The E3 ubiquitin ligase RNF146 promotes colorectal cancer by activating the Wnt/β-catenin pathway via ubiquitination of Axin1.

PubMed

Shen, Jiangli; Yu, Zhaohui; Li, Na

2018-06-20

The E3 ubiquitin ligase ring finger protein 146 (RNF146) has been implicated in tumor development. However, the role and clinical significance of RNF146 in colorectal cancer (CRC) remain unknown. In this study, we reported for the first time that RNF146 was upregulated in CRC tissues as well as in cell lines. Further, RNF146 expression was independent prognostic factor for poor outcome of CRC patients. RNF146 knockdown in cell lines inhibited cell growth, promoted cell apoptosis in vitro and suppressed colorectal tumor growth in vivo. Mechanistic investigations revealed that RNF146 exerted oncogenic role through ubiquitination of Axin1 to activate β-catenin signalling. In addition, RNF146 expression was positively correlated with β-catenin expression in CRC tissues. Collectively, our data suggest that RNF146 might function as a oncogene in human CRC, and represent a promising prognostic factor and a valuable therapeutic target for CRC. Copyright © 2018. Published by Elsevier Inc.

Ubiquitin-like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway.

PubMed

Oka, Yasuyoshi; Bekker-Jensen, Simon; Mailand, Niels

2015-05-12

Ubiquitin and ubiquitin-like proteins (UBLs) function in a wide array of cellular processes. UBL5 is an atypical UBL that does not form covalent conjugates with cellular proteins and which has a known role in modulating pre-mRNA splicing. Here, we report an unexpected involvement of human UBL5 in promoting the function of the Fanconi anemia (FA) pathway for repair of DNA interstrand crosslinks (ICLs), mediated by a specific interaction with the central FA pathway component FANCI. UBL5-deficient cells display spliceosome-independent reduction of FANCI protein stability, defective FANCI function in response to DNA damage and hypersensitivity to ICLs. By mapping the sequence determinants underlying UBL5-FANCI binding, we generated separation-of-function mutants to demonstrate that key aspects of FA pathway function, including FANCI-FANCD2 heterodimerization, FANCD2 and FANCI monoubiquitylation and maintenance of chromosome stability after ICLs, are compromised when the UBL5-FANCI interaction is selectively inhibited by mutations in either protein. Together, our findings establish UBL5 as a factor that promotes the functionality of the FA DNA repair pathway. © 2015 The Authors.

CNOT4-Mediated Ubiquitination of Influenza A Virus Nucleoprotein Promotes Viral RNA Replication

PubMed Central

Lin, Yu-Chen; Jeng, King-Song

2017-01-01

ABSTRACT Influenza A virus (IAV) RNA segments are individually packaged with viral nucleoprotein (NP) and RNA polymerases to form a viral ribonucleoprotein (vRNP) complex. We previously reported that NP is a monoubiquitinated protein which can be deubiquitinated by a cellular ubiquitin protease, USP11. In this study, we identified an E3 ubiquitin ligase, CNOT4 (Ccr4-Not transcription complex subunit 4), which can ubiquitinate NP. We found that the levels of viral RNA, protein, viral particles, and RNA polymerase activity in CNOT4 knockdown cells were lower than those in the control cells upon IAV infection. Conversely, overexpression of CNOT4 rescued viral RNP activity. In addition, CNOT4 interacted with the NP in the cell. An in vitro ubiquitination assay also showed that NP could be ubiquitinated by in vitro-translated CNOT4, but ubiquitination did not affect the protein stability of NP. Significantly, CNOT4 increased NP ubiquitination, whereas USP11 decreased it. Mass spectrometry analysis of ubiquitinated NP revealed multiple ubiquitination sites on the various lysine residues of NP. Three of these, K184, K227, and K273, are located on the RNA-binding groove of NP. Mutations of these sites to arginine reduced viral RNA replication. These results indicate that CNOT4 is a ubiquitin ligase of NP, and ubiquitination of NP plays a positive role in viral RNA replication. PMID:28536288

The Ubiquitin Ligase CHIP Prevents SirT6 Degradation through Noncanonical Ubiquitination

PubMed Central

Ronnebaum, Sarah M.; Wu, Yaxu; McDonough, Holly

2013-01-01

The ubiquitin ligase CHIP (carboxyl terminus of Hsp70-interacting protein) regulates protein quality control, and CHIP deletion accelerates aging and reduces the life span in mice. Here, we reveal a mechanism for CHIP's influence on longevity by demonstrating that CHIP stabilizes the sirtuin family member SirT6, a lysine deacetylase/ADP ribosylase involved in DNA repair, metabolism, and longevity. In CHIP-deficient cells, SirT6 protein half-life is substantially reduced due to increased proteasome-mediated degradation, but CHIP overexpression in these cells increases SirT6 protein expression without affecting SirT6 transcription. CHIP noncanonically ubiquitinates SirT6 at K170, which stabilizes SirT6 and prevents SirT6 canonical ubiquitination by other ubiquitin ligases. In CHIP-depleted cells, SirT6 K170 mutation increases SirT6 half-life and prevents proteasome-mediated degradation. The global decrease in SirT6 expression in the absence of CHIP is associated with decreased SirT6 promoter occupancy, which increases histone acetylation and promotes downstream gene transcription in CHIP-depleted cells. Cells lacking CHIP are hypersensitive to DNA-damaging agents, but DNA repair and cell viability are rescued by enforced expression of SirT6. The discovery of this CHIP-SirT6 interaction represents a novel protein-stabilizing mechanism and defines an intersection between protein quality control and epigenetic regulation to influence pathways that regulate the biology of aging. PMID:24043303

Diggin’ on U(biquitin): A Novel Method for the Identification of Physiological E3 Ubiquitin Ligase Substrates

PubMed Central

Rubel, Carrie E.; Schisler, Jonathan C.; Hamlett, Eric D.; DeKroon, Robert M.; Gautel, Mathias; Alzate, Oscar; Patterson, Cam

2013-01-01

The ubiquitin-proteasome system (UPS) plays a central role in maintaining protein homeostasis, emphasized by a myriad of diseases that are associated with altered UPS function such as cancer, muscle-wasting, and neurodegeneration. Protein ubiquitination plays a central role in both the promotion of proteasomal degradation as well as cellular signaling through regulation of the stability of transcription factors and other signaling molecules. Substrate specificity is a critical regulatory step of ubiquitination and is mediated by ubiquitin ligases. Recent studies implicate ubiquitin ligases in multiple models of cardiac diseases such as cardiac hypertrophy, atrophy, and ischemia/reperfusion injury, both in a cardioprotective and maladaptive role. Therefore, identifying physiological substrates of cardiac ubiquitin ligases provides both mechanistic insights into heart disease as well as possible therapeutic targets. Current methods identifying substrates for ubiquitin ligases rely heavily upon non-physiologic in vitro methods, impeding the unbiased discovery of physiological substrates in relevant model systems. Here we describe a novel method for identifying ubiquitin ligase substrates utilizing Tandem Ubiquitin Binding Entities (TUBE) technology, two-dimensional differential in gel electrophoresis (2-D DIGE), and mass spectrometry, validated by the identification of both known and novel physiological substrates of the ubiquitin ligase MuRF1 in primary cardiomyocytes. This method can be applied to any ubiquitin ligase, both in normal and disease model systems, in order to identify relevant physiological substrates under various biological conditions, opening the door to a clearer mechanistic understanding of ubiquitin ligase function and broadening their potential as therapeutic targets. PMID:23695782

Terminating protein ubiquitination: Hasta la vista, ubiquitin.

PubMed

Stringer, Daniel K; Piper, Robert C

2011-09-15

Ubiquitination is a post-translational modification that generally directs proteins for degradation by the proteasome or by lysosomes. However, ubiquitination has been implicated in many other cellular processes, including transcriptional regulation, DNA repair, regulation of protein-protein interactions and association with ubiquitin-binding scaffolds. Ubiquitination is a dynamic process. Ubiquitin is added to proteins by E3 ubiquitin ligases as a covalent modification to one or multiple lysine residues as well as non-lysine amino acids. Ubiquitin itself contains seven lysines, each of which can also be ubiquitinated, leading to polyubiquitin chains that are best characterized for linkages occurring through K48 and K63. Ubiquitination can also be reversed by the action of deubiquitination enzymes (DUbs). Like E3 ligases, DUbs play diverse and critical roles in cells. ( 1) Ubiquitin is expressed as a fusion protein, as a linear repeat or as a fusion to ribosomal subunits, and DUbs are necessary to liberate free ubiquitin, making them the first enzyme of the ubiquitin cascade. Proteins destined for degradation by the proteasome or by lysosomes are deubiquitinated prior to their degradation, which allows ubiquitin to be recycled by the cell, contributing to the steady-state pool of free ubiquitin. Proteins destined for degradation by lysosomes are also acted upon by both ligases and DUbs. Deubiquitination can also act as a means to prevent protein degradation, and many proteins are thought to undergo rounds of ubiquitination and deubiquitination, ultimately resulting in either the degradation or stabilization of those proteins. Despite years of study, examining the effects of the ubiquitination of proteins remains quite challenging. This is because the methods that are currently being employed to study ubiquitination are limiting. Here, we briefly examine current strategies to study the effects of ubiquitination and describe an additional novel approach that we have

AGEs-RAGE system down-regulates Sirt1 through the ubiquitin-proteasome pathway to promote FN and TGF-β1 expression in male rat glomerular mesangial cells.

PubMed

Huang, Kai-Peng; Chen, Cheng; Hao, Jie; Huang, Jun-Ying; Liu, Pei-Qing; Huang, He-Qing

2015-01-01

We previously demonstrated that advanced glycation-end products (AGEs) promote the pathological progression of diabetic nephropathy by decreasing silent information regulator 2-related protein 1 (Sirt1) expression in glomerular mesangial cells (GMCs). Here, we investigated whether AGEs-receptor for AGEs (RAGE) system down-regulated Sirt1 expression through ubiquitin-proteasome pathway and whether Sirt1 ubiquitination affected fibronectin (FN) and TGF-β1, 2 fibrotic indicators in GMCs. Sirt1 was polyubiquitinated and subsequently degraded by proteasome. AGEs increased Sirt1 ubiquitination and proteasome-mediated degradation, shortened Sirt1 half-life, and promoted FN and TGF-β1 expression. Ubiquitin-specific protease 22 (USP22) reduced Sirt1 ubiquitination and degradation and decreased FN and TGF-β1 expression in GMCs under both basal and AGEs-treated conditions. USP22 depletion enhanced Sirt1 degradation and displayed combined effects with AGEs to further promote FN and TGF-β1 expression. RAGE functioned crucial mediating roles in these processes via its C-terminal cytosolic domain. Inhibiting Sirt1 by EX-527 substantially suppressed the down-regulation of FN and TGF-β1 resulting from USP22 overexpression under both normal and AGEs-treated conditions, eventually leading to their up-regulation in GMCs. These results indicated that the AGEs-RAGE system increased the ubiquitination and subsequent proteasome-mediated degradation of Sirt1 by reducing USP22 level, and AGEs-RAGE-USP22-Sirt1 formed a cascade pathway that regulated FN and TGF-β1 level, which participated in the pathological progression of diabetic nephropathy.

The E3 ubiquitin ligase NEDD4 enhances killing of membrane-perturbing intracellular bacteria by promoting autophagy

PubMed Central

Pei, Gang; Buijze, Hellen; Liu, Haipeng; Moura-Alves, Pedro; Goosmann, Christian; Brinkmann, Volker; Kawabe, Hiroshi; Dorhoi, Anca; Kaufmann, Stefan H. E.

2017-01-01

ABSTRACT The E3 ubiquitin ligase NEDD4 has been intensively studied in processes involved in viral infections, such as virus budding. However, little is known about its functions in bacterial infections. Our investigations into the role of NEDD4 in intracellular bacterial infections demonstrate that Mycobacterium tuberculosis and Listeria monocytogenes, but not Mycobacterium bovis BCG, replicate more efficiently in NEDD4 knockdown macrophages. In parallel, NEDD4 knockdown or knockout impaired basal macroautophagy/autophagy, as well as infection-induced autophagy. Conversely, NEDD4 expression promoted autophagy in an E3 catalytic activity-dependent manner, thereby restricting intracellular Listeria replication. Mechanistic studies uncovered that endogenous NEDD4 interacted with BECN1/Beclin 1 and this interaction increased during Listeria infection. Deficiency of NEDD4 resulted in elevated K48-linkage ubiquitination of endogenous BECN1. Further, NEDD4 mediated K6- and K27- linkage ubiquitination of BECN1, leading to elevated stability of BECN1 and increased autophagy. Thus, NEDD4 participates in killing of intracellular bacterial pathogens via autophagy by sustaining the stability of BECN1. PMID:29251248

SKP2- and OTUD1-regulated non-proteolytic ubiquitination of YAP promotes YAP nuclear localization and activity.

PubMed

Yao, Fan; Zhou, Zhicheng; Kim, Jongchan; Hang, Qinglei; Xiao, Zhenna; Ton, Baochau N; Chang, Liang; Liu, Na; Zeng, Liyong; Wang, Wenqi; Wang, Yumeng; Zhang, Peijing; Hu, Xiaoyu; Su, Xiaohua; Liang, Han; Sun, Yutong; Ma, Li

2018-06-11

Dysregulation of YAP localization and activity is associated with pathological conditions such as cancer. Although activation of the Hippo phosphorylation cascade is known to cause cytoplasmic retention and inactivation of YAP, emerging evidence suggests that YAP can be regulated in a Hippo-independent manner. Here, we report that YAP is subject to non-proteolytic, K63-linked polyubiquitination by the SCF SKP2 E3 ligase complex (SKP2), which is reversed by the deubiquitinase OTUD1. The non-proteolytic ubiquitination of YAP enhances its interaction with its nuclear binding partner TEAD, thereby inducing YAP's nuclear localization, transcriptional activity, and growth-promoting function. Independently of Hippo signaling, mutation of YAP's K63-linkage specific ubiquitination sites K321 and K497, depletion of SKP2, or overexpression of OTUD1 retains YAP in the cytoplasm and inhibits its activity. Conversely, overexpression of SKP2 or loss of OTUD1 leads to nuclear localization and activation of YAP. Altogether, our study sheds light on the ubiquitination-mediated, Hippo-independent regulation of YAP.

Ubiquitin conjugating enzyme E2-N and sequestosome-1 (p62) are components of the ubiquitination process mediated by the malin-laforin E3-ubiquitin ligase complex.

PubMed

Sánchez-Martín, Pablo; Romá-Mateo, Carlos; Viana, Rosa; Sanz, Pascual

2015-12-01

Lafora disease (LD, OMIM254780, ORPHA501) is a rare neurodegenerative form of epilepsy related to mutations in two proteins: laforin, a dual specificity phosphatase, and malin, an E3-ubiquitin ligase. Both proteins form a functional complex, where laforin recruits specific substrates to be ubiquitinated by malin. However, little is known about the mechanism driving malin-laforin mediated ubiquitination of its substrates. In this work we present evidence indicating that the malin-laforin complex interacts physically and functionally with the ubiquitin conjugating enzyme E2-N (UBE2N). This binding determines the topology of the chains that the complex is able to promote in the corresponding substrates (mainly K63-linked polyubiquitin chains). In addition, we demonstrate that the malin-laforin complex interacts with the selective autophagy adaptor sequestosome-1 (p62). Binding of p62 to the malin-laforin complex allows its recognition by LC3, a component of the autophagosomal membrane. In addition, p62 enhances the ubiquitinating activity of the malin-laforin E3-ubiquitin ligase complex. These data enrich our knowledge on the mechanism of action of the malin-laforin complex as an E3-ubiquitin ligase and reinforces the role of this complex in targeting substrates toward the autophagy pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

Riplet/RNF135, a RING finger protein, ubiquitinates RIG-I to promote interferon-beta induction during the early phase of viral infection.

PubMed

Oshiumi, Hiroyuki; Matsumoto, Misako; Hatakeyama, Shigetsugu; Seya, Tsukasa

2009-01-09

RIG-I (retinoic acid-inducible gene-I), a cytoplasmic RNA helicase, interacts with IPS-1/MAVS/Cardif/VISA, a protein on the outer membrane of mitochondria, to signal the presence of virus-derived RNA and induce type I interferon production. Activation of RIG-I requires the ubiquitin ligase, TRIM25, which mediates lysine 63-linked polyubiquitination of the RIG-I N-terminal CARD-like region. However, how this modification proceeds for activation of IPS-1 by RIG-I remains unclear. Here we identify an alternative factor, Riplet/RNF135, that promotes RIG-I activation independent of TRIM25. The Riplet/RNF135 protein consists of an N-terminal RING finger domain, C-terminal SPRY and PRY motifs, and shows sequence similarity to TRIM25. Immunoprecipitation analyses demonstrated that the C-terminal helicase and repressor domains of RIG-I interact with the Riplet/RNF135 C-terminal region, whereas the CARD-like region of RIG-I is dispensable for this interaction. Riplet/RNF135 promotes lysine 63-linked polyubiquitination of the C-terminal region of RIG-I, modification of which differs from the N-terminal ubiquitination by TRIM25. Overexpression and knockdown analyses revealed that Riplet/RNF135 promotes RIG-I-mediated interferon-beta promoter activation and inhibits propagation of the negative-strand RNA virus, vesicular stomatitis virus. Our data suggest that Riplet/RNF135 is a novel factor of the RIG-I pathway that is involved in the evoking of human innate immunity against RNA virus infection, and activates RIG-I through ubiquitination of its C-terminal region. We infer that a variety of RIG-I-ubiquitinating molecular complexes sustain RIG-I activation to modulate RNA virus replication in the cytoplasm.

