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Eun Hee Koh, Yong Chen, David A Bader, Mark P Hamilton, Bin He, Brian York, Shingo Kajimura, Sean E McGuire, Sean M Hartig
The acquisition of beige adipocyte features by white fat cells corresponds to protection against obesity-induced metabolic diseases in humans and animal models of type 2 diabetes. In adipose tissue, expression of the E2-SUMO ligase Ubiquitin Carrier Protein 9 (Ubc9) is positively correlated with markers of insulin resistance and corresponds with impaired browning of human white adipocytes. However, the molecular regulation of Ubc9 expression in adipocytes and other cells remains unclear. In this study, we demonstrated the mRNA and protein expression of Ubc9 are regulated by the microRNA miRNA-30a (miR-30a) in human subcutaneous adipocytes...
October 10, 2016: Journal of Biological Chemistry
Ching-Yi Tsai, Faith C H Li, Carol H Y Wu, Alice Y W Chang, Samuel H H Chan
BACKGROUND: Small ubiquitin-related modifier (SUMO) is a group of proteins that participates in post-translational modifications. One known SUMO target is the transcription factor nuclear factor-kB (NF-kB) that plays a pivotal role in many disease processes; sumoylation inactivates NF-kB by conjugation with inhibitors of NF-kB (IkB). Our laboratory demonstrated previously that transcriptional upregulation of nitric oxide synthase II (NOS II) by NF-kB, leading to nitrosative stress by the formation of peroxynitrite in the rostral ventrolateral medulla (RVLM), underpins the defunct brain stem cardiovascular regulation that precedes brain death...
2016: Journal of Biomedical Science
Tobias Ritterhoff, Hrishikesh Das, Yuqing Hao, Volkan Sakin, Annette Flotho, Andreas Werner, Frauke Melchior
One of the few proteins that have SUMO E3 ligase activity is the 358 kDa nucleoporin RanBP2 (Nup358). While small fragments of RanBP2 can stimulate SUMOylation in vitro, the physiologically relevant E3 ligase is a stable multi-subunit complex comprised of RanBP2, SUMOylated RanGAP1, and Ubc9. Here, we provide a detailed protocol to in vitro reconstitute the RanBP2 SUMO E3 ligase complex. With the exception of RanBP2, reconstitution involves untagged full-length proteins. We describe the bacterial expression and purification of all complex components, namely an 86 kDa His-tagged RanBP2 fragment, the SUMO E2-conjugating enzyme Ubc9, RanGAP1, and SUMO1, and we provide a protocol for quantitative SUMOylation of RanGAP1...
2016: Methods in Molecular Biology
Franziska Wetzel, Sonnhild Mittag, Misael Cano-Cortina, Tobias Wagner, Oliver H Krämer, Rainer Niedenthal, Lorenza Gonzalez-Mariscal, Otmar Huber
The zonula occludens (ZO)-2 protein links tight junctional transmembrane proteins to the actin cytoskeleton and associates with splicing and transcription factors in the nucleus. Multiple posttranslational modifications control the intracellular distribution of ZO-2. Here, we report that ZO-2 is a target of the SUMOylation machinery and provide evidence on how this modification may affect its cellular distribution and function. We show that ZO-2 associates with the E2 SUMO-conjugating enzyme Ubc9 and with SUMO-deconjugating proteases SENP1 and SENP3...
September 7, 2016: Cellular and Molecular Life Sciences: CMLS
Manish K Gupta, Jeffrey Robbins
Cardiac proteins are subject to continuous stress and these intrinsic and extrinsic factors, both physiological and pathological can lead to protein misfolding. If the protein quality control (PQC) pathways are in any way compromised or their activities diminished, intracellular aggregates can form and a proteotoxic environment is generated, which contributes to cardiac disease and heart failure. We studied the role that SUMO post-translational modification plays in a proteotoxic cardiac environment. SUMOylation can have an integral role in controlling flux through the ubiquitin-proteasome system, and expression of the SUMO (small ubiquitin-like modifier) E2 enzyme UBE2I/UBC9 improves cardiac PQC...
