT-0 complex that comprises the STAM and Hrs proteins. Previous studies have shown that the presence of the STAM protein increases the efficiency of Lys63-linked polyubiquitin chain cleavage by AMSH, one of the deubiquitinating enzyme involved in lysosomal degradation...
October 7, 2016: Journal of Molecular Biology
Sabrina Torre, Maria J Polyak, David Langlais, Nassima Fodil, James M Kennedy, Irena Radovanovic, Joanne Berghout, Gabriel A Leiva-Torres, Connie M Krawczyk, Subburaj Ilangumaran, Karen Mossman, Chen Liang, Klaus-Peter Knobeloch, Luke M Healy, Jack Antel, Nathalie Arbour, Alexandre Prat, Jacek Majewski, Mark Lathrop, Silvia M Vidal, Philippe Gros
Genes and pathways in which inactivation dampens tissue inflammation present new opportunities for understanding the pathogenesis of common human inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis and multiple sclerosis. We identified a mutation in the gene encoding the deubiquitination enzyme USP15 (Usp15(L749R)) that protected mice against both experimental cerebral malaria (ECM) induced by Plasmodium berghei and experimental autoimmune encephalomyelitis (EAE). Combining immunophenotyping and RNA sequencing in brain (ECM) and spinal cord (EAE) revealed that Usp15(L749R)-associated resistance to neuroinflammation was linked to dampened type I interferon responses in situ...
October 10, 2016: Nature Immunology
Simon Hauri, Federico Comoglio, Makiko Seimiya, Moritz Gerstung, Timo Glatter, Klaus Hansen, Ruedi Aebersold, Renato Paro, Matthias Gstaiger, Christian Beisel
Polycomb group (PcG) proteins are major determinants of gene silencing and epigenetic memory in higher eukaryotes. Here, we systematically mapped the human PcG complexome using a robust affinity purification mass spectrometry approach. Our high-density protein interaction network uncovered a diverse range of PcG complexes. Moreover, our analysis identified PcG interactors linking them to the PcG system, thus providing insight into the molecular function of PcG complexes and mechanisms of recruitment to target genes...
October 4, 2016: Cell Reports
Leena Ackermann, Michael Schell, Wojciech Pokrzywa, Éva Kevei, Anton Gartner, Björn Schumacher, Thorsten Hoppe
Multiple protein ubiquitination events at DNA double-strand breaks (DSBs) regulate damage recognition, signaling and repair. It has remained poorly understood how the repair process of DSBs is coordinated with the apoptotic response. Here, we identified the E4 ubiquitin ligase UFD-2 as a mediator of DNA-damage-induced apoptosis in a genetic screen in Caenorhabditis elegans. We found that, after initiation of homologous recombination by RAD-51, UFD-2 forms foci that contain substrate-processivity factors including the ubiquitin-selective segregase CDC-48 (p97), the deubiquitination enzyme ATX-3 (Ataxin-3) and the proteasome...
September 26, 2016: Nature Structural & Molecular Biology
Emilio Lecona, Oscar Fernandez-Capetillo
Post-translational modifications regulate each step of DNA replication to ensure the faithful transmission of genetic information. In this context, we recently showed that deubiquitination of SUMO2/3 and SUMOylated proteins by USP7 helps to create a SUMO-rich and ubiquitin-low environment around replisomes that is necessary to maintain the activity of replication forks and for new origin firing. We propose that a two-flag system mediates the collective concentration of factors at sites of DNA replication, whereby SUMO and Ubiquitinated-SUMO would constitute "stay" or "go" signals respectively for replisome and accessory factors...
September 26, 2016: BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology
(no author information available yet)
USP7 deubiquitinates MYCN to enhance its stability and transcriptional activity in neuroblastoma.
September 23, 2016: Cancer Discovery
Bindu Nanduri, Leslie A Shack, Aswathy N Rai, William B Epperson, Wes Baumgartner, Ty B Schmidt, Mariola J Edelmann
To develop a reproducible tissue lysis method that retains enzyme function for activity-based protein profiling, we compared four different methods to obtain protein extracts from bovine lung tissue: focused ultrasonication, standard sonication, mortar & pestle method, and homogenization combined with standard sonication. Focused ultrasonication and mortar & pestle methods were sufficiently effective for activity-based profiling of deubiquitinases in tissue, and focused ultrasonication also had the fastest processing time...