The mechanism of linkage-specific ubiquitin chain elongation by a single-subunit E2

PubMed Central

Wickliffe, Katherine E.; Lorenz, Sonja; Wemmer, David E.; Kuriyan, John; Rape, Michael

2011-01-01

Ubiquitin chains of different topologies trigger distinct functional consequences, including protein degradation and reorganization of complexes. The assembly of most ubiquitin chains is promoted by E2s, yet how these enzymes achieve linkage specificity is poorly understood. We have discovered that the K11-specific Ube2S orients the donor ubiquitin through an essential non-covalent interaction that occurs in addition to the thioester bond at the E2 active site. The E2-donor ubiquitin complex transiently recognizes the acceptor ubiquitin, primarily through electrostatic interactions. The recognition of the acceptor ubiquitin surface around Lys11, but not around other lysines, generates a catalytically competent active site, which is composed of residues of both Ube2S and ubiquitin. Our studies suggest that monomeric E2s promote linkage-specific ubiquitin chain formation through substrate-assisted catalysis. PMID:21376237

BAG3 Expression in Glioblastoma Cells Promotes Accumulation of Ubiquitinated Clients in an Hsp70-dependent Manner*

PubMed Central

Gentilella, Antonio; Khalili, Kamel

2011-01-01

Disposal of damaged proteins and protein aggregates is a prerequisite for the maintenance of cellular homeostasis and impairment of this disposal can lead to a broad range of pathological conditions, most notably in brain-associated disorders including Parkinson and Alzheimer diseases, and cancer. In this respect, the Protein Quality Control (PQC) pathway plays a central role in the clearance of damaged proteins. The Hsc/Hsp70-co-chaperone BAG3 has been described as a new and critical component of the PQC in several cellular contexts. For example, the expression of BAG3 in the rodent brain correlates with the engagement of protein degradation machineries in response to proteotoxic stress. Nevertheless, little is known about the molecular events assisted by BAG3. Here we show that ectopic expression of BAG3 in glioblastoma cells leads to the activation of an HSF1-driven stress response, as attested by transcriptional activation of BAG3 and Hsp70. BAG3 overexpression determines an accumulation of ubiquitinated proteins and this event requires the N-terminal region, WW domain of BAG3 and the association of BAG3 with Hsp70. The ubiquitination mainly occurs on BAG3-client proteins and the inhibition of proteasomal activity results in a further accumulation of ubiquitinated clients. At the cellular level, overexpression of BAG3 in glioblastoma cell lines, but not in non-glial cells, results in a remarkable decrease in colony formation capacity and this effect is reverted when the binding of BAG3 to Hsp70 is impaired. These observations provide the first evidence for an involvement of BAG3 in the ubiquitination and turnover of its partners. PMID:21233200

Proteostasis regulation by the ubiquitin system.

PubMed

Bett, John S

2016-10-15

Cells have developed an evolutionary obligation to survey and maintain proteome fidelity and avoid the possible toxic consequences of protein misfolding and aggregation. Disturbances to protein homoeostasis (proteostasis) can result in severe cellular phenotypes and are closely linked with the accumulation of microscopically visible deposits of aggregated proteins. These include inclusion bodies found in AD (Alzheimer's disease), HD (Huntington's disease) and ALS (amyotrophic lateral sclerosis) patient neurons. Protein aggregation is intimately linked with the ubiquitin and ubiquitin-like post-translational modifier system, which manages cellular protein folding stress and promotes the restoration of proteostasis. This is achieved in large part through the action of the UPS (ubiquitin-proteasome system), which is responsible for directing the proteasomal destruction of misfolded and damaged proteins tagged with ubiquitin chains. There are other less well understood ways in which ubiquitin family members can help to maintain proteostasis that complement, but are independent of, the UPS. This article discusses our current understanding of how the ubiquitin family regulates the protein misfolding pathways that threaten proteome fidelity, and how this is achieved by the key players in this process. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Armadillo Repeat Containing 8α Binds to HRS and Promotes HRS Interaction with Ubiquitinated Proteins

PubMed Central

Tomaru, Koji; Ueda, Atsuhisa; Suzuki, Takeyuki; Kobayashi, Nobuaki; Yang, Jun; Yamamoto, Masaki; Takeno, Mitsuhiro; Kaneko, Takeshi; Ishigatsubo, Yoshiaki

2010-01-01

Recently, we reported that a complex with an essential role in the degradation of Fructose-1,6-bisphosphatase in yeast is well conserved in mammalian cells; we named this mammalian complex C-terminal to the Lissencephaly type-1-like homology (CTLH) complex. Although the function of the CTLH complex remains unclear, here we used yeast two-hybrid screening to isolate Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) as a protein binding to a key component of CTLH complex, Armadillo repeat containing 8 (ARMc8) α. The association was confirmed by a yeast two-hybrid assay and a co-immunoprecipitation assay. The proline-rich domain of HRS was essential for the association. As demonstrated through immunofluorescence microscopy, ARMc8α co-localized with HRS. ARMc8α promoted the interaction of HRS with various ubiquitinated proteins through the ubiquitin-interacting motif. These findings suggest that HRS mediates protein endosomal trafficking partly through its interaction with ARMc8α. PMID:20224683

Ubiquitin modifications

PubMed Central

Swatek, Kirby N; Komander, David

2016-01-01

Protein ubiquitination is a dynamic multifaceted post-translational modification involved in nearly all aspects of eukaryotic biology. Once attached to a substrate, the 76-amino acid protein ubiquitin is subjected to further modifications, creating a multitude of distinct signals with distinct cellular outcomes, referred to as the 'ubiquitin code'. Ubiquitin can be ubiquitinated on seven lysine (Lys) residues or on the N-terminus, leading to polyubiquitin chains that can encompass complex topologies. Alternatively or in addition, ubiquitin Lys residues can be modified by ubiquitin-like molecules (such as SUMO or NEDD8). Finally, ubiquitin can also be acetylated on Lys, or phosphorylated on Ser, Thr or Tyr residues, and each modification has the potential to dramatically alter the signaling outcome. While the number of distinctly modified ubiquitin species in cells is mind-boggling, much progress has been made to characterize the roles of distinct ubiquitin modifications, and many enzymes and receptors have been identified that create, recognize or remove these ubiquitin modifications. We here provide an overview of the various ubiquitin modifications present in cells, and highlight recent progress on ubiquitin chain biology. We then discuss the recent findings in the field of ubiquitin acetylation and phosphorylation, with a focus on Ser65-phosphorylation and its role in mitophagy and Parkin activation. PMID:27012465

An ubiquitin-binding molecule can work as an inhibitor of ubiquitin processing enzymes and ubiquitin receptors.

PubMed

Nguyen, Thanh; Ho, Minh; Ghosh, Ambarnil; Kim, Truc; Yun, Sun Il; Lee, Seung Seo; Kim, Kyeong Kyu

2016-10-07

The ubiquitin pathway plays a critical role in regulating diverse biological processes, and its dysregulation is associated with various diseases. Therefore, it is important to have a tool that can control the ubiquitin pathway in order to improve understanding of this pathway and to develop therapeutics against relevant diseases. We found that Chicago Sky Blue 6B binds directly to the β-groove, a major interacting surface of ubiquitin. Hence, it could successfully inhibit the enzymatic activity of ubiquitin processing enzymes and the binding of ubiquitin to the CXCR4, a cell surface ubiquitin receptor. Furthermore, we demonstrated that this ubiquitin binding chemical could effectively suppress the ubiquitin induced cancer cell migration by blocking ubiquitin-CXCR4 interaction. Current results suggest that ubiquitin binding molecules can be developed as inhibitors of ubiquitin-protein interactions, which will have the value not only in unveiling the biological role of ubiquitin but also in treating related diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Ufd2p synthesizes branched ubiquitin chains to promote the degradation of substrates modified with atypical chains

PubMed Central

Liu, Chao; Liu, Weixiao; Ye, Yihong; Li, Wei

2017-01-01

Ubiquitination of a subset of proteins by ubiquitin chain elongation factors (E4), represented by Ufd2p in Saccharomyces cerevisiae, is a pivotal regulator for many biological processes. However, the mechanism of Ufd2p-mediated ubiquitination is largely unclear. Here, we show that Ufd2p catalyses K48-linked multi-monoubiquitination on K29-linked ubiquitin chains assembled by the ubiquitin ligase (Ufd4p), resulting in branched ubiquitin chains. This reaction depends on the interaction of K29-linked ubiquitin chains with two N-terminal loops of Ufd2p. Only following the addition of K48-linked ubiquitin to substrates modified with K29-linked ubiquitin chains, can the substrates be escorted to the proteasome for degradation. We demonstrate that this ubiquitin chain linkage switching reaction is essential for ERAD, oleic acid and acid pH resistance in yeast. Thus, our results suggest that Ufd2p functions by switching ubiquitin chain linkages to allow the degradation of proteins modified with a ubiquitin linkage, which is normally not targeted to the proteasome. PMID:28165462

The mechanism of OTUB1-mediated inhibition of ubiquitination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wiener, Reuven; Zhang, Xiangbin; Wang, Tao

2013-04-08

Histones are ubiquitinated in response to DNA double-strand breaks (DSB), promoting recruitment of repair proteins to chromatin. UBC13 (also known as UBE2N) is a ubiquitin-conjugating enzyme (E2) that heterodimerizes with UEV1A (also known as UBE2V1) and synthesizes K63-linked polyubiquitin (K63Ub) chains at DSB sites in concert with the ubiquitin ligase (E3), RNF168 (ref. 3). K63Ub synthesis is regulated in a non-canonical manner by the deubiquitinating enzyme, OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), which binds preferentially to the UBC13-Ub thiolester. Residues amino-terminal to the OTU domain, which had been implicated in ubiquitin binding, are required for binding to UBC13-Ub andmore » inhibition of K63Ub synthesis. Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes. We unexpectedly find that OTUB1 binding to UBC13-Ub is allosterically regulated by free ubiquitin, which binds to a second site in OTUB1 and increases its affinity for UBC13-Ub, while at the same time disrupting interactions with UEV1A in a manner that depends on the OTUB1 N terminus. Crystal structures of an OTUB1-UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13-Ub conjugate show that binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13-Ub to OTUB1. The donor ubiquitin thus cannot interact with the E2 enzyme, which has been shown to be important for ubiquitin transfer. The N-terminal helix of OTUB1 is positioned to interfere with UEV1A binding to UBC13, as well as with attack on the thiolester by an acceptor ubiquitin, thereby inhibiting K63Ub synthesis. OTUB1 binding also occludes the RING E3 binding site on UBC13, thus providing a further component of inhibition. The general features of the inhibition mechanism explain how

Aggregation Pathways of Native-Like Ubiquitin Promoted by Single-Point Mutation, Metal Ion Concentration, and Dielectric Constant of the Medium.

PubMed

Fermani, Simona; Calvaresi, Matteo; Mangini, Vincenzo; Falini, Giuseppe; Bottoni, Andrea; Natile, Giovanni; Arnesano, Fabio

2018-03-15

Ubiquitin-positive protein aggregates are biomarkers of neurodegeneration, but the molecular mechanism responsible for their formation and accumulation is still unclear. Possible aggregation pathways of human ubiquitin (hUb) promoted by both intrinsic and extrinsic factors, are here investigated. By a computational analysis, two different hUb dimers are indicated as possible precursors of amyloid-like structures, but their formation is disfavored by an electrostatic repulsion involving Glu16 and other carboxylate residues present at the dimer interface. Experimental data on the E16V mutant of hUb shows that this single-point mutation, although not affecting the overall protein conformation, promotes protein aggregation. It is sufficient to shift the same mutation by only two residues (E18V) to regain the behavior of wild-type hUb. The neutralization of Glu16 negative charge by a metal ion and a decrease of the dielectric constant of the medium by addition of trifluoroethanol (TFE), also promote hUb aggregation. The outcomes of this research have important implications for the prediction of physiological parameters that favor aggregate formation. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex

DOE PAGES

Brown, Nicholas G.; VanderLinden, Ryan; Watson, Edmond R.; ...

2015-03-30

For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2~Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaboratesmore » with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING–E2~Ub catalytic modules such as APC11–UBCH10~Ub collide with distally tethered disordered substrates remains poorly understood. In this paper, we report structural mechanisms of UBCH10 recruitment to APC CDH1 and substrate ubiquitination. Unexpectedly, in addition to binding APC11’s RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APC CDH1–UBCH10~Ub–substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin–RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin–RING–E2 interactions establish APC’s specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. Finally, we propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3–E2~Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.« less

Functional characterization of Anaphase Promoting Complex/Cyclosome (APC/C) E3 ubiquitin ligases in tumorigenesis

PubMed Central

Zhang, Jinfang; Wan, Lixin; Dai, Xiangpeng; Sun, Yi; Wei, Wenyi

2014-01-01

The Anaphase Promoting Complex/Cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that primarily governs cell cycle progression. APC/C is composed of at least 14 core subunits and recruits its substrates for ubiquitination via one of the two adaptor proteins, Cdc20 or Cdh1, in M or M/early G1 phase, respectively. Furthermore, recent studies have shed light on crucial functions for APC/C in maintaining genomic integrity, neuronal differentiation, cellular metabolism and tumorigenesis. To gain better insight into the in vivo physiological functions of APC/C in regulating various cellular processes, particularly development and tumorigenesis, a number of mouse models of APC/C core subunits, coactivators or inhibitors have been established and characterized. However, due to their essential role in cell cycle regulation, most of the germline knockout mice targeting the APC/C pathway are embryonic lethal, indicating the need for generating conditional knockout mouse models to assess the role in tumorigenesis for each APC/C signaling component in specific tissues. In this review, we will first provide a brief introduction of the ubiquitin-proteasome system (UPS) and the biochemical activities and cellular functions of the APC/C E3 ligase. We will then focus primarily on characterizing genetic mouse models used to understand the physiological roles of each APC/C signaling component in embryogenesis, cell proliferation, development and carcinogenesis. Finally, we discuss future research directions to further elucidate the physiological contributions of APC/C components during tumorigenesis and validate their potentials as a novel class of anti-cancer targets. PMID:24569229

The Ubiquitin Code in the Ubiquitin-Proteasome System and Autophagy.

PubMed

Kwon, Yong Tae; Ciechanover, Aaron

2017-11-01

The conjugation of the 76 amino acid protein ubiquitin to other proteins can alter the metabolic stability or non-proteolytic functions of the substrate. Once attached to a substrate (monoubiquitination), ubiquitin can itself be ubiquitinated on any of its seven lysine (Lys) residues or its N-terminal methionine (Met1). A single ubiquitin polymer may contain mixed linkages and/or two or more branches. In addition, ubiquitin can be conjugated with ubiquitin-like modifiers such as SUMO or small molecules such as phosphate. The diverse ways to assemble ubiquitin chains provide countless means to modulate biological processes. We overview here the complexity of the ubiquitin code, with an emphasis on the emerging role of linkage-specific degradation signals (degrons) in the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system (hereafter autophagy). Copyright © 2017 Elsevier Ltd. All rights reserved.

cAMP signalling decreases p300 protein levels by promoting its ubiquitin/proteasome dependent degradation via Epac and p38 MAPK in lung cancer cells.

PubMed

Jeong, Min-Jae; Kim, Eui-Jun; Cho, Eun-Ah; Ye, Sang-Kyu; Kang, Gyeong Hoon; Juhnn, Yong-Sung

2013-05-02

The transcriptional coactivator p300 functions as a histone acetyltransferase and a scaffold for transcription factors. We investigated the effect of cAMP signalling on p300 expression. The activation of cAMP signalling by the expression of constitutively active Gαs or by treatment with isoproterenol decreased the p300 protein expression in lung cancer cells. Isoproterenol promoted the ubiquitination and subsequent proteasomal degradation of p300 in an Epac-dependent manner. Epac promoted p300 degradation by inhibiting the activity of p38 MAPK. It is concluded that cAMP signalling decreases the level of the p300 protein by promoting its ubiquitin-proteasome dependent degradation, which is mediated by Epac and p38 MAPK, in lung cancer cells. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Ubiquitin Proteasome System in Parkinson Disease: a keeper or a witness?