August 29, 2016: Autophagy
Maria Lauda Tomasi, Komal Ramani, Minjung Ryoo
Lipopolysaccharide (LPS), a bacterial endotoxin, induces inflammation in macrophages via activation of NF-κB signaling. Sumoylation is a post-translational modification mediated by the small ubiquitin-like modifier, SUMO. Ubiquitin-conjugating enzyme 9 (UBC9) is the only known SUMO conjugating enzyme. LPS treatment lowers SUMO-1 and UBC9 mRNA levels in primary astrocytes. UBC9 can degrade NF-κB inhibitor α (Ikbα) via a SUMO2/3-ubiquitin pathway. However, UBC9 may also promote Ikbα stability by SUMO-1 conjugation that further regulates NF-κB signaling...
September 2016: American Journal of Pathology
María Juliana Rodríguez-García, Andrés García-Reina, Vilmar Machado, José Galián
Ubiquitin and small ubiquitin-like modifiers (SUMO) are post-translational modifiers essential in a variety of cellular processes, including gametogenesis. SUMO-conjugating enzyme (UBC9) and the ubiquitin ribosomal fusion protein UBS27 have been characterised in several model species. However, their expression in coleopteran remains unstudied. In this study, UBC9 and UBS27 genes have been characterised in the tiger beetle Cicindela campestris for the first time. Bioinformatic analysis showed that the Cc-UBC9 gene encoded a 159 amino acid protein with a predicted molecular weight of 18...
August 11, 2016: Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology
Elżbieta Wieczorek, Sylwia Kędracka-Krok, Katarzyna Sołtys, Urszula Jankowska, Rafał Hołubowicz, Justyna Seliga, Andrzej Ożyhar
Ageing and mutations of transthyretin (TTR), the thyroid hormones and retinol transporting protein lead to amyloidosis by destabilizing the structure of TTR. Because protein structure is regulated through posttranslational modifications, we investigated the Small Ubiquitin-like Modifier (SUMO)ylation of TTR. We chose the widely used Ubc9 fusion-directed SUMOylation system, which is based on a fusion of the SUMOylation substrate of interest with Ubc9, a sole SUMO conjugating enzyme. Surprisingly, despite our presumptions, we found that Ubc9 fused to TTR was SUMOylated at a unique set of lysine residues...
2016: PloS One
Xukun Bi, Jiale Song, Jing Gao, Juanjuan Zhao, Meihui Wang, Corey A Scipione, Marlys L Koschinsky, Zhao V Wang, Shiming Xu, Guosheng Fu
The liver X receptor (LXR) is a cholesterol-sensing nuclear receptor that has an established function in lipid metabolism; however, its role in inflammation is elusive. In this study, we showed that the LXR agonist GW3965 exhibited potent anti-inflammatory activity by suppressing the firm adhesion of monocytes to endothelial cells. To further address the mechanisms underlying the inhibition of inflammatory cell infiltration, we evaluated the effects of LXR agonist on interleukin-8 (IL-8) secretion and nuclear factor-kappa B (NF-κB) activation in human umbilical vein endothelial cells (HUVECs)...
August 4, 2016: Journal of Cellular and Molecular Medicine
Christèle Maison, Delphine Bailly, Jean-Pierre Quivy, Geneviève Almouzni
The trimethylation of histone H3 on lysine 9 (H3K9me3) - a mark recognized by HP1 that depends on the Suv39h lysine methyltransferases (KMTs) - has provided a basis for the reader/writer model to explain HP1 accumulation at pericentric heterochromatin in mammals. Here, we identify the Suv39h1 paralog, as a unique enhancer of HP1α sumoylation both in vitro and in vivo. The region responsible for promoting HP1α sumoylation (aa1-167) is distinct from the KMT catalytic domain and mediates binding to Ubc9. Tethering the 1-167 domain of Suv39h1 to pericentric heterochromatin, but not mutants unable to bind Ubc9, accelerates the de novo targeting of HP1α to these domains...