September 20, 2016: Analytical Biochemistry
Shreya Dharadhar, Marcello Clerici, Willem J van Dijk, Alexander Fish, Titia K Sixma
Regulation of deubiquitinating enzyme (DUB) activity is an essential step for proper function of cellular ubiquitin signals. UAF1 is a WD40 repeat protein, which binds and activates three important DUBs, USP1, USP12 and USP46. Here, we report the crystal structure of the USP12-Ub/UAF1 complex at a resolution of 2.8Å and of UAF1 at 2.3Å. In the complex we find two potential sites for UAF1 binding, analogous to what was seen in a USP46/UAF1 complex. In line with these observed dual binding states, we show here that USP12/UAF1 complex has 1:2 stoichiometry in solution, with a two-step binding at 4nM and 325nM respectively...
September 17, 2016: Journal of Structural Biology
Xing Mao, Weili Luo, Jianyong Sun, Nianji Yang, Linda Wei Zhang, Zhonghua Zhao, Zhigang Zhang, Huijuan Wu
Mesangial proliferative glomerulonephritis is characterized by proliferation of mesangial cells (MCs) and transforming growth factor-β (TGF-β)-dependent stimulation of abnormal extracellular matrix (ECM) accumulation. We previously showed that Decorin--a leucine-rich proteoglycan inhibiting the progression of glomerulonephritis and glomerular sclerosis--can be degraded by the ubiquitin-proteasome pathway and deubiquitinated and stabilized by ubiquitin-specific processing protease 2-69(Usp2-69). Usp2-69 is highly expressed in the kidney and has been implicated in the regulation of cell proliferation and apoptosis...
September 15, 2016: Experimental and Molecular Pathology
Lei Yi, Yan Cui, Qingfu Xu, Yugang Jiang
The substrates and mechanisms of ubiquitin specific peptidase 7 (USP7) in glioma remain unclear. Lysine‑specific demethylase 1 (LSD1) may undergo proteasomal degradation; however, a reciprocal mechanism that stabilizes LSD1 in glioma has not been dertermined. Here co-immunoprecipitation and GST pull-down assays revealed that LSD1 is associated with USP7 in vivo and in vitro. USP7 inhibited LSD1 ubiquitination and stabilized LSD1 in A172 and T98G cells. MTT, EdU proliferation, flow cytometry and Transwell assays indicated that LSD1 played a critical role in the proliferation and invasion of glioblastoma (GBM) cells...
September 16, 2016: Oncology Reports
Kapil Sareen-Khanna, Joan Papillon, Simon S Wing, Andrey V Cybulsky
Kidney cell injury may be associated with protein misfolding and induction of endoplasmic reticulum (ER) stress. Examples include complement-induced glomerular epithelial cell (GEC)/podocyte injury in membranous nephropathy, and ischemia-reperfusion injury. Renal cell injury can also result from mutations in integral proteins, which lead to their misfolding and accumulation. Certain nephrin missense mutants misfold, accumulate in the ER, and induce ER stress. We examined if enhancement of ubiquitin-proteasome system (UPS) function may facilitate proteostasis and confer protection against injury...
September 14, 2016: American Journal of Physiology. Renal Physiology
Lili Xie, Aihong Li, Jiabing Shen, Maohong Cao, Xiaojin Ning, Debin Yuan, Yuteng Ji, Hongmei Wang, Kaifu Ke
OTUB1 is a member of deubiquitinating enzymes, which was shown as a proteasome-associated DUB to be involved in the proteins Ub-dependent degradation. Previous studies have indicated that OTUB1 was expressed in brain. But its distribution and function in the brain remain unclear. In this study, we explored the roles of OTUB1 protein in the pathophysiology of intracerebral hemorrhage (ICH). From the results of Western blot, immunohistochemistry, and immunofluorescence, we found an obvious up-regulation of OTUB1 in neurons adjacent to the hematoma after ICH...
November 2016: Molecular and Cellular Biochemistry
Cheng-Yuan Lai, Yu-Cheng Ho, Ming-Chun Hsieh, Hsueh-Hsiao Wang, Jen-Kun Cheng, Yat-Pang Chau, Hsien-Yu Peng
UNLABELLED: Spinal plasticity, a key process mediating neuropathic pain development, requires ubiquitination-dependent protein turnover. Presynaptic active zone proteins have a crucial role in regulating vesicle exocytosis, which is essential for synaptic plasticity. Nevertheless, the mechanism for ubiquitination-regulated turnover of presynaptic active zone proteins in the progression of spinal plasticity-associated neuropathic pain remains unclear. Here, after research involving Sprague Dawley rats, we reported that spinal nerve ligation (SNL), in addition to causing allodynia, enhances the Rab3-interactive molecule-1α (RIM1α), a major active zone protein presumed to regulate neural plasticity, specifically in the synaptic plasma membranes (SPMs) of the ipsilateral dorsal horn...
September 14, 2016: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
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