PubMed Central

Martins-Branco, Diogo; Esteves, Ana R.; Santos, Daniel; Arduino, Daniela M.; Swerdlow, Russell H.; Oliveira, Catarina R.; Januario, Cristina; Cardoso, Sandra M.

2014-01-01

Objective The aim of this work was to evaluate the role of Ubiquitin-Proteasome System (UPS) on mitochondrial-driven alpha-synuclein (aSN) clearance in in vitro, ex vivo and in vivo Parkinson disease (PD) cellular models. Method We used SH-SY5Y ndufa2 knock-down (KD) cells, PD cybrids and peripheral blood mononuclear cells (PBMC) from patients meeting the diagnostic criteria for PD. We quantified aSN aggregation, proteasome activity and protein ubiquitination levels. In PBMC of PD patients population we evaluated aSN levels in plasma and the influence of several demographic characteristics in the above mentioned determinations. Results We found that ubiquitin-independent proteasome activity was up-regulated in SH-SY5Y ndufa2 KD cells while a down regulation was observed in PD cybrids and PBMC. Moreover, we observed an increase in protein ubiquitination that correlates with a decrease in ubiquitin-dependent proteasome activity. Accordingly, proteasome inhibition prevented ubiquitin-dependent aSN clearance. Ubiquitin-independent proteasome activity was positively correlated with ubiquitination in PBMC. We also report a negative correlation of chymotrypsin-like activity with age in control and late-onset PD groups. Total ubiquitin content is positively correlated with aSN oligomers levels, which leads to an age-dependent increase of aSN ubiquitination in LOPD. Moreover, aSN levels are increased in the plasma of PD patients. Interpretation aSN oligomers are ubiquitinated and we identified an ubiquitin-dependent clearance insufficiency with accumulation of both aSN and ubiquitin. However, SH-SY5Y ndufa2 KD cells showed a significant up-regulation of ubiquitin-independent proteasomal enzymatic activity that could mean a cell rescue attempt. Moreover, we identified that UPS function is age-dependent in PBMC. PMID:22921536

Ubiquitin proteasome system in Parkinson's disease: a keeper or a witness?

PubMed

Martins-Branco, Diogo; Esteves, Ana R; Santos, Daniel; Arduino, Daniela M; Swerdlow, Russell H; Oliveira, Catarina R; Januario, Cristina; Cardoso, Sandra M

2012-12-01

The aim of this work was to evaluate the role of ubiquitin-proteasome system (UPS) on mitochondrial-driven alpha-synuclein (aSN) clearance in in vitro, ex vivo and in vivo Parkinson's disease (PD) cellular models. We used SH-SY5Y ndufa2 knock-down (KD) cells, PD cybrids and peripheral blood mononuclear cells (PBMC) from patients meeting the diagnostic criteria for PD. We quantified aSN aggregation, proteasome activity and protein ubiquitination levels. In PBMC of PD patient population we evaluated the aSN levels in the plasma and the influence of several demographic characteristics in the above mentioned determinations. We found that ubiquitin-independent proteasome activity was up-regulated in SH-SY5Y ndufa2 KD cells while a downregulation was observed in PD cybrids and PBMC. Moreover, we observed an increase in protein ubiquitination that correlates with a decrease in ubiquitin-dependent proteasome activity. Accordingly, proteasome inhibition prevented ubiquitin-dependent aSN clearance. Ubiquitin-independent proteasome activity was positively correlated with ubiquitination in PBMC. We also report a negative correlation of chymotrypsin-like activity with age in control and late-onset PD groups. Total ubiquitin content is positively correlated with aSN oligomer levels, which leads to an age-dependent increase of aSN ubiquitination in LOPD. Moreover, aSN levels are increased in the plasma of PD patients. aSN oligomers are ubiquitinated and we identified a ubiquitin-dependent clearance insufficiency with the accumulation of both aSN and ubiquitin. However, SH-SY5Y ndufa2 KD cells showed a significant up-regulation of ubiquitin-independent proteasomal enzymatic activity that could mean a cell rescue attempt. Moreover, we identified that UPS function is age-dependent in PBMC. Copyright © 2012 Elsevier Inc. All rights reserved.

Phospho-ubiquitin: upending the PINK–Parkin–ubiquitin cascade

PubMed Central

Matsuda, Noriyuki

2016-01-01

Mitochondria with decreased membrane potential are characterized by defects in protein import into the matrix and impairments in high-efficiency synthesis of ATP. These low-quality mitochondria are marked with ubiquitin for selective degradation. Key factors in this mechanism are PTEN-induced putative kinase 1 (PINK1, a mitochondrial kinase) and Parkin (a ubiquitin ligase), disruption of which has been implicated in predisposition to Parkinson’s disease. Previously, the clearance of damaged mitochondria had been thought to be the end result of a simple cascading reaction of PINK1–Parkin–ubiquitin. However, in the past year, several research groups including ours unexpectedly revealed that Parkin regulation is mediated by PINK1-dependent phosphorylation of ubiquitin. These results overturned the simple hierarchy that posited PINK1 and ubiquitin as the upstream and downstream factors of Parkin, respectively. Although ubiquitylation is well-known as a post-translational modification, it has recently become clear that ubiquitin itself can be modified, and that this modification unexpectedly converts ubiquitin to a factor that functions in retrograde signalling. PMID:26839319

A decrease in ubiquitination and resulting prolonged life-span of KIT underlies the KIT overexpression-mediated imatinib resistance of KIT mutation-driven canine mast cell tumor cells.

PubMed

Kobayashi, Masato; Kuroki, Shiori; Kurita, Sena; Miyamoto, Ryo; Tani, Hiroyuki; Tamura, Kyoichi; Bonkobara, Makoto

2017-10-01

Overexpression of KIT is one of the mechanisms that contributes to imatinib resistance in KIT mutation-driven tumors. Here, the mechanism underlying this overexpression of KIT was investigated using an imatinib-sensitive canine mast cell tumor (MCT) line CoMS, which has an activating mutation in KIT exon 11. A KIT-overexpressing imatinib-resistant subline, rCoMS1, was generated from CoMS cells by their continuous exposure to increasing concentrations of imatinib. Neither a secondary mutation nor upregulated transcription of KIT was detected in rCoMS1 cells. A decrease in KIT ubiquitination, a prolonged KIT life-span, and KIT overexpression were found in rCoMS1 cells. These events were suppressed by withdrawal of imatinib and were re-induced by re‑treatment with imatinib. These findings suggest that imatinib elicited overexpression of KIT via suppression of its ubiquitination. These results also indicated that imatinib-induced overexpression of KIT in rCoMS1 cells was not a permanently acquired feature but was a reversible response of the cells. Moreover, the pan deubiquitinating enzyme inhibitor PR619 prevented imatinib induction of KIT overexpression, suggesting that the imatinib-induced decrease in KIT ubiquitination could be mediated by upregulation and/or activation of deubiquitinating enzyme(s). It may be possible that a similar mechanism of KIT overexpression underlies the acquisition of imatinib resistance in some human tumors that are driven by KIT mutation.

The Anaphase-Promoting Complex (APC) ubiquitin ligase affects chemosensory behavior in C. elegans.

PubMed

Wang, Julia; Jennings, Alexandra K; Kowalski, Jennifer R

2016-01-01

The regulation of fundamental aspects of neurobiological function has been linked to the ubiquitin signaling system (USS), which regulates the degradation and activity of proteins and is catalyzed by E1, E2, and E3 enzymes. The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that controls diverse developmental and signaling processes in post-mitotic neurons; however, potential roles for the APC in sensory function have yet to be explored. In this study, we examined the effect of the APC ubiquitin ligase on chemosensation in Caenorhabditis elegans by testing chemotaxis to the volatile odorants, diacetyl, pyrazine, and isoamyl alcohol, to which wild-type worms are attracted. Animals with loss of function mutations in either of two alleles (g48 and ye143) of the gene encoding the APC subunit EMB-27 APC6 showed increased chemotaxis towards diacetyl and pyrazine, odorants sensed by AWA neurons, but exhibited normal chemotaxis to isoamyl alcohol, which is sensed by AWC neurons. The statistically significant increase in chemotaxis in the emb-27 APC6 mutants suggests that the APC inhibits AWA-mediated chemosensation in C. elegans. Increased chemotaxis to pyrazine was also seen with mutants lacking another essential APC subunit, MAT-2 APC1; however, mat-2 APC1 mutants exhibited wild type responses to diacetyl. The difference in responsiveness of these two APC subunit mutants may be due to differential strength of these hypomorphic alleles or may indicate the presence of functional sub-complexes of the APC at work in this process. These findings are the first evidence for APC-mediated regulation of chemosensation and lay the groundwork for further studies aimed at identifying the expression levels, function, and targets of the APC in specific sensory neurons. Because of the similarity between human and C. elegans nervous systems, the role of the APC in sensory neurons may also advance our understanding of human sensory function and disease.

Calmodulin promotes matrix metalloproteinase 9 production and cell migration by inhibiting the ubiquitination and degradation of TBC1D3 oncoprotein in human breast cancer cells.

PubMed

Zhao, Huzi; Zhang, Lina; Zhang, Yongchen; Zhao, Lei; Wan, Qing; Wang, Bei; Bu, Xiaodong; Wan, Meiling; Shen, Chuanlu

2017-05-30

The hominoid oncoprotein TBC1D3 enhances growth factor (GF) signaling and GF signaling, conversely, induces the ubiquitination and subsequent degradation of TBC1D3. However, little is known regarding the regulation of this degradation, and the role of TBC1D3 in the progression of tumors has also not been defined. In the present study, we demonstrated that calmodulin (CaM), a ubiquitous cellular calcium sensor, specifically interacted with TBC1D3 in a Ca2+-dependent manner and inhibited GF signaling-induced ubiquitination and degradation of the oncoprotein in both cytoplasm and nucleus of human breast cancer cells. The CaM-interacting site of TBC1D3 was mapped to amino acids 157~171, which comprises two 1-14 hydrophobic motifs and one lysine residue (K166). Deletion of these motifs was shown to abolish interaction between TBC1D3 and CaM. Surprisingly, this deletion mutation caused inability of GF signaling to induce the ubiquitination and subsequent degradation of TBC1D3. In agreement with this, we identified lysine residue 166 within the CaM-interacting motifs of TBC1D3 as the actual site for the GF signaling-induced ubiquitination using mutational analysis. Point mutation of this lysine residue exhibited the same effect on TBC1D3 as the deletion mutant, suggesting that CaM inhibits GF signaling-induced degradation of TBC1D3 by occluding its ubiquitination at K166. Notably, we found that TBC1D3 promoted the expression and activation of MMP-9 and the migration of MCF-7 cells. Furthermore, interaction with CaM considerably enhanced such effect of TBC1D3. Taken together, our work reveals a novel model by which CaM promotes cell migration through inhibiting the ubiquitination and degradation of TBC1D3.

Phospho-ubiquitin: upending the PINK-Parkin-ubiquitin cascade.

PubMed

Matsuda, Noriyuki

2016-04-01

Mitochondria with decreased membrane potential are characterized by defects in protein import into the matrix and impairments in high-efficiency synthesis of ATP. These low-quality mitochondria are marked with ubiquitin for selective degradation. Key factors in this mechanism are PTEN-induced putative kinase 1 (PINK1, a mitochondrial kinase) and Parkin (a ubiquitin ligase), disruption of which has been implicated in predisposition to Parkinson's disease. Previously, the clearance of damaged mitochondria had been thought to be the end result of a simple cascading reaction of PINK1-Parkin-ubiquitin. However, in the past year, several research groups including ours unexpectedly revealed that Parkin regulation is mediated by PINK1-dependent phosphorylation of ubiquitin. These results overturned the simple hierarchy that posited PINK1 and ubiquitin as the upstream and downstream factors of Parkin, respectively. Although ubiquitylation is well-known as a post-translational modification, it has recently become clear that ubiquitin itself can be modified, and that this modification unexpectedly converts ubiquitin to a factor that functions in retrograde signalling. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Auto-ubiquitination of Mdm2 Enhances Its Substrate Ubiquitin Ligase Activity*

PubMed Central

Ranaweera, Ruchira S.; Yang, Xiaolu

2013-01-01

The RING domain E3 ubiquitin ligase Mdm2 is the master regulator of the tumor suppressor p53. It targets p53 for proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Like most E3 ligases, Mdm2 can also ubiquitinate itself. How Mdm2 auto-ubiquitination may influence its substrate ubiquitin ligase activity is undefined. Here we show that auto-ubiquitination of Mdm2 is an activating event. Mdm2 that has been conjugated to polyubiquitin chains, but not to single ubiquitins, exhibits substantially enhanced activity to polyubiquitinate p53. Mechanistically, auto-ubiquitination of Mdm2 facilitates the recruitment of the E2 ubiquitin-conjugating enzyme. This occurs through noncovalent interactions between the ubiquitin chains on Mdm2 and the ubiquitin binding domain on E2s. Mutations that diminish the noncovalent interactions render auto-ubiquitination unable to stimulate Mdm2 substrate E3 activity. These results suggest a model in which polyubiquitin chains on an E3 increase the local concentration of E2 enzymes and permit the processivity of substrate ubiquitination. They also support the notion that autocatalysis may be a prevalent mode for turning on the activity of latent enzymes. PMID:23671280

Ubiquitin C-Terminal Hydrolase L1 (UCH-L1) Promotes Hippocampus-Dependent Memory via Its Deubiquitinating Effect on TrkB.

PubMed

Guo, Yun-Yun; Lu, Yi; Zheng, Yuan; Chen, Xiao-Rong; Dong, Jun-Lu; Yuan, Rong-Rong; Huang, Shu-Hong; Yu, Hui; Wang, Yue; Chen, Zhe-Yu; Su, Bo

2017-06-21

Multiple studies have established that brain-derived neurotrophic factor (BDNF) plays a critical role in the regulation of synaptic plasticity via its receptor, TrkB. In addition to being phosphorylated, TrkB has also been demonstrated to be ubiquitinated. However, the mechanisms of TrkB ubiquitination and its biological functions remain poorly understood. In this study, we demonstrate that ubiquitin C-terminal hydrolase L1 (UCH-L1) promotes contextual fear conditioning learning and memory via the regulation of ubiquitination of TrkB. We provide evidence that UCH-L1 can deubiquitinate TrkB directly. K460 in the juxtamembane domain of TrkB is the primary ubiquitination site and is regulated by UCH-L1. By using a peptide that competitively inhibits the association between UCH-L1 and TrkB, we show that the blockade of UCH-L1-regulated TrkB deubiquitination leads to increased BDNF-induced TrkB internalization and consequently directs the internalized TrkB to the degradation pathway, resulting in increased degradation of surface TrkB and attenuation of TrkB activation and its downstream signaling pathways. Moreover, injection of the peptide into the DG region of mice impairs hippocampus-dependent memory. Together, our results suggest that the ubiquitination of TrkB is a mechanism that controls its downstream signaling pathways via the regulation of its endocytosis and postendocytic trafficking and that UCH-L1 mediates the deubiquitination of TrkB and could be a potential target for the modulation of hippocampus-dependent memory. SIGNIFICANCE STATEMENT Ubiquitin C-terminal hydrolase L1 (UCH-L1) has been demonstrated to play important roles in the regulation of synaptic plasticity and learning and memory. TrkB, the receptor for brain-derived neurotrophic factor, has also been shown to be a potent regulator of synaptic plasticity. In this study, we demonstrate that UCH-L1 functions as a deubiquitinase for TrkB. The blockage of UCH-L1-regulated deubiquitination of Trk

Ubiquitin fusion expression and tissue-dependent targeting of hG-CSF in transgenic tobacco

PubMed Central

2011-01-01

Background Human granulocyte colony-stimulating factor (hG-CSF) is an important human cytokine which has been widely used in oncology and infection protection. To satisfy clinical needs, expression of recombinant hG-CSF has been studied in several organisms, including rice cell suspension culture and transient expression in tobacco leaves, but there was no published report on its expression in stably transformed plants which can serve as a more economical expression platform with potential industrial application. Results In this study, hG-CSF expression was investigated in transgenic tobacco leaves and seeds in which the accumulation of hG-CSF could be enhanced through fusion with ubiquitin by up to 7 fold in leaves and 2 fold in seeds, leading to an accumulation level of 2.5 mg/g total soluble protein (TSP) in leaves and 1.3 mg/g TSP in seeds, relative to hG-CSF expressed without a fusion partner. Immunoblot analysis showed that ubiquitin was processed from the final protein product, and ubiquitination was up-regulated in all transgenic plants analyzed. Driven by CaMV 35S promoter and phaseolin signal peptide, hG-CSF was observed to be secreted into apoplast in leaves but deposited in protein storage vacuole (PSV) in seeds, indicating that targeting of the hG-CSF was tissue-dependent in transgenic tobacco. Bioactivity assay showed that hG-CSF expressed in both seeds and leaves was bioactive to support the proliferation of NFS-60 cells. Conclusions In this study, the expression of bioactive hG-CSF in transgenic plants was improved through ubiquitin fusion strategy, demonstrating that protein expression can be enhanced in both plant leaves and seeds through fusion with ubiquitin and providing a typical case of tissue-dependent expression of recombinant protein in transgenic plants. PMID:21985646