2016: Nature Communications
Xiao Ding, Aibo Wang, Xiaopeng Ma, Maud Demarque, Wei Jin, Huawei Xin, Anne Dejean, Chen Dong
Foxp3-expressing regulatory T (Treg) cells are essential for immune tolerance; however, the molecular mechanisms underlying Treg cell expansion and function are still not well understood. SUMOylation is a protein post-translational modification characterized by covalent attachment of SUMO moieties to lysines. UBC9 is the only E2 conjugating enzyme involved in this process, and loss of UBC9 completely abolishes the SUMOylation pathway. Here, we report that selective deletion of Ubc9 within the Treg lineage results in fatal early-onset autoimmunity similar to Foxp3 mutant mice...
July 26, 2016: Cell Reports
Aileen Y Alontaga, Nigus D Ambaye, Yi-Jia Li, Ramir Vega, Chih-Hong Chen, Krzysztof P Bzymek, John C Williams, Weidong Hu, Yuan Chen
Post-translational modifications by the small ubiquitin-like modifiers (SUMO), in particular the formation of poly-SUMO-2 and -3 chains, regulates essential cellular functions and its aberration leads to life-threatening diseases (Geoffroy and Hay, 2009) [1]. It was shown previously that the non-covalent interaction between SUMO and the conjugating enzyme (E2) for SUMO, known as Ubc9, is required for poly-SUMO-2/3 chain formation (Knipscheer et al., 2007) [2]. However, the structure of SUMO-Ubc9 non-covalent complex, by itself, could not explain how the poly-SUMO-2/3 chain forms and consequently a Ubc9 homodimer, although never been observed, was proposed for poly-SUMO-2/3 chain formation (Knipscheer et al...
June 2016: Data in Brief
Eleni Arvaniti, Athina Vakrakou, Valeria Kaltezioti, Athanasios Stergiopoulos, Niki Prakoura, Panagiotis K Politis, Aristidis Charonis
BACKGROUND: Renal fibrosis is a common histological finding present in many pathologies; however, key signaling pathways and molecular determinants involved in the development of fibrosis are not fully known yet. Previous findings have established a causative role of calreticulin's up-regulation during the development of renal fibrosis while its down-regulation exhibited a protective effect against fibrosis. Therefore, the mechanism of its up-regulation needs to be explored. METHODS: Bioinformatics analyses of the calreticulin gene promoter combined with transcriptional assays and in vivo chromatin immunoprecipitation experiments in the Unilateral Ureteric Obstruction (UUO) model of renal fibrosis, indicated that NR5A2 is a critical regulator of calreticulin expression...
September 2016: Biochimica et Biophysica Acta
Denise E Bustard, Lindsay G Ball, Jennifer A Cobb
The Smc5/6 complex in Saccharomyces cerevisiae contains six essential non-Smc elements, Nse1-6. With the exception of Nse2 (also known as Mms21), which is an E3 small ubiquitin-like modifier (SUMO) ligase, very little is understood about the role of these components or their contribution to Smc5/6 functionality. Our characterization of Nse5 establishes a previously unidentified relationship between the Smc5/6 complex and factors of the SUMO pathway. Nse5 physically associates with the E2 conjugating enzyme, Ubc9, where contacts are stabilized by non-covalent interactions with SUMO...
2016: Biology Open
Silvia Schwartz, Mauro Truglio, Maxwell J Scott, Helen L Fitzsimons
HDAC4 is a potent memory repressor with overexpression of wild type or a nuclear-restricted mutant resulting in memory deficits. Interestingly, reduction of HDAC4 also impairs memory via an as yet unknown mechanism. Although histone deacetylase family members are important mediators of epigenetic mechanisms in neurons, HDAC4 is predominantly cytoplasmic in the brain and there is increasing evidence for interactions with nonhistone proteins, suggesting HDAC4 has roles beyond transcriptional regulation. To that end, we performed a genetic interaction screen in Drosophila and identified 26 genes that interacted with HDAC4, including Ubc9, the sole SUMO E2-conjugating enzyme...