Protein tyrosine kinase regulation by ubiquitination: Critical roles of Cbl-family ubiquitin ligases

PubMed Central

Mohapatra, Bhopal; Ahmad, Gulzar; Nadeau, Scott; Zutshi, Neha; An, Wei; Scheffe, Sarah; Dong, Lin; Feng, Dan; Goetz, Benjamin; Arya, Priyanka; Bailey, Tameka A.; Palermo, Nicholas; Borgstahl, Gloria E.O.; Natarajan, Amarnath; Raja, Srikumar M.; Naramura, Mayumi; Band, Vimla; Band, Hamid

2012-01-01

Protein tyrosine kinases (PTKs) coordinate a broad spectrum of cellular responses to extracellular stimuli and cell–cell interactions during development, tissue homeostasis, and responses to environmental challenges. Thus, an understanding of the regulatory mechanisms that ensure physiological PTK function and potential aberrations of these regulatory processes during diseases such as cancer are of broad interest in biology and medicine. Aside from the expected role of phospho-tyrosine phosphatases, recent studies have revealed a critical role of covalent modification of activated PTKs with ubiquitin as a critical mechanism of their negative regulation. Members of the Cbl protein family (Cbl, Cbl-b and Cbl-c in mammals) have emerged as dominant “activated PTK-selective” ubiquitin ligases. Structural, biochemical and cell biological studies have established that Cbl protein-dependent ubiquitination targets activated PTKs for degradation either by facilitating their endocytic sorting into lysosomes or by promoting their proteasomal degradation. This mechanism also targets PTK signaling intermediates that become associated with Cbl proteins in a PTK activation-dependent manner. Cellular and animal studies have established that the relatively broadly expressed mammalian Cbl family members Cbl and Cbl-b play key physiological roles, including their critical functions to prevent the transition of normal immune responses into autoimmune disease and as tumor suppressors; the latter function has received validation from human studies linking mutations in Cbl to human leukemia. These newer insights together with embryonic lethality seen in mice with a combined deletion of Cbl and Cbl-b genes suggest an unappreciated role of the Cbl family proteins, and by implication the ubiquitin-dependent control of activated PTKs, in stem/progenitor cell maintenance. Future studies of existing and emerging animal models and their various cell lineages should help test the broader

A Perturbed Ubiquitin Landscape Distinguishes Between Ubiquitin in Trafficking and in Proteolysis*

PubMed Central

Ziv, Inbal; Matiuhin, Yulia; Kirkpatrick, Donald S.; Erpapazoglou, Zoi; Leon, Sebastien; Pantazopoulou, Marina; Kim, Woong; Gygi, Steven P.; Haguenauer-Tsapis, Rosine; Reis, Noa; Glickman, Michael H.; Kleifeld, Oded

2011-01-01

Any of seven lysine residues on ubiquitin can serve as the base for chain-extension, resulting in a sizeable spectrum of ubiquitin modifications differing in chain length or linkage type. By optimizing a procedure for rapid lysis, we charted the profile of conjugated cellular ubiquitin directly from whole cell extract. Roughly half of conjugated ubiquitin (even at high molecular weights) was nonextended, consisting of monoubiquitin modifications and chain terminators (endcaps). Of extended ubiquitin, the primary linkages were via Lys48 and Lys63. All other linkages were detected, contributing a relatively small portion that increased at lower molecular weights. In vivo expression of lysineless ubiquitin (K0 Ub) perturbed the ubiquitin landscape leading to elevated levels of conjugated ubiquitin, with a higher mono-to-poly ratio. Affinity purification of these trapped conjugates identified a comprehensive list of close to 900 proteins including novel targets. Many of the proteins enriched by K0 ubiquitination were membrane-associated, or involved in cellular trafficking. Prime among them are components of the ESCRT machinery and adaptors of the Rsp5 E3 ubiquitin ligase. Ubiquitin chains associated with these substrates were enriched for Lys63 linkages over Lys48, indicating that K0 Ub is unevenly distributed throughout the ubiquitinome. Biological assays validated the interference of K0 Ub with protein trafficking and MVB sorting, minimally affecting Lys48-dependent turnover of proteasome substrates. We conclude that despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery—the ubiquitin-proteasome system and the ubiquitin trafficking system—were unevenly perturbed by expression of K0 ubiquitin. PMID:21427232

RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination.

PubMed

Inano, Shojiro; Sato, Koichi; Katsuki, Yoko; Kobayashi, Wataru; Tanaka, Hiroki; Nakajima, Kazuhiro; Nakada, Shinichiro; Miyoshi, Hiroyuki; Knies, Kerstin; Takaori-Kondo, Akifumi; Schindler, Detlev; Ishiai, Masamichi; Kurumizaka, Hitoshi; Takata, Minoru

2017-06-01

RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype. Copyright © 2017 Elsevier Inc. All rights reserved.

Ubiquitin-Dependent Degradation of Mitochondrial Proteins Regulates Energy Metabolism.

PubMed

Lavie, Julie; De Belvalet, Harmony; Sonon, Sessinou; Ion, Ana Madalina; Dumon, Elodie; Melser, Su; Lacombe, Didier; Dupuy, Jean-William; Lalou, Claude; Bénard, Giovanni

2018-06-05

The ubiquitin proteasome system (UPS) regulates many cellular functions by degrading key proteins. Notably, the role of UPS in regulating mitochondrial metabolic functions is unclear. Here, we show that ubiquitination occurs in different mitochondrial compartments, including the inner mitochondrial membrane, and that turnover of several metabolic proteins is UPS dependent. We specifically detailed mitochondrial ubiquitination and subsequent UPS-dependent degradation of succinate dehydrogenase subunit A (SDHA), which occurred when SDHA was minimally involved in mitochondrial energy metabolism. We demonstrate that SDHA ubiquitination occurs inside the organelle. In addition, we show that the specific inhibition of SDHA degradation by UPS promotes SDHA-dependent oxygen consumption and increases ATP, malate, and citrate levels. These findings suggest that the mitochondrial metabolic machinery is also regulated by the UPS. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

The Ubiquitin Ligase RNF125 Targets Innate Immune Adaptor Protein TRIM14 for Ubiquitination and Degradation.

PubMed

Jia, Xue; Zhou, Hongli; Wu, Chao; Wu, Qiankun; Ma, Shichao; Wei, Congwen; Cao, Ye; Song, Jingdong; Zhong, Hui; Zhou, Zhuo; Wang, Jianwei

2017-06-15

Tripartite motif-containing 14 (TRIM14) is a mitochondrial adaptor that facilitates innate immune signaling. Upon virus infection, the expression of TRIM14 is significantly induced, which stimulates the production of type-I IFNs and proinflammatory cytokines. As excessive immune responses lead to harmful consequences, TRIM14-mediated signaling needs to be tightly balanced. In this study, we identify really interesting new gene-type zinc finger protein 125 (RNF125) as a negative regulator of TRIM14 in the innate antiviral immune response. Overexpression of RNF125 inhibits TRIM14-mediated antiviral response, whereas knockdown of RNF125 has the opposite effect. RNF125 interacts with TRIM14 and acts as an E3 ubiquitin ligase that catalyzes TRIM14 ubiquitination. RNF125 promotes K48-linked polyubiquitination of TRIM14 and mediates its degradation via the ubiquitin-proteasome pathway. Consequently, wild-type mouse embryonic fibroblasts show significantly reduced TRIM14 protein levels in late time points of viral infection, whereas TRIM14 protein is retained in RNF125-deficient mouse embryonic fibroblasts. Collectively, our data suggest that RNF125 plays a new role in innate immune response by regulating TRIM14 ubiquitination and degradation. Copyright © 2017 by The American Association of Immunologists, Inc.

Isolation and characterization of an ubiquitin extension protein gene (JcUEP) promoter from Jatropha curcas.

PubMed

Tao, Yan-Bin; He, Liang-Liang; Niu, Long-Jian; Xu, Zeng-Fu

2015-04-01

The JcUEP promoter is active constitutively in the bio-fuel plant Jatropha curcas , and is an alternative to the widely used CaMV35S promoter for driving constitutive overexpression of transgenes in Jatropha. Well-characterized promoters are required for transgenic breeding of Jatropha curcas, a biofuel feedstock with great potential for production of bio-diesel and bio-jet fuel. In this study, an ubiquitin extension protein gene from Jatropha, designated JcUEP, was identified to be ubiquitously expressed. Thus, we isolated a 1.2 kb fragment of the 5' flanking region of JcUEP and evaluated its activity as a constitutive promoter in Arabidopsis and Jatropha using the β-glucuronidase (GUS) reporter gene. As expected, histochemical GUS assay showed that the JcUEP promoter was active in all Arabidopsis and Jatropha tissues tested. We also compared the activity of the JcUEP promoter with that of the cauliflower mosaic virus 35S (CaMV35S) promoter, a well-characterized constitutive promoter conferring strong transgene expression in dicot species, in various tissues of Jatropha. In a fluorometric GUS assay, the two promoters showed similar activities in stems, mature leaves and female flowers; while the CaMV35S promoter was more effective than the JcUEP promoter in other tissues, especially young leaves and inflorescences. In addition, the JcUEP promoter retained its activity under stress conditions in low temperature, high salt, dehydration and exogenous ABA treatments. These results suggest that the plant-derived JcUEP promoter could be an alternative to the CaMV35S promoter for driving constitutive overexpression of transgenes in Jatropha and other plants.

Quantifying ubiquitin signaling.

PubMed

Ordureau, Alban; Münch, Christian; Harper, J Wade

2015-05-21

Ubiquitin (UB)-driven signaling systems permeate biology, and are often integrated with other types of post-translational modifications (PTMs), including phosphorylation. Flux through such pathways is dictated by the fractional stoichiometry of distinct modifications and protein assemblies as well as the spatial organization of pathway components. Yet, we rarely understand the dynamics and stoichiometry of rate-limiting intermediates along a reaction trajectory. Here, we review how quantitative proteomic tools and enrichment strategies are being used to quantify UB-dependent signaling systems, and to integrate UB signaling with regulatory phosphorylation events, illustrated with the PINK1/PARKIN pathway. A key feature of ubiquitylation is that the identity of UB chain linkage types can control downstream processes. We also describe how proteomic and enzymological tools can be used to identify and quantify UB chain synthesis and linkage preferences. The emergence of sophisticated quantitative proteomic approaches will set a new standard for elucidating biochemical mechanisms of UB-driven signaling systems. Copyright © 2015 Elsevier Inc. All rights reserved.

Quantifying Ubiquitin Signaling

PubMed Central

Ordureau, Alban; Münch, Christian; Harper, J. Wade

2015-01-01

Ubiquitin (UB)-driven signaling systems permeate biology, and are often integrated with other types of post-translational modifications (PTMs), most notably phosphorylation. Flux through such pathways is typically dictated by the fractional stoichiometry of distinct regulatory modifications and protein assemblies as well as the spatial organization of pathway components. Yet, we rarely understand the dynamics and stoichiometry of rate-limiting intermediates along a reaction trajectory. Here, we review how quantitative proteomic tools and enrichment strategies are being used to quantify UB-dependent signaling systems, and to integrate UB signaling with regulatory phosphorylation events. A key regulatory feature of ubiquitylation is that the identity of UB chain linkage types can control downstream processes. We also describe how proteomic and enzymological tools can be used to identify and quantify UB chain synthesis and linkage preferences. The emergence of sophisticated quantitative proteomic approaches will set a new standard for elucidating biochemical mechanisms of UB-driven signaling systems. PMID:26000850

Ubiquitin-dependent Regulation of Phospho-AKT Dynamics by the Ubiquitin E3 Ligase, NEDD4-1, in the Insulin-like Growth Factor-1 Response*

PubMed Central

Fan, Chuan-Dong; Lum, Michelle A.; Xu, Chao; Black, Jennifer D.; Wang, Xinjiang

2013-01-01

AKT is a critical effector kinase downstream of the PI3K pathway that regulates a plethora of cellular processes including cell growth, death, differentiation, and migration. Mechanisms underlying activated phospho-AKT (pAKT) translocation to its action sites remain unclear. Here we show that NEDD4-1 is a novel E3 ligase that specifically regulates ubiquitin-dependent trafficking of pAKT in insulin-like growth factor (IGF)-1 signaling. NEDD4-1 physically interacts with AKT and promotes HECT domain-dependent ubiquitination of exogenous and endogenous AKT. NEDD4-1 catalyzes K63-type polyubiquitin chain formation on AKT in vitro. Plasma membrane binding is the key step for AKT ubiquitination by NEDD4-1 in vivo. Ubiquitinated pAKT translocates to perinuclear regions, where it is released into the cytoplasm, imported into the nucleus, or coupled with proteasomal degradation. IGF-1 signaling specifically stimulates NEDD4-1-mediated ubiquitination of pAKT, without altering total AKT ubiquitination. A cancer-derived plasma membrane-philic mutant AKT(E17K) is more effectively ubiquitinated by NEDD4-1 and more efficiently trafficked into the nucleus compared with wild type AKT. This study reveals a novel mechanism by which a specific E3 ligase is required for ubiquitin-dependent control of pAKT dynamics in a ligand-specific manner. PMID:23195959

Aβ-Induced Synaptic Alterations Require the E3 Ubiquitin Ligase Nedd4-1.

PubMed

Rodrigues, Elizabeth M; Scudder, Samantha L; Goo, Marisa S; Patrick, Gentry N

2016-02-03

Alzheimer's disease (AD) is a neurodegenerative disease in which patients experience progressive cognitive decline. A wealth of evidence suggests that this cognitive impairment results from synaptic dysfunction in affected brain regions caused by cleavage of amyloid precursor protein into the pathogenic peptide amyloid-β (Aβ). Specifically, it has been shown that Aβ decreases surface AMPARs, dendritic spine density, and synaptic strength, and also alters synaptic plasticity. The precise molecular mechanisms by which this occurs remain unclear. Here we demonstrate a role for ubiquitination in Aβ-induced synaptic dysfunction in cultured rat neurons. We find that Aβ promotes the ubiquitination of AMPARs, as well as the redistribution and recruitment of Nedd4-1, a HECT E3 ubiquitin ligase we previously demonstrated to target AMPARs for ubiquitination and degradation. Strikingly, we show that Nedd4-1 is required for Aβ-induced reductions in surface AMPARs, synaptic strength, and dendritic spine density. Our findings, therefore, indicate an important role for Nedd4-1 and ubiquitin in the synaptic alterations induced by Aβ. Synaptic changes in Alzheimer's disease (AD) include surface AMPAR loss, which can weaken synapses. In a cell culture model of AD, we found that AMPAR loss correlates with increased AMPAR ubiquitination. In addition, the ubiquitin ligase Nedd4-1, known to ubiquitinate AMPARs, is recruited to synapses in response to Aβ. Strikingly, reducing Nedd4-1 levels in this model prevented surface AMPAR loss and synaptic weakening. These findings suggest that, in AD, Nedd4-1 may ubiquitinate AMPARs to promote their internalization and weaken synaptic strength, similar to what occurs in Nedd4-1's established role in homeostatic synaptic scaling. This is the first demonstration of Aβ-mediated control of a ubiquitin ligase to regulate surface AMPAR expression. Copyright © 2016 the authors 0270-6474/16/361590-06$15.00/0.

Human Papillomavirus Type 16 E6 Induces Self-Ubiquitination of the E6AP Ubiquitin-Protein Ligase

PubMed Central

Kao, Wynn H.; Beaudenon, Sylvie L.; Talis, Andrea L.; Huibregtse, Jon M.; Howley, Peter M.

2000-01-01

The E6 protein of the high-risk human papillomaviruses (HPVs) and the cellular ubiquitin-protein ligase E6AP form a complex which causes the ubiquitination and degradation of p53. We show here that HPV16 E6 promotes the ubiquitination and degradation of E6AP itself. The half-life of E6AP is shorter in HPV-positive cervical cancer cells than in HPV-negative cervical cancer cells, and E6AP is stabilized in HPV-positive cancer cells when expression of the viral oncoproteins is repressed. Expression of HPV16 E6 in cells results in a threefold decrease in the half-life of transfected E6AP. E6-mediated degradation of E6AP requires (i) the binding of E6 to E6AP, (ii) the catalytic activity of E6AP, and (iii) activity of the 26S proteasome, suggesting that E6-E6AP interaction results in E6AP self-ubiquitination and degradation. In addition, both in vitro and in vivo experiments indicate that E6AP self-ubiquitination results primarily from an intramolecular transfer of ubiquitin from the active-site cysteine to one or more lysine residues; however, intermolecular transfer can also occur in the context of an E6-mediated E6AP multimer. Finally, we demonstrate that an E6 mutant that is able to immortalize human mammary epithelial cells but is unable to degrade p53 retains its ability to bind and degrade E6AP, raising the possibility that E6-mediated degradation of E6AP contributes to its ability to transform mammalian cells. PMID:10864652

Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System*

PubMed Central

Singh, Rajesh K.; Zerath, Sylvia; Kleifeld, Oded; Scheffner, Martin; Glickman, Michael H.; Fushman, David

2012-01-01

Of all ubiquitin-like proteins, Rub1 (Nedd8 in mammals) is the closest kin of ubiquitin. We show via NMR that structurally, Rub1 and ubiquitin are fundamentally similar as well. Despite these profound similarities, the prevalence of Rub1/Nedd8 and of ubiquitin as modifiers of the proteome is starkly different, and their attachments to specific substrates perform different functions. Recently, some proteins, including p53, p73, EGFR, caspase-7, and Parkin, have been shown to be modified by both Rub1/Nedd8 and ubiquitin within cells. To understand whether and how it might be possible to distinguish among the same target protein modified by Rub1 or ubiquitin or both, we examined whether ubiquitin receptors can differentiate between Rub1 and ubiquitin. Surprisingly, Rub1 interacts with proteasome ubiquitin-shuttle proteins comparably to ubiquitin but binds more weakly to a proteasomal ubiquitin receptor Rpn10. We identified Rub1-ubiquitin heteromers in yeast and Nedd8-Ub heteromers in human cells. We validate that in human cells and in vitro, human Rub1 (Nedd8) forms chains with ubiquitin where it acts as a chain terminator. Interestingly, enzymatically assembled K48-linked Rub1-ubiquitin heterodimers are recognized by various proteasomal ubiquitin shuttles and receptors comparably to K48-linked ubiquitin homodimers. Furthermore, these heterologous chains are cleaved by COP9 signalosome or 26S proteasome. A derubylation function of the proteasome expands the repertoire of its enzymatic activities. In contrast, Rub1 conjugates may be somewhat resilient to the actions of other canonical deubiquitinating enzymes. Taken together, these findings suggest that once Rub1/Nedd8 is channeled into ubiquitin pathways, it is recognized essentially like ubiquitin. PMID:23105008

E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6

PubMed Central

Hibbert, Richard G.; Huang, Anding; Boelens, Rolf; Sixma, Titia K.

2011-01-01

In ubiquitin conjugation, different combinations of E2 and E3 enzymes catalyse either monoubiquitination or ubiquitin chain formation. The E2/E3 complex Rad6/Rad18 exclusively monoubiquitinates the proliferating cell nuclear antigen (PCNA) to signal for “error prone” DNA damage tolerance, whereas a different set of conjugation enzymes is required for ubiquitin chain formation on PCNA. Here we show that human E2 enzyme Rad6b is intrinsically capable of catalyzing ubiquitin chain formation. This activity is prevented during PCNA ubiquitination by the interaction of Rad6 with E3 enzyme Rad18. Using NMR and X-ray crystallography we show that the R6BD of Rad18 inhibits this activity by competing with ubiquitin for a noncovalent “backside” binding site on Rad6. Our findings provide mechanistic insights into how E3 enzymes can regulate the ubiquitin conjugation process. PMID:21422291

Parkin targets HIF-1α for ubiquitination and degradation to inhibit breast tumor progression.

PubMed

Liu, Juan; Zhang, Cen; Zhao, Yuhan; Yue, Xuetian; Wu, Hao; Huang, Shan; Chen, James; Tomsky, Kyle; Xie, Haiyang; Khella, Christen A; Gatza, Michael L; Xia, Dajing; Gao, Jimin; White, Eileen; Haffty, Bruce G; Hu, Wenwei; Feng, Zhaohui

2017-11-28

Mutations in E3 ubiquitin ligase Parkin have been linked to familial Parkinson's disease. Accumulating evidence suggests that Parkin is a tumor suppressor, but the underlying mechanism is poorly understood. Here we show that Parkin is an E3 ubiquitin ligase for hypoxia-inducible factor 1α (HIF-1α). Parkin interacts with HIF-1α and promotes HIF-1α degradation through ubiquitination, which in turn inhibits metastasis of breast cancer cells. Parkin downregulation in breast cancer cells promotes metastasis, which can be inhibited by targeting HIF-1α with RNA interference or the small-molecule inhibitor YC-1. We further identify lysine 477 (K477) of HIF-1α as a major ubiquitination site for Parkin. K477R HIF-1α mutation and specific cancer-associated Parkin mutations largely abolish the functions of Parkin to ubiquitinate HIF-1α and inhibit cancer metastasis. Importantly, Parkin expression is inversely correlated with HIF-1α expression and metastasis in breast cancer. Our results reveal an important mechanism for Parkin in tumor suppression and HIF-1α regulation.

Roles of mono-ubiquitinated Smad4 in the formation of Smad transcriptional complexes.

PubMed

Wang, Bei; Suzuki, Hiroyuki; Kato, Mitsuyasu

2008-11-14

TGF-beta activates receptor-regulated Smad (R-Smad) through phosphorylation by type I receptors. Activated R-Smad binds to Smad4 and the complex translocates into the nucleus and stimulates the transcription of target genes through association with co-activators including p300. It is not clear, however, how activated Smad complexes are removed from target genes. In this study, we show that TGF-beta enhances the mono-ubiquitination of Smad4. Smad4 mono-ubiquitination was promoted by p300 and suppressed by the c-Ski co-repressor. Smad4 mono-ubiquitination disrupted the interaction with Smad2 in the presence of constitutively active TGF-beta type I receptor. Furthermore, mono-ubiquitinated Smad4 was not found in DNA-binding Smad complexes. A Smad4-Ubiquitin fusion protein, which mimics mono-ubiquitinated Smad4, enhanced localization to the cytoplasm. These results suggest that mono-ubiquitination of Smad4 occurs in the transcriptional activator complex and facilitates the turnover of Smad complexes at target genes.

RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks.

PubMed

Elia, Andrew E H; Wang, David C; Willis, Nicholas A; Boardman, Alexander P; Hajdu, Ildiko; Adeyemi, Richard O; Lowry, Elizabeth; Gygi, Steven P; Scully, Ralph; Elledge, Stephen J

2015-10-15

We have used quantitative proteomics to profile ubiquitination in the DNA damage response (DDR). We demonstrate that RPA, which functions as a protein scaffold in the replication stress response, is multiply ubiquitinated upon replication fork stalling. Ubiquitination of RPA occurs on chromatin, involves sites outside its DNA binding channel, does not cause proteasomal degradation, and increases under conditions of fork collapse, suggesting a role in repair at stalled forks. We demonstrate that the E3 ligase RFWD3 mediates RPA ubiquitination. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination (HR) at stalled replication forks. Mutational analysis suggests that multisite ubiquitination of the entire RPA complex is responsible for repair at stalled forks. Multisite protein group sumoylation is known to promote HR in yeast. Our findings reveal a similar requirement for multisite protein group ubiquitination during HR at stalled forks in mammalian cells. Copyright © 2015 Elsevier Inc. All rights reserved.

The Rewiring of Ubiquitination Targets in a Pathogenic Yeast Promotes Metabolic Flexibility, Host Colonization and Virulence

PubMed Central

Childers, Delma S.; Raziunaite, Ingrida; Mol Avelar, Gabriela; Mackie, Joanna; Budge, Susan; Stead, David; Gow, Neil A. R.; Lenardon, Megan D.; Ballou, Elizabeth R.; MacCallum, Donna M.; Brown, Alistair J. P.

2016-01-01

Efficient carbon assimilation is critical for microbial growth and pathogenesis. The environmental yeast Saccharomyces cerevisiae is “Crabtree positive”, displaying a rapid metabolic switch from the assimilation of alternative carbon sources to sugars. Following exposure to sugars, this switch is mediated by the transcriptional repression of genes (carbon catabolite repression) and the turnover (catabolite inactivation) of enzymes involved in the assimilation of alternative carbon sources. The pathogenic yeast Candida albicans is Crabtree negative. It has retained carbon catabolite repression mechanisms, but has undergone posttranscriptional rewiring such that gluconeogenic and glyoxylate cycle enzymes are not subject to ubiquitin-mediated catabolite inactivation. Consequently, when glucose becomes available, C. albicans can continue to assimilate alternative carbon sources alongside the glucose. We show that this metabolic flexibility promotes host colonization and virulence. The glyoxylate cycle enzyme isocitrate lyase (CaIcl1) was rendered sensitive to ubiquitin-mediated catabolite inactivation in C. albicans by addition of a ubiquitination site. This mutation, which inhibits lactate assimilation in the presence of glucose, reduces the ability of C. albicans cells to withstand macrophage killing, colonize the gastrointestinal tract and cause systemic infections in mice. Interestingly, most S. cerevisiae clinical isolates we examined (67%) have acquired the ability to assimilate lactate in the presence of glucose (i.e. they have become Crabtree negative). These S. cerevisiae strains are more resistant to macrophage killing than Crabtree positive clinical isolates. Moreover, Crabtree negative S. cerevisiae mutants that lack Gid8, a key component of the Glucose-Induced Degradation complex, are more resistant to macrophage killing and display increased virulence in immunocompromised mice. Thus, while Crabtree positivity might impart a fitness advantage for

MARCH2 regulates autophagy by promoting CFTR ubiquitination and degradation and PIK3CA-AKT-MTOR signaling.

PubMed

Xia, Dan; Qu, Liujing; Li, Ge; Hongdu, Beiqi; Xu, Chentong; Lin, Xin; Lou, Yaxin; He, Qihua; Ma, Dalong; Chen, Yingyu

2016-09-01

MARCH2 (membrane-associated RING-CH protein 2), an E3 ubiquitin ligase, is mainly associated with the vesicle trafficking. In the present study, for the first time, we demonstrated that MARCH2 negatively regulates autophagy. Our data indicated that overexpression of MARCH2 impaired autophagy, as evidenced by attenuated levels of LC3B-II and impaired degradation of endogenous and exogenous autophagic substrates. By contrast, loss of MARCH2 expression had the opposite effects. In vivo experiments demonstrate that MARCH2 knockout mediated autophagy results in an inhibition of tumorigenicity. Further investigation revealed that the induction of autophagy by MARCH2 deficiency was mediated through the PIK3CA-AKT-MTOR signaling pathway. Additionally, we found that MARCH2 interacts with CFTR (cystic fibrosis transmembrane conductance regulator), promotes the ubiquitination and degradation of CFTR, and inhibits CFTR-mediated autophagy in tumor cells. The functional PDZ domain of MARCH2 is required for the association with CFTR. Thus, our study identified a novel negative regulator of autophagy and suggested that the physical and functional connection between the MARCH2 and CFTR in different conditions will be elucidated in the further experiments.

Myostatin promotes the wasting of human myoblast cultures through promoting ubiquitin-proteasome pathway-mediated loss of sarcomeric proteins.

PubMed

Lokireddy, Sudarsanareddy; Mouly, Vincent; Butler-Browne, Gillian; Gluckman, Peter D; Sharma, Mridula; Kambadur, Ravi; McFarlane, Craig

2011-12-01

Myostatin is a negative regulator of skeletal muscle growth and in fact acts as a potent inducer of "cachectic-like" muscle wasting in mice. The mechanism of action of myostatin in promoting muscle wasting has been predominantly studied in murine models. Despite numerous reports linking elevated levels of myostatin to human skeletal muscle wasting conditions, little is currently known about the signaling mechanism(s) through which myostatin promotes human skeletal muscle wasting. Therefore, in this present study we describe in further detail the mechanisms behind myostatin regulation of human skeletal muscle wasting using an in vitro human primary myotube atrophy model. Treatment of human myotube populations with myostatin promoted dramatic myotubular atrophy. Mechanistically, myostatin-induced myotube atrophy resulted in reduced p-AKT concomitant with the accumulation of active dephosphorylated Forkhead Box-O (FOXO1) and FOXO3. We further show that addition of myostatin results in enhanced activation of atrogin-1 and muscle-specific RING finger protein 1 (MURF1) and reduced expression of both myosin light chain (MYL) and myosin heavy chain (MYH). In addition, we found that myostatin-induced loss of MYL and MYH proteins is dependent on the activity of the proteasome and mediated via SMAD3-dependent regulation of FOXO1 and atrogin-1. Therefore, these data suggest that the mechanism through which myostatin promotes muscle wasting is very well conserved between species, and that myostatin-induced human myotube atrophy is mediated through inhibition of insulin-like growth factor (IGF)/phosphoinositide 3-kinase (PI3-K)/AKT signaling and enhanced activation of the ubiquitin-proteasome pathway and elevated protein degradation.

A Novel Strategy to Isolate Ubiquitin Conjugates Reveals Wide Role for Ubiquitination during Neural Development*

PubMed Central

Franco, Maribel; Seyfried, Nicholas T.; Brand, Andrea H.; Peng, Junmin; Mayor, Ugo

2011-01-01

Ubiquitination has essential roles in neuronal development and function. Ubiquitin proteomics studies on yeast and HeLa cells have proven very informative, but there still is a gap regarding neuronal tissue-specific ubiquitination. In an organism context, direct evidence for the ubiquitination of neuronal proteins is even scarcer. Here, we report a novel proteomics strategy based on the in vivo biotinylation of ubiquitin to isolate ubiquitin conjugates from the neurons of Drosophila melanogaster embryos. We confidently identified 48 neuronal ubiquitin substrates, none of which was yet known to be ubiquitinated. Earlier proteomics and biochemical studies in non-neuronal cell types had identified orthologs to some of those but not to others. The identification here of novel ubiquitin substrates, those with no known ubiquitinated ortholog, suggests that proteomics studies must be performed on neuronal cells to identify ubiquitination pathways not shared by other cell types. Importantly, several of those newly found neuronal ubiquitin substrates are key players in synaptogenesis. Mass spectrometry results were validated by Western blotting to confirm that those proteins are indeed ubiquitinated in the Drosophila embryonic nervous system and to elucidate whether they are mono- or polyubiquitinated. In addition to the ubiquitin substrates, we also identified the ubiquitin carriers that are active during synaptogenesis. Identifying endogenously ubiquitinated proteins in specific cell types, at specific developmental stages, and within the context of a living organism will allow understanding how the tissue-specific function of those proteins is regulated by the ubiquitin system. PMID:20861518

OTUB1 Co-opts Lys48-Linked Ubiquitin Recognition to Suppress E2 Enzyme Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Juang, Yu-Chi; Landry, Marie-Claude; Sanches, Mario

2012-03-26

Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibitedmore » E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.« less

Roles of mono-ubiquitinated Smad4 in the formation of Smad transcriptional complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Bei; Suzuki, Hiroyuki; Kato, Mitsuyasu

2008-11-14

TGF-{beta} activates receptor-regulated Smad (R-Smad) through phosphorylation by type I receptors. Activated R-Smad binds to Smad4 and the complex translocates into the nucleus and stimulates the transcription of target genes through association with co-activators including p300. It is not clear, however, how activated Smad complexes are removed from target genes. In this study, we show that TGF-{beta} enhances the mono-ubiquitination of Smad4. Smad4 mono-ubiquitination was promoted by p300 and suppressed by the c-Ski co-repressor. Smad4 mono-ubiquitination disrupted the interaction with Smad2 in the presence of constitutively active TGF-{beta} type I receptor. Furthermore, mono-ubiquitinated Smad4 was not found in DNA-binding Smadmore » complexes. A Smad4-Ubiquitin fusion protein, which mimics mono-ubiquitinated Smad4, enhanced localization to the cytoplasm. These results suggest that mono-ubiquitination of Smad4 occurs in the transcriptional activator complex and facilitates the turnover of Smad complexes at target genes.« less

Ubiquitin turnover and endocytic trafficking in yeast are regulated by Ser57 phosphorylation of ubiquitin

PubMed Central

Lee, Sora; Tumolo, Jessica M; Ehlinger, Aaron C; Jernigan, Kristin K; Qualls-Histed, Susan J; Hsu, Pi-Chiang; McDonald, W Hayes; Chazin, Walter J

2017-01-01

Despite its central role in protein degradation little is known about the molecular mechanisms that sense, maintain, and regulate steady state concentration of ubiquitin in the cell. Here, we describe a novel mechanism for regulation of ubiquitin homeostasis that is mediated by phosphorylation of ubiquitin at the Ser57 position. We find that loss of Ppz phosphatase activity leads to defects in ubiquitin homeostasis that are at least partially attributable to elevated levels of Ser57 phosphorylated ubiquitin. Phosphomimetic mutation at the Ser57 position of ubiquitin conferred increased rates of endocytic trafficking and ubiquitin turnover. These phenotypes are associated with bypass of recognition by endosome-localized deubiquitylases - including Doa4 which is critical for regulation of ubiquitin recycling. Thus, ubiquitin homeostasis is significantly impacted by the rate of ubiquitin flux through the endocytic pathway and by signaling pathways that converge on ubiquitin itself to determine whether it is recycled or degraded in the vacuole. PMID:29130884

Robust Lys63-Linked Ubiquitination of RIG-I Promotes Cytokine Eruption in Early Influenza B Virus Infection

PubMed Central

Jiang, Jingwen; Fan, Wenhui; Zheng, Weinan; Yu, Meng; Chen, Can; Sun, Lei; Bi, Yuhai; Ding, Chan; Gao, George F.