July 2016: Genetics
Nicolas Stankovic-Valentin, Katarzyna Drzewicka, Cornelia König, Elmar Schiebel, Frauke Melchior
To sense and defend against oxidative stress, cells depend on signal transduction cascades involving redox-sensitive proteins. We previously identified SUMO (small ubiquitin-related modifier) enzymes as downstream effectors of reactive oxygen species (ROS). Hydrogen peroxide transiently inactivates SUMO E1 and E2 enzymes by inducing a disulfide bond between their catalytic cysteines. How important their oxidation is in light of many other redox-regulated proteins has however been unclear. To selectively disrupt this redox switch, we identified a catalytically fully active SUMO E2 enzyme variant (Ubc9 D100A) with strongly reduced propensity to maintain a disulfide with the E1 enzyme in vitro and in cells...
June 15, 2016: EMBO Journal
Tobias Ritterhoff, Hrishikesh Das, Götz Hofhaus, Rasmus R Schröder, Annette Flotho, Frauke Melchior
Continuous cycles of nucleocytoplasmic transport require disassembly of transport receptor/Ran-GTP complexes in the cytoplasm. A basic disassembly mechanism in all eukaryotes depends on soluble RanGAP and RanBP1. In vertebrates, a significant fraction of RanGAP1 stably interacts with the nucleoporin RanBP2 at a binding site that is flanked by FG-repeats and Ran-binding domains, and overlaps with RanBP2's SUMO E3 ligase region. Here, we show that the RanBP2/RanGAP1*SUMO1/Ubc9 complex functions as an autonomous disassembly machine with a preference for the export receptor Crm1...
2016: Nature Communications
Yi-Han Dai, Liang-Yi Hung, Ruo-Yu Chen, Chien-Hsien Lai, Kung-Chao Chang
Diffuse large B-cell lymphoma (DLBCL), the most common lymphoma, shows either no response or development of resistance to further treatment in 30% of the patients that warrants the development of novel drugs. We have reported that ON 01910.Na (rigosertib), a multikinase inhibitor, is selectively cytotoxic for DLBCL and induces more hyperphosphorylation and sumoylation of Ran GTPase-activating protein 1 (RanGAP1) in DLBCL cells than in non-neoplastic lymphoblastoid cell line. However, the exact mechanism of rigosertib-induced cell death in DLBCL remains to be clarified...
September 2016: Translational Research: the Journal of Laboratory and Clinical Medicine
Manish K Gupta, Patrick M McLendon, James Gulick, Jeanne James, Kamel Khalili, Jeffrey Robbins
RATIONALE: SUMOylation plays an important role in cardiac function and can be protective against cardiac stress. Recent studies show that SUMOylation is an integral part of the ubiquitin proteasome system, and expression of the small ubiquitin-like modifier (SUMO) E2 enzyme UBC9 improves cardiac protein quality control. However, the precise role of SUMOylation on other protein degradation pathways, particularly autophagy, remains undefined in the heart. OBJECTIVE: To determine whether SUMOylation affects cardiac autophagy and whether this effect is protective in a mouse model of proteotoxic cardiac stress...
June 10, 2016: Circulation Research
William M Hewitt, George T Lountos, Katherine Zlotkowski, Samuel D Dahlhauser, Lindsey B Saunders, Danielle Needle, Joseph E Tropea, Chendi Zhan, Guanghong Wei, Buyong Ma, Ruth Nussinov, David S Waugh, John S Schneekloth
Conjugation of the small ubiquitin-like modifier (SUMO) to protein substrates is an important disease-associated posttranslational modification, although few inhibitors of this process are known. Herein, we report the discovery of an allosteric small-molecule binding site on Ubc9, the sole SUMO E2 enzyme. An X-ray crystallographic screen was used to identify two distinct small-molecule fragments that bind to Ubc9 at a site distal to its catalytic cysteine. These fragments and related compounds inhibit SUMO conjugation in biochemical assays with potencies of 1...
May 4, 2016: Angewandte Chemie
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