2016-01-01

ABSTRACT Influenza A and B virus infections both cause a host innate immunity response. Here, we report that the robust production of type I and III interferons (IFNs), IFN-stimulated genes, and proinflammatory factors can be induced by influenza B virus rather than influenza A virus infection in alveolar epithelial (A549) cells during early infection. This response is mainly dependent on the retinoic acid-inducible gene I (RIG-I)-mediated signaling pathway. Infection by influenza B virus promotes intense Lys63-linked ubiquitination of RIG-I, resulting in cytokine eruption. It is known that the influenza A virus NS1 protein (NS1-A) interacts with RIG-I and TRIM25 to suppress the activation of RIG-I-mediated signaling. However, the present results indicate that the influenza B virus NS1 protein (NS1-B) is unable to interact with RIG-I but engages in the formation of a RIG-I/TRIM25/NS1-B ternary complex. Furthermore, we demonstrate that the N-terminal RNA-binding domain (RBD) of NS1-B is responsible for interaction with TRIM25 and that this interaction blocks the inhibitory effect of the NS1-B C-terminal effector domain (TED) on RIG-I ubiquitination. Our findings reveal a novel mechanism for the host cytokine response to influenza B virus infection through regulatory interplay between host and viral proteins. IMPORTANCE Influenza B virus generally causes local mild epidemics but is occasionally lethal to individuals. Existing studies describe the broad characteristics of influenza B virus epidemiology and pathology. However, to develop better prevention and treatments for the disease, determining the concrete molecular mechanisms of pathogenesis becomes pivotal to understand how the host reacts to the challenge of influenza B virus. Thus, we aimed to characterize the host innate immune response to influenza B virus infection. Here, we show that vigorous Lys63-linked ubiquitination of RIG-I and cytokine eruption dependent on RIG-I-mediated signal transduction are

Robust Lys63-Linked Ubiquitination of RIG-I Promotes Cytokine Eruption in Early Influenza B Virus Infection.

PubMed

Jiang, Jingwen; Li, Jing; Fan, Wenhui; Zheng, Weinan; Yu, Meng; Chen, Can; Sun, Lei; Bi, Yuhai; Ding, Chan; Gao, George F; Liu, Wenjun

2016-07-15

Influenza A and B virus infections both cause a host innate immunity response. Here, we report that the robust production of type I and III interferons (IFNs), IFN-stimulated genes, and proinflammatory factors can be induced by influenza B virus rather than influenza A virus infection in alveolar epithelial (A549) cells during early infection. This response is mainly dependent on the retinoic acid-inducible gene I (RIG-I)-mediated signaling pathway. Infection by influenza B virus promotes intense Lys63-linked ubiquitination of RIG-I, resulting in cytokine eruption. It is known that the influenza A virus NS1 protein (NS1-A) interacts with RIG-I and TRIM25 to suppress the activation of RIG-I-mediated signaling. However, the present results indicate that the influenza B virus NS1 protein (NS1-B) is unable to interact with RIG-I but engages in the formation of a RIG-I/TRIM25/NS1-B ternary complex. Furthermore, we demonstrate that the N-terminal RNA-binding domain (RBD) of NS1-B is responsible for interaction with TRIM25 and that this interaction blocks the inhibitory effect of the NS1-B C-terminal effector domain (TED) on RIG-I ubiquitination. Our findings reveal a novel mechanism for the host cytokine response to influenza B virus infection through regulatory interplay between host and viral proteins. Influenza B virus generally causes local mild epidemics but is occasionally lethal to individuals. Existing studies describe the broad characteristics of influenza B virus epidemiology and pathology. However, to develop better prevention and treatments for the disease, determining the concrete molecular mechanisms of pathogenesis becomes pivotal to understand how the host reacts to the challenge of influenza B virus. Thus, we aimed to characterize the host innate immune response to influenza B virus infection. Here, we show that vigorous Lys63-linked ubiquitination of RIG-I and cytokine eruption dependent on RIG-I-mediated signal transduction are induced by virus

The Ubiquitin-associated Domain of Cellular Inhibitor of Apoptosis Proteins Facilitates Ubiquitylation*

PubMed Central

Budhidarmo, Rhesa; Day, Catherine L.

2014-01-01

The cellular inhibitor of apoptosis (cIAP) proteins are essential RING E3 ubiquitin ligases that regulate apoptosis and inflammatory responses. cIAPs contain a ubiquitin-associated (UBA) domain that binds ubiquitin and is implicated in the regulation of cell survival and proteasomal degradation. Here we show that mutation of the MGF and LL motifs in the UBA domain of cIAP1 caused unfolding and increased cIAP1 multimonoubiquitylation. By developing a UBA mutant that disrupted ubiquitin binding but not the structure of the UBA domain, we found that the UBA domain enhances cIAP1 and cIAP2 ubiquitylation. We demonstrate that the UBA domain binds to the UbcH5b∼Ub conjugate, and this promotes RING domain-dependent monoubiquitylation. This study establishes ubiquitin-binding modules, such as the UBA domain, as important regulatory modules that can fine tune the activity of E3 ligases. PMID:25065467

Molecular piracy: manipulation of the ubiquitin system by Kaposi's sarcoma-associated herpesvirus.

PubMed

Fujimuro, Masahiro; Hayward, S Diane; Yokosawa, Hideyoshi

2007-01-01

Ubiquitination, one of several post-translational protein modifications, plays a key role in the regulation of cellular events, including protein degradation, signal transduction, endocytosis, protein trafficking, apoptosis and immune responses. Ubiquitin attachment at the lysine residue of cellular factors acts as a signal for endocytosis and rapid degradation by the 26S proteasome. It has recently been observed that viruses, especially oncogenic herpesviruses, utilise molecular piracy by encoding their own proteins to interfere with regulation of cell signalling. Kaposi's sarcoma- associated herpesvirus (KSHV) manipulates the ubiquitin system to facilitate cell proliferation, anti-apoptosis and evasion from immunity. In this review, we will describe the strategies used by KSHV at distinct stages of the viral life-cycle to control the ubiquitin system and promote oncogenesis and viral persistence. (c) 2007 John Wiley & Sons, Ltd.

Divergence in Ubiquitin Interaction and Catalysis among the Ubiquitin-Specific Protease Family Deubiquitinating Enzymes.

PubMed

Tencer, Adam H; Liang, Qin; Zhuang, Zhihao

2016-08-23

Deubiquitinating enzymes (DUBs) are responsible for reversing mono- and polyubiquitination of proteins and play essential roles in numerous cellular processes. Close to 100 human DUBs have been identified and are classified into five families, with the ubiquitin-specific protease (USP) family being the largest (>50 members). The binding of ubiquitin (Ub) to USP is strikingly different from that observed for the DUBs in the ubiquitin C-terminal hydrolase (UCH) and ovarian tumor domain protease (OTU) families. We generated a panel of mutant ubiquitins and used them to probe the ubiquitin's interaction with a number of USPs. Our results revealed a remarkable divergence of USP-Ub interactions among the USP catalytic domains. Our double-mutant cycle analysis targeting the ubiquitin residues located in the tip, the central body, and the tail of ubiquitin also demonstrated different crosstalk among the USP-Ub interactions. This work uncovered intriguing divergence in the ubiquitin-binding mode in the USP family DUBs and raised the possibility of targeting the ubiquitin-binding hot spots on USPs for selective inhibition of USPs by small molecule antagonists.

Intrinsic Flexibility of Ubiquitin on Proliferating Cell Nuclear Antigen (PCNA) in Translesion Synthesis*

PubMed Central

Hibbert, Richard G.; Sixma, Titia K.

2012-01-01

Ubiquitin conjugation provides a crucial signaling role in hundreds of cellular pathways; however, a structural understanding of ubiquitinated substrates is lacking. One important substrate is monoubiquitinated PCNA (PCNA-Ub), which signals for recruitment of damage-tolerant polymerases in the translesion synthesis (TLS) pathway of DNA damage avoidance. We use a novel and efficient enzymatic method to produce PCNA-Ub at high yield with a native isopeptide bond and study its Usp1/UAF1-dependent deconjugation. In solution we find that the ubiquitin moiety is flexible relative to the PCNA, with its hydrophobic patch mostly accessible for recruitment of TLS polymerases, which promotes the interaction with polymerase η. The studies are a prototype for the nature of the ubiquitin modification. PMID:22989887

Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation

PubMed Central

Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan MF; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul MK

2015-01-01

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser65)—which lies within its ubiquitin-like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of

Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation.

PubMed

Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan M F; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul M K

2015-08-01

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser(65))--which lies within its ubiquitin-like domain (Ubl)--and indirectly through phosphorylation of ubiquitin at Ser(65). How Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin(Phospho-Ser65) binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser(65) by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin(Phospho-Ser65), thereby promoting Parkin Ser(65) phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser(65) phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin(Phospho-Ser65) to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser(65). Finally, purified Parkin maximally phosphorylated at Ser(65) in vitro cannot be further activated by the addition of ubiquitin(Phospho-Ser65). Our results thus suggest that a major role of ubiquitin(Phospho-Ser65) is to promote PINK1-mediated phosphorylation of Parkin at Ser(65), leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser(65)-binding pocket on the surface of Parkin that is critical for the ubiquitin(Phospho-Ser65) interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin(Phospho-Ser65), which could aid in the development of Parkin

Rad6–Bre1-mediated H2B ubiquitination regulates telomere replication by promoting telomere-end resection

PubMed Central

Wu, Zhenfang; Liu, Jun; Zhang, Qiong-Di; Lv, De-Kang; Wu, Nian-Feng

2017-01-01

Abstract Rad6 and Bre1, ubiquitin-conjugating E2 and E3 enzymes respectively, are responsible for histone H2B lysine 123 mono-ubiquitination (H2Bub1) in Saccharomyces cerevisiae. Previous studies have shown that Rad6 and Bre1 regulate telomere length and recombination. However, the underlying molecular mechanism remains largely unknown. Here we report that H2BK123 mutation results in telomere shortening, while inactivation of Ubp8 and/or Ubp10, deubiquitinases of H2Bub1, leads to telomere lengthening in Rad6–Bre1-dependent manner. In telomerase-deficient cells, inactivation of Rad6–Bre1 pathway retards telomere shortening rate and the onset of senescence, while deletion of UBP8 and/or UBP10 accelerates senescence. Thus, Rad6–Bre1 pathway regulates both telomere length and recombination through its role in H2Bub1. Additionally, inactivation of both Rad6–Bre1–H2Bub1 and Mre11–Rad50–Xrs2 (MRX) pathways causes synthetic growth defects and telomere shortening in telomerase-proficient cells, and significantly accelerates senescence and eliminates type II telomere recombination in telomerase-deficient cells. Furthermore, RAD6 or BRE1 deletion, or H2BK123R mutation decreases the accumulation of ssDNA at telomere ends. These results support the model that Rad6–Bre1–H2Bub1 cooperates with MRX to promote telomere-end resection and thus positively regulates both telomerase- and recombination-dependent telomere replication. This study provides a mechanistic link between histone H2B ubiquitination and telomere replication. PMID:28180293

Cycles of Ubiquitination and Deubiquitination Critically Regulate Growth Factor-Mediated Activation of Akt Signaling

PubMed Central

Yang, Wei-Lei; Jin, Guoxiang; Li, Chien-Feng; Jeong, Yun Seong; Moten, Asad; Xu, Dazhi; Feng, Zizhen; Chen, Wei; Cai, Zhen; Darnay, Bryant; Gu, Wei; Lin, Hui-Kuan

2013-01-01

K63-linked ubiquitination of Akt is a posttranslational modification that plays a critical role in growth factor-mediated membrane recruitment and activation of Akt. Although E3 ligases involved in growth factor-induced Akt ubiquitination have been defined, the deubiquitinating enzyme (DUB) that triggers deubiquitination of Akt and the function of Akt deubiquitination remain largely unclear. Here, we showed that CYLD was a DUB for Akt and suppressed growth factor-mediated Akt ubiquitination and activation. CYLD directly removed ubiquitin moieties on Akt under serum-starved conditions. CYLD dissociated from Akt upon growth factor stimulation, thereby allowing E3 ligases to induce ubiquitination and activation of Akt. CYLD deficiency also promoted cancer cell proliferation, survival, glucose uptake and growth of prostate tumors. Our findings reveal the crucial role of cycles of ubiquitination and deubiquitination of Akt in its membrane recruitment and activation, and further identifies CYLD as a molecular switch for these processes. PMID:23300340

Regulation of Ubiquitin Enzymes in the TGF-β Pathway.

PubMed

Iyengar, Prasanna Vasudevan

2017-04-20

The transforming growth factor-β (TGF-β) pathway has a tumor suppressor role in normal and premalignant cells but promotes oncogenesis in advanced cancer cells. Components of the pathway are tightly controlled by ubiquitin modifying enzymes and aberrations in these enzymes are frequently observed to dysregulate the pathway causing diseases such as bone disorders, cancer and metastasis. These enzymes and their counterparts are increasingly being tested as druggable targets, and thus a deeper understanding of the enzymes is required. This review summarizes the roles of specific ubiquitin modifying enzymes in the TGF-β pathway and how they are regulated.

Covalent ISG15 conjugation to CHIP promotes its ubiquitin E3 ligase activity and inhibits lung cancer cell growth in response to type I interferon.

PubMed

Yoo, Lang; Yoon, A-Rum; Yun, Chae-Ok; Chung, Kwang Chul

2018-01-24

The carboxyl terminus of Hsp70-interacting protein (CHIP) acts as a ubiquitin E3 ligase and a link between the chaperones Hsp70/90 and the proteasome system, playing a vital role in maintaining protein homeostasis. CHIP regulates a number of proteins involved in a myriad of physiological and pathological processes, but the underlying mechanism of action via posttranslational modification has not been extensively explored. In this study, we investigated a novel modulatory mode of CHIP and its effect on CHIP enzymatic activity. ISG15, an ubiquitin-like modifier, is induced by type I interferon (IFN) stimulation and can be conjugated to target proteins (ISGylation). Here we demonstrated that CHIP may be a novel target of ISGylation in HEK293 cells stimulated with type I IFN. We also found that Lys143/144/145 and Lys287 residues in CHIP are important for and target residues of ISGylation. Moreover, ISGylation promotes the E3 ubiquitin ligase activity of CHIP, subsequently causing a decrease in levels of oncogenic c-Myc, one of its many ubiquitination targets, in A549 lung cancer cells and inhibiting A549 cell and tumor growth. In conclusion, the present study demonstrates that covalent ISG15 conjugation produces a novel CHIP regulatory mode that enhances the tumor-suppressive activity of CHIP, thereby contributing to the antitumor effect of type I IFN.

Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3

PubMed Central

Tang, Liu-Ya; Yamashita, Motozo; Coussens, Nathan P; Tang, Yi; Wang, Xiangchun; Li, Cuiling; Deng, Chu-Xia; Cheng, Steven Y; Zhang, Ying E

2011-01-01

TGF-β signalling is regulated by post-translational modifications of Smad proteins to translate quantitative difference in ligand concentration into proportional transcriptional output. Previous studies in cell culture systems suggested that Smad ubiquitination regulatory factors (Smurfs) act in this regulation by targeting Smads for proteasomal degradation, but whether this mechanism operates under physiological conditions is not clear. Here, we generated mice harbouring a target-disrupted Smurf2 allele. Using primary mouse embryonic fibroblasts and dermal fibroblasts, we show that TGF-β-mediated, Smad-dependent transcriptional responses are elevated in the absence of Smurf2. Instead of promoting poly-ubiquitination and degradation, we show that Smurf2 actually induces multiple mono-ubiquitination of Smad3 in vivo. Phosphorylation of T179, immediately upstream of the Smad3 PY motif, enhances Smurf2 and Smad3 interaction and Smad3 ubiquitination. We have mapped Smurf2-induced Smad3 ubiquitination sites to lysine residues at the MH2 domain, and demonstrate that Smad3 ubiquitination inhibits the formation of Smad3 complexes. Thus, our data support a model in which Smurf2 negatively regulates TGF-β signalling by attenuating the activity of Smad3 rather than promoting its degradation. PMID:22045334

CBL family E3 ubiquitin ligases control JAK2 ubiquitination and stability in hematopoietic stem cells and myeloid malignancies

PubMed Central

Lv, Kaosheng; Jiang, Jing; Donaghy, Ryan; Riling, Christopher R.; Cheng, Ying; Chandra, Vemika; Rozenova, Krasimira; An, Wei; Mohapatra, Bhopal C.; Goetz, Benjamin T.; Pillai, Vinodh; Han, Xu; Todd, Emily A.; Jeschke, Grace R.; Langdon, Wallace Y.; Kumar, Suresh; Hexner, Elizabeth O.

2017-01-01

Janus kinase 2 (JAK2) is a central kinase in hematopoietic stem/progenitor cells (HSPCs), and its uncontrolled activation is a prominent oncogenic driver of hematopoietic neoplasms. However, molecular mechanisms underlying the regulation of JAK2 have remained elusive. Here we report that the Casitas B-cell lymphoma (CBL) family E3 ubiquitin ligases down-regulate JAK2 stability and signaling via the adaptor protein LNK/SH2B3. We demonstrated that depletion of CBL/CBL-B or LNK abrogated JAK2 ubiquitination, extended JAK2 half-life, and enhanced JAK2 signaling and cell growth in human cell lines as well as primary murine HSPCs. Built on these findings, we showed that JAK inhibitor (JAKi) significantly reduced aberrant HSPCs and mitigated leukemia development in a mouse model of aggressive myeloid leukemia driven by loss of Cbl and Cbl-b. Importantly, primary human CBL mutated (CBLmut) leukemias exhibited increased JAK2 protein levels and signaling and were hypersensitive to JAKi. Loss-of-function mutations in CBL E3 ubiquitin ligases are found in a wide range of myeloid malignancies, which are diseases without effective treatment options. Hence, our studies reveal a novel signaling axis that regulates JAK2 in normal and malignant HSPCs and suggest new therapeutic strategies for treating CBLmut myeloid malignancies. PMID:28611190

FBXO11 promotes ubiquitination of the Snail family of transcription factors in cancer progression and epidermal development.

PubMed

Jin, Yue; Shenoy, Anitha K; Doernberg, Samuel; Chen, Hao; Luo, Huacheng; Shen, Huangxuan; Lin, Tong; Tarrash, Miriam; Cai, Qingsong; Hu, Xin; Fiske, Ryan; Chen, Ting; Wu, Lizi; Mohammed, Kamal A; Rottiers, Veerle; Lee, Siu Sylvia; Lu, Jianrong

2015-06-28

The Snail family of transcription factors are core inducers of epithelial-to-mesenchymal transition (EMT). Here we show that the F-box protein FBXO11 recognizes and promotes ubiquitin-mediated degradation of multiple Snail family members including Scratch. The association between FBXO11 and Snai1 in vitro is independent of Snai1 phosphorylation. Overexpression of FBXO11 in mesenchymal cells reduces Snail protein abundance and cellular invasiveness. Conversely, depletion of endogenous FBXO11 in epithelial cancer cells causes Snail protein accumulation, EMT, and tumor invasion, as well as loss of estrogen receptor expression in breast cancer cells. Expression of FBXO11 is downregulated by EMT-inducing signals TGFβ and nickel. In human cancer, high FBXO11 levels correlate with expression of epithelial markers and favorable prognosis. The results suggest that FBXO11 sustains the epithelial state and inhibits cancer progression. Inactivation of FBXO11 in mice leads to neonatal lethality, epidermal thickening, and increased Snail protein levels in epidermis, validating that FBXO11 is a physiological ubiquitin ligase of Snail. Moreover, in C. elegans, the FBXO11 mutant phenotype is attributed to the Snail factors as it is suppressed by inactivation/depletion of Snail homologs. Collectively, these findings suggest that the FBXO11-Snail regulatory axis is evolutionarily conserved and critically governs carcinoma progression and mammalian epidermal development. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications.

PubMed

Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

2014-01-01

Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling.

Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications

PubMed Central

Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

2014-01-01

Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling. PMID:25628960

E3 ubiquitin ligase SP1 regulates peroxisome biogenesis in Arabidopsis

DOE PAGES

Pan, Ronghui; Satkovich, John; Hu, Jianping

2016-10-31

Peroxisomes are ubiquitous eukaryotic organelles that play pivotal roles in a suite of metabolic processes and often act coordinately with other organelles, such as chloroplasts and mitochondria. Peroxisomes import proteins to the peroxisome matrix by peroxins (PEX proteins), but how the function of the PEX proteins is regulated is poorly understood. In this study, we identified the Arabidopsis RING (really interesting new gene) type E3 ubiquitin ligase SP1 [suppressor of plastid protein import locus 1 (ppi1) 1] as a peroxisome membrane protein with a regulatory role in peroxisome protein import. SP1 interacts physically with the two components of the peroxisomemore » protein docking complex PEX13–PEX14 and the (RING)-finger peroxin PEX2. Loss of SP1 function suppresses defects of the pex14-2 and pex13-1 mutants, and SP1 is involved in the degradation of PEX13 and possibly PEX14 and all three RING peroxins. An in vivo ubiquitination assay showed that SP1 has the ability to promote PEX13 ubiquitination. Our study has revealed that, in addition to its previously reported function in chloroplast biogenesis, SP1 plays a role in peroxisome biogenesis. The same E3 ubiquitin ligase promotes the destabilization of components of two distinct protein-import machineries, indicating that degradation of organelle biogenesis factors by the ubiquitin–proteasome system may constitute an important regulatory mechanism in coordinating the biogenesis of metabolically linked organelles in eukaryotes.« less

The emerging complexity of ubiquitin architecture.

PubMed

Ohtake, Fumiaki; Tsuchiya, Hikaru

2017-02-01

Ubiquitylation is an essential post-translational modification (PTM) of proteins with diverse cellular functions. Polyubiquitin chains with different topologies have different cellular roles, and are referred to as a 'ubiquitin code'. Recent studies have begun to reveal that more complex ubiquitin architectures function as important signals in several biological pathways. These include PTMs of ubiquitin itself, such as acetylated ubiquitin and phospho-ubiquitin. Moreover, important roles for heterogeneous polyubiquitin chains, such as mixed or branched chains, have been reported, which significantly increase the diversity of the ubiquitin code. In this review, we describe mass spectrometry-based methods to characterize the ubiquitin signal. We also describe recent advances in our understanding of complex ubiquitin architectures, including our own findings concerning ubiquitin acetylation and branching within polyubiquitin chains. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Chapter Seven - When Phosphorylation Encounters Ubiquitination: A Balanced Perspective on IGF-1R Signaling.

PubMed

Girnita, L; Takahashi, S-I; Crudden, C; Fukushima, T; Worrall, C; Furuta, H; Yoshihara, H; Hakuno, F; Girnita, A

2016-01-01

Cell-surface receptors govern the critical information passage from outside to inside the cell and hence control important cellular decisions such as survival, growth, and differentiation. These receptors, structurally grouped into different families, utilize common intracellular signaling-proteins and pathways, yet promote divergent biological consequences. In rapid processing of extracellular signals to biological outcomes, posttranslational modifications offer a repertoire of protein processing options. Protein ubiquitination was originally identified as a signal for protein degradation through the proteasome system. It is now becoming increasingly recognized that both ubiquitin and ubiquitin-like proteins, all evolved from a common ubiquitin structural superfold, are used extensively by the cell and encompass signal tags for many different cellular fates. In this chapter we examine the current understanding of the ubiquitin regulation surrounding the insulin-like growth factor and insulin signaling systems, major members of the larger family of receptor tyrosine kinases (RTKs) and key regulators of fundamental physiological and pathological states. Copyright © 2016 Elsevier Inc. All rights reserved.

Atomic structure of the APC/C and its mechanism of protein ubiquitination

PubMed Central

Yang, Jing; McLaughlin, Stephen H.; Barford, David

2015-01-01

The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex, and interphase inhibitor Emi1 ensures the correct order and timing of distinct cell cycle transitions. Here, we used cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module, and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of experiments to further investigate APC/C functions in vivo. PMID:26083744

Ubiquitination of the Dishevelled DIX domain blocks its head-to-tail polymerization

PubMed Central

Madrzak, Julia; Fiedler, Marc; Johnson, Christopher M.; Ewan, Richard; Knebel, Axel; Bienz, Mariann; Chin, Jason W.

2015-01-01

Dishevelled relays Wnt signals from the plasma membrane to different cytoplasmic effectors. Its signalling activity depends on its DIX domain, which undergoes head-to-tail polymerization to assemble signalosomes. The DIX domain is ubiquitinated in vivo at multiple lysines, which can be antagonized by various deubiquitinases (DUBs) including the CYLD tumour suppressor that attenuates Wnt signalling. Here, we generate milligram quantities of pure human Dvl2 DIX domain mono-ubiquitinated at two lysines (K54 and K58) by genetically encoded orthogonal protection with activated ligation (GOPAL), to investigate their effect on DIX polymerization. We show that the ubiquitination of DIX at K54 blocks its polymerization in solution, whereas DIX58-Ub remains oligomerization-competent. DUB profiling identified 28 DUBs that cleave DIX-ubiquitin conjugates, half of which prefer, or are specific for, DIX54-Ub, including Cezanne and CYLD. These DUBs thus have the potential to promote Dvl polymerization and signalosome formation, rather than antagonize it as previously thought for CYLD. PMID:25907794

Screening promoters for Anthurium transformation using transient expression

USDA-ARS?s Scientific Manuscript database

Different promoters and tissue types were evaluated for transient '-glucoronidase (GUS) expression in Anthurium andreanum Hort. ‘Marian Seefurth’ following microprojectile bombardment. Plasmids containing the Ubiquitin 2, Actin 1, Cytochrome C1 from rice, Ubiquitin 1 from maize and 35 S promoter fr...

Ubiquitination in Periodontal Disease: A Review.

PubMed

Tsuchida, Sachio; Satoh, Mamoru; Takiwaki, Masaki; Nomura, Fumio

2017-07-10

Periodontal disease (periodontitis) is a chronic inflammatory condition initiated by microbial infection that leads to gingival tissue destruction and alveolar bone resorption. The periodontal tissue's response to dental plaque is characterized by the accumulation of polymorphonuclear leukocytes, macrophages, and lymphocytes, all of which release inflammatory mediators and cytokines to orchestrate the immunopathogenesis of periodontal disease. Ubiquitination is achieved by a mechanism that involves a number of factors, including an ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin-protein ligase. Ubiquitination is a post-translational modification restricted to eukaryotes that are involved in essential host processes. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Increasing numbers of recent reports have provided evidence that many approaches are delivering promising reports for discovering the relationship between ubiquitination and periodontal disease. The scope of this review was to investigate recent progress in the discovery of ubiquitinated protein in diseased periodontium and to discuss the ubiquitination process in periodontal diseases.

Ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) is a potential tumour suppressor in prostate cancer and is frequently silenced by promoter methylation

PubMed Central

2011-01-01

Background We have previously reported significant downregulation of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in prostate cancer (PCa) compared to the surrounding benign tissue. UCHL1 plays an important role in ubiquitin system and different cellular processes such as cell proliferation and differentiation. We now show that the underlying mechanism of UCHL1 downregulation in PCa is linked to its promoter hypermethylation. Furthermore, we present evidences that UCHL1 expression can affect the behavior of prostate cancer cells in different ways. Results Methylation specific PCR analysis results showed a highly methylated promoter region for UCHL1 in 90% (18/20) of tumor tissue compared to 15% (3/20) of normal tissues from PCa patients. Pyrosequencing results confirmed a mean methylation of 41.4% in PCa whereas only 8.6% in normal tissues. To conduct functional analysis of UCHL1 in PCa, UCHL1 is overexpressed in LNCaP cells whose UCHL1 expression is normally suppressed by promoter methylation and found that UCHL1 has the ability to decrease the rate of cell proliferation and suppresses anchorage-independent growth of these cells. In further analysis, we found evidence that exogenous expression of UCHL1 suppress LNCaP cells growth probably via p53-mediated inhibition of Akt/PKB phosphorylation and also via accumulation of p27kip1 a cyclin dependant kinase inhibitor of cell cycle regulating proteins. Notably, we also observed that exogenous expression of UCHL1 induced a senescent phenotype that was detected by using the SA-ß-gal assay and might be due to increased p14ARF, p53, p27kip1 and decreased MDM2. Conclusion From these results, we propose that UCHL1 downregulation via promoter hypermethylation plays an important role in various molecular aspects of PCa biology, such as morphological diversification and regulation of proliferation. PMID:21999842

Non-degradative Ubiquitination of Protein Kinases

PubMed Central

Ball, K. Aurelia; Johnson, Jeffrey R.; Lewinski, Mary K.; Guatelli, John; Verschueren, Erik; Krogan, Nevan J.; Jacobson, Matthew P.

2016-01-01

Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well. PMID:27253329

RAP80, ubiquitin and SUMO in the DNA damage response.

PubMed

Lombardi, Patrick M; Matunis, Michael J; Wolberger, Cynthia

2017-08-01

A decade has passed since the first reported connection between RAP80 and BRCA1 in DNA double-strand break repair. Despite the initial identification of RAP80 as a factor localizing BRCA1 to DNA double-strand breaks and potentially promoting homologous recombination, there is increasing evidence that RAP80 instead suppresses homologous recombination to fine-tune the balance of competing DNA repair processes during the S/G 2 phase of the cell cycle. RAP80 opposes homologous recombination by inhibiting DNA end-resection and sequestering BRCA1 into the BRCA1-A complex. Ubiquitin and SUMO modifications of chromatin at DNA double-strand breaks recruit RAP80, which contains distinct sequence motifs that recognize ubiquitin and SUMO. Here, we review RAP80's role in repressing homologous recombination at DNA double-strand breaks and how this role is facilitated by its ability to bind ubiquitin and SUMO modifications.

Ubiquitin in Motion: Structural Studies of the Ubiquitin-Conjugating Enzyme~Ubiquitin Conjugate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pruneda, Jonathan N.; Stoll, Kate E.; Bolton, Laura J.

2011-03-15

Ubiquitination of proteins provides a powerful and versatile post-translational signal in the eukaryotic cell. The formation of a thioester bond between ubiquitin (Ub) and the active site of a ubiquitin-conjugating enzyme (E2) is critical for the transfer of Ub to substrates. Assembly of a functional ubiquitin ligase (E3) complex poised for Ub transfer involves recognition and binding of an E2~Ub conjugate. Therefore, full characterization of the structure and dynamics of E2~Ub conjugates is required for further mechanistic understanding of Ub transfer reactions. Here we present characterization of the dynamic behavior of E2~Ub conjugates of two human enzymes, UbcH5c~Ub and Ubc13~Ub,more » in solution as determined by nuclear magnetic resonance and small-angle X-ray scattering. Within each conjugate, Ub retains great flexibility with respect to the E2, indicative of highly dynamic species that adopt manifold orientations. The population distribution of Ub conformations is dictated by the identity of the E2: the UbcH5c~Ub conjugate populates an array of extended conformations, and the population of Ubc13~Ub conjugates favors a closed conformation in which the hydrophobic surface of Ub faces helix 2 of Ubc13. Finally, we propose that the varied conformations adopted by Ub represent available binding modes of the E2~Ub species and thus provide insight into the diverse E2~Ub protein interactome, particularly with regard to interaction with Ub ligases.« less

Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4)*

PubMed Central

Hendriks, Ivo A.; Schimmel, Joost; Eifler, Karolin; Olsen, Jesper V.; Vertegaal, Alfred C. O.

2015-01-01

Ring finger protein 4 (RNF4) is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO interaction motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach, we identified the deubiquitinating enzyme ubiquitin-specific protease 11 (USP11), a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO-targeted ubiquitin ligase/protease pair with a role in the DDR. PMID:25969536

Proteasomal Ubiquitin Receptor RPN-10 Controls Sex Determination in Caenorhabditis elegans

PubMed Central

Shimada, Masumi; Kanematsu, Kenji; Tanaka, Keiji; Yokosawa, Hideyoshi

2006-01-01

The ubiquitin-binding RPN-10 protein serves as a ubiquitin receptor that delivers client proteins to the 26S proteasome. Although ubiquitin recognition is an essential step for proteasomal destruction, deletion of the rpn-10 gene in yeast does not influence viability, indicating redundancy of the substrate delivery pathway. However, their specificity and biological relevance in higher eukaryotes is still enigmatic. We report herein that knockdown of the rpn-10 gene, but not any other proteasome subunit genes, sexually transforms hermaphrodites to females by eliminating hermaphrodite spermatogenesis in Caenorhabditis elegans. The feminization phenotype induced by deletion of the rpn-10 gene was rescued by knockdown of tra-2, one of sexual fate decision genes promoting female development, and its downstream target tra-1, indicating that the TRA-2–mediated sex determination pathway is crucial for the Δrpn-10–induced sterile phenotype. Intriguingly, we found that co-knockdown of rpn-10 and functionally related ubiquitin ligase ufd-2 overcomes the germline-musculinizing effect of fem-3(gf). Furthermore, TRA-2 proteins accumulated in rpn-10-defective worms. Our results show that the RPN-10–mediated ubiquitin pathway is indispensable for control of the TRA-2–mediated sex-determining pathway. PMID:17050737

TRAF6-Mediated SM22α K21 Ubiquitination Promotes G6PD Activation and NADPH Production, Contributing to GSH Homeostasis and VSMC Survival In Vitro and In Vivo.

PubMed

Dong, Li-Hua; Li, Liang; Song, Yu; Duan, Zhi-Li; Sun, Shao-Guang; Lin, Yan-Ling; Miao, Sui-Bing; Yin, Ya-Juan; Shu, Ya-Nan; Li, Huan; Chen, Peng; Zhao, Li-Li; Han, Mei

2015-09-25

Vascular smooth muscle cell (VSMC) survival under stressful conditions is integral to promoting vascular repair, but facilitates plaque stability during the development of atherosclerosis. The cytoskeleton-associated smooth muscle (SM) 22α protein is involved in the regulation of VSMC phenotypes, whereas the pentose phosphate pathway plays an essential role in cell proliferation through the production of dihydronicotinamide adenine dinucleotide phosphate. To identify the relationship between dihydronicotinamide adenine dinucleotide phosphate production and SM22α activity in the development and progression of vascular diseases. We showed that the expression and activity of glucose-6-phosphate dehydrogenase (G6PD) are promoted in platelet-derived growth factor (PDGF)-BB-induced proliferative VSMCs. PDGF-BB induced G6PD membrane translocation and activation in an SM22α K21 ubiquitination-dependent manner. Specifically, the ubiquitinated SM22α interacted with G6PD and mediated G6PD membrane translocation. Furthermore, we found that tumor necrosis factor receptor-associated factor (TRAF) 6 mediated SM22α K21 ubiquitination in a K63-linked manner on PDGF-BB stimulation. Knockdown of TRAF6 decreased the membrane translocation and activity of G6PD, in parallel with reduced SM22α K21 ubiquitination. Elevated levels of activated G6PD consequent to PDGF-BB induction led to increased dihydronicotinamide adenine dinucleotide phosphate generation through stimulation of the pentose phosphate pathway, which enhanced VSMC viability and reduced apoptosis in vivo and in vitro via glutathione homeostasis. We provide evidence that TRAF6-induced SM22α ubiquitination maintains VSMC survival through increased G6PD activity and dihydronicotinamide adenine dinucleotide phosphate production. The TRAF6-SM22α-G6PD pathway is a novel mechanism underlying the association between glucose metabolism and VSMC survival, which is beneficial for vascular repair after injury but facilitates

Cigarette smoke destabilizes NLRP3 protein by promoting its ubiquitination.

PubMed

Han, SeungHye; Jerome, Jacob A; Gregory, Alyssa D; Mallampalli, Rama K

2017-01-05

Cigarette smoke suppresses innate immunity, making smokers more susceptible to infection. The NLRP3 inflammasome is a multi-protein complex that releases interleukin (IL) -1β and IL -18. These cytokines are critical for a timely host response to pathogens. Whether cigarette smoke affects NLRP3 protein levels, and its ability to form an inflammasome, is not known. Using the human monocyte THP1 cell line and C57BL/6 mice, we show that cigarette smoke decreases NLRP3 levels in cells by increasing ubiquitin-mediated proteasomal processing. Half-life of NLRP3 is shortened with the exposure to cigarette smoke extract. Cigarette smoke extract reduces cellular NLRP3 protein abundance in the presence of lipopolysaccharide, a known inducer of NLRP3 protein, thereby decreasing the formation of NLRP3 inflammasomes. The release of IL-1β and IL-18 by inflammasome activation is also decreased with the exposure to cigarette smoke extract both in THP1 cells and primary human peripheral blood macrophages. Cigarette smoke extract decreased NLRP3 protein abundance via increased ubiquitin-mediated proteasomal processing. The release of IL-1β and IL-18 is also decreased with cigarette smoke extract. Our findings may provide mechanistic insights on immunosuppression in smokers and unique opportunities to develop a strategy to modulate immune function.

Exploring the Ubiquitin-Proteasome Protein Degradation Pathway in Yeast

ERIC Educational Resources Information Center

Will, Tamara J.; McWatters, Melissa K.; McQuade, Kristi L.

2006-01-01

This article describes an undergraduate biochemistry laboratory investigating the ubiquitin-proteasome pathway in yeast. In this exercise, the enzyme beta-galactosidase (beta-gal) is expressed in yeast under the control of a stress response promoter. Following exposure to heat stress to induce beta-gal expression, cycloheximide is added to halt…

Differences in hypothalamic type 2 deiodinase ubiquitination explain localized sensitivity to thyroxine

PubMed Central

Werneck de Castro, Joao Pedro; Fonseca, Tatiana L.; Ueta, Cintia B.; McAninch, Elizabeth A.; Abdalla, Sherine; Wittmann, Gabor; Lechan, Ronald M.; Gereben, Balazs; Bianco, Antonio C.

2015-01-01

The current treatment for patients with hypothyroidism is levothyroxine (L-T4) along with normalization of serum thyroid-stimulating hormone (TSH). However, normalization of serum TSH with L-T4 monotherapy results in relatively low serum 3,5,3′-triiodothyronine (T3) and high serum thyroxine/T3 (T4/T3) ratio. In the hypothalamus-pituitary dyad as well as the rest of the brain, the majority of T3 present is generated locally by T4 deiodination via the type 2 deiodinase (D2); this pathway is self-limited by ubiquitination of D2 by the ubiquitin ligase WSB-1. Here, we determined that tissue-specific differences in D2 ubiquitination account for the high T4/T3 serum ratio in adult thyroidectomized (Tx) rats chronically implanted with subcutaneous L-T4 pellets. While L-T4 administration decreased whole-body D2-dependent T4 conversion to T3, D2 activity in the hypothalamus was only minimally affected by L-T4. In vivo studies in mice harboring an astrocyte-specific Wsb1 deletion as well as in vitro analysis of D2 ubiquitination driven by different tissue extracts indicated that D2 ubiquitination in the hypothalamus is relatively less. As a result, in contrast to other D2-expressing tissues, the hypothalamus is wired to have increased sensitivity to T4. These studies reveal that tissue-specific differences in D2 ubiquitination are an inherent property of the TRH/TSH feedback mechanism and indicate that only constant delivery of L-T4 and L-T3 fully normalizes T3-dependent metabolic markers and gene expression profiles in Tx rats. PMID:25555216

Drosophila BRUCE inhibits apoptosis through non-lysine ubiquitination of the IAP-antagonist REAPER

PubMed Central

Domingues, C; Ryoo, H D

2012-01-01

Active caspases execute apoptosis to eliminate superfluous or harmful cells in animals. In Drosophila, living cells prevent uncontrolled caspase activation through an inhibitor of apoptosis protein (IAP) family member, dIAP1, and apoptosis is preceded by the expression of IAP-antagonists, such as Reaper, Hid and Grim. Strong genetic modifiers of this pathway include another IAP family gene encoding an E2 ubiquitin conjugating enzyme domain, dBruce. Although the genetic effects of dBruce mutants are well documented, molecular targets of its encoded protein have remained elusive. Here, we report that dBruce targets Reaper for ubiquitination through an unconventional mechanism. Specifically, we show that dBruce physically interacts with Reaper, dependent upon Reaper's IAP-binding (IBM) and GH3 motifs. Consistently, Reaper levels were elevated in a dBruce −/− background. Unexpectedly, we found that dBruce also affects the levels of a mutant form of Reaper without any internal lysine residues, which normally serve as conventional ubiquitin acceptor sites. Furthermore, we were able to biochemically detect ubiquitin conjugation on lysine-deficient Reaper proteins, and knockdown of dBruce significantly reduced the extent of this ubiquitination. Our results indicate that dBruce inhibits apoptosis by promoting IAP-antagonist ubiquitination on unconventional acceptor sites. PMID:21886178

Ubiquitin in health and disease.

PubMed

Mayer, R J; Arnold, J; László, L; Landon, M; Lowe, J

1991-06-13

Studies in recent years have shown that ubiquitin has increasingly important functions in eukaryotic cells; roles which were previously not suspected in healthy and diseased cells. The interplay between molecular pathological and molecular cell biological findings has indicated that ubiquitin may be pivotal in the cell stress response in chronic degenerative and viral diseases. Furthermore, the studies have led to the notion that ubiquitination may not only serve as a signal for nonlysosomal protein degradation but may be a unifying covalent protein modification for the major intracellular protein catabolic systems; these can act to identify proteins for cytosolic proteinases or direct intact and fragmented proteins into the lysosome system for breakdown to amino acids. This unifying role could explain why ubiquitin is restricted to eukaryotic cells, which possess extensive endomembrane systems in addition to a nuclear envelope. Protein ubiquitination is a feature of most filamentous inclusions and certain other intracellular conglomerates that are found in some degenerative and viral diseases. The detection of ubiquitin-protein conjugates is not of great diagnostic importance in these diseases. Protein ubiquitination is not only essential for the normal physiological turnover of proteins but appears to have been adapted as part of an intracellular surveillance system that can be activated by altered, damaged, or foreign proteins and organelles. The purpose of this system is to isolate and eliminate these noxious structures from the cell: as a cytoprotective mechanism this appears to have evolved in the cell akin perhaps to an 'intracellular immune system'. Other heat shock proteins such as hsp 70 may be involved in this process. It is apparent that ubiquitin has a role in embryonic development. Protein ubiquitination is presumably involved in the reorganisation of cytoplasm that accompanies cell differentiation. Ubiquitin is also necessary for the gross

Ubiquitination in Periodontal Disease: A Review

PubMed Central

Tsuchida, Sachio; Satoh, Mamoru; Takiwaki, Masaki; Nomura, Fumio

2017-01-01

Periodontal disease (periodontitis) is a chronic inflammatory condition initiated by microbial infection that leads to gingival tissue destruction and alveolar bone resorption. The periodontal tissue’s response to dental plaque is characterized by the accumulation of polymorphonuclear leukocytes, macrophages, and lymphocytes, all of which release inflammatory mediators and cytokines to orchestrate the immunopathogenesis of periodontal disease. Ubiquitination is achieved by a mechanism that involves a number of factors, including an ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin–protein ligase. Ubiquitination is a post-translational modification restricted to eukaryotes that are involved in essential host processes. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Increasing numbers of recent reports have provided evidence that many approaches are delivering promising reports for discovering the relationship between ubiquitination and periodontal disease. The scope of this review was to investigate recent progress in the discovery of ubiquitinated protein in diseased periodontium and to discuss the ubiquitination process in periodontal diseases. PMID:28698506

Internalization and vacuolar targeting of the brassinosteroid hormone receptor BRI1 are regulated by ubiquitination.

PubMed

Martins, Sara; Dohmann, Esther M N; Cayrel, Anne; Johnson, Alexander; Fischer, Wolfgang; Pojer, Florence; Satiat-Jeunemaître, Béatrice; Jaillais, Yvon; Chory, Joanne; Geldner, Niko; Vert, Grégory

2015-01-21

Brassinosteroids are plant steroid hormones that control many aspects of plant growth and development, and are perceived at the cell surface by the plasma membrane-localized receptor kinase BRI1. Here we show that BRI1 is post-translationally modified by K63 polyubiquitin chains in vivo. Using both artificial ubiquitination of BRI1 and generation of an ubiquitination-defective BRI1 mutant form, we demonstrate that ubiquitination promotes BRI1 internalization from the cell surface and is essential for its recognition at the trans-Golgi network/early endosomes (TGN/EE) for vacuolar targeting. Finally, we demonstrate that the control of BRI1 protein dynamics by ubiquitination is an important control mechanism for brassinosteroid responses in plants. Altogether, our results identify ubiquitination and K63-linked polyubiquitin chain formation as a dual targeting signal for BRI1 internalization and sorting along the endocytic pathway, and highlight its role in hormonally controlled plant development.

Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, James M.; Korzhnev, Dmitry M.; Ceccarelli, Derek F.

2012-10-23

The Par-1/MARK protein kinases play a pivotal role in establishing cellular polarity. This family of kinases contains a unique domain architecture, in which a ubiquitin-associated (UBA) domain is located C-terminal to the kinase domain. We have used a combination of x-ray crystallography and NMR dynamics experiments to understand the interaction of the human (h) MARK3 UBA domain with the adjacent kinase domain as compared with ubiquitin. The x-ray crystal structure of the linked hMARK3 kinase and UBA domains establishes that the UBA domain forms a stable intramolecular interaction with the N-terminal lobe of the kinase domain. However, solution-state NMR studiesmore » of the isolated UBA domain indicate that it is highly dynamic, undergoing conformational transitions that can be explained by a folding-unfolding equilibrium. NMR titration experiments indicated that the hMARK3 UBA domain has a detectable but extremely weak affinity for mono ubiquitin, which suggests that conformational instability of the isolated hMARK3 UBA domain attenuates binding to ubiquitin despite the presence of residues typically involved in ubiquitin recognition. Our data identify a molecular mechanism through which the hMARK3 UBA domain has evolved to bind the kinase domain, in a fashion that stabilizes an open conformation of the N- and C-terminal lobes, at the expense of its capacity to engage ubiquitin. These results may be relevant more generally to the 30% of UBA domains that lack significant ubiquitin-binding activity, and they suggest a unique mechanism by which interaction domains may evolve new binding properties.« less

UUCD: a family-based database of ubiquitin and ubiquitin-like conjugation.

PubMed

Gao, Tianshun; Liu, Zexian; Wang, Yongbo; Cheng, Han; Yang, Qing; Guo, Anyuan; Ren, Jian; Xue, Yu

2013-01-01

In this work, we developed a family-based database of UUCD (http://uucd.biocuckoo.org) for ubiquitin and ubiquitin-like conjugation, which is one of the most important post-translational modifications responsible for regulating a variety of cellular processes, through a similar E1 (ubiquitin-activating enzyme)-E2 (ubiquitin-conjugating enzyme)-E3 (ubiquitin-protein ligase) enzyme thioester cascade. Although extensive experimental efforts have been taken, an integrative data resource is still not available. From the scientific literature, 26 E1s, 105 E2s, 1003 E3s and 148 deubiquitination enzymes (DUBs) were collected and classified into 1, 3, 19 and 7 families, respectively. To computationally characterize potential enzymes in eukaryotes, we constructed 1, 1, 15 and 6 hidden Markov model (HMM) profiles for E1s, E2s, E3s and DUBs at the family level, separately. Moreover, the ortholog searches were conducted for E3 and DUB families without HMM profiles. Then the UUCD database was developed with 738 E1s, 2937 E2s, 46 631 E3s and 6647 DUBs of 70 eukaryotic species. The detailed annotations and classifications were also provided. The online service of UUCD was implemented in PHP + MySQL + JavaScript + Perl.

Viral Mimicry to Usurp Ubiquitin and SUMO Host Pathways

PubMed Central

Wimmer, Peter; Schreiner, Sabrina

2015-01-01

Posttranslational modifications (PTMs) of proteins include enzymatic changes by covalent addition of cellular regulatory determinants such as ubiquitin (Ub) and small ubiquitin-like modifier (SUMO) moieties. These modifications are widely used by eukaryotic cells to control the functional repertoire of proteins. Over the last decade, it became apparent that the repertoire of ubiquitiylation and SUMOylation regulating various biological functions is not restricted to eukaryotic cells, but is also a feature of human virus families, used to extensively exploit complex host-cell networks and homeostasis. Intriguingly, besides binding to host SUMO/Ub control proteins and interfering with the respective enzymatic cascade, many viral proteins mimic key regulatory factors to usurp this host machinery and promote efficient viral outcomes. Advanced detection methods and functional studies of ubiquitiylation and SUMOylation during virus-host interplay have revealed that human viruses have evolved a large arsenal of strategies to exploit these specific PTM processes. In this review, we highlight the known viral analogs orchestrating ubiquitin and SUMO conjugation events to subvert and utilize basic enzymatic pathways. PMID:26343706

Cyclophilin A-regulated ubiquitination is critical for RIG-I-mediated antiviral immune responses.

PubMed

Liu, Wei; Li, Jing; Zheng, Weinan; Shang, Yingli; Zhao, Zhendong; Wang, Shanshan; Bi, Yuhai; Zhang, Shuang; Xu, Chongfeng; Duan, Ziyuan; Zhang, Lianfeng; Wang, Yue L; Jiang, Zhengfan; Liu, Wenjun; Sun, Lei

2017-06-08

RIG-I is a key cytosolic pattern recognition receptor that interacts with MAVS to induce type I interferons (IFNs) against RNA virus infection. In this study, we found that cyclophilin A (CypA), a peptidyl-prolyl cis/trans isomerase, functioned as a critical positive regulator of RIG-I-mediated antiviral immune responses. Deficiency of CypA impaired RIG-I-mediated type I IFN production and promoted viral replication in human cells and mice. Upon Sendai virus infection, CypA increased the interaction between RIG-I and its E3 ubiquitin ligase TRIM25, leading to enhanced TRIM25-mediated K63-linked ubiquitination of RIG-I that facilitated recruitment of RIG-I to MAVS. In addition, CypA and TRIM25 competitively interacted with MAVS, thereby inhibiting TRIM25-induced K48-linked ubiquitination of MAVS. Taken together, our findings reveal an essential role of CypA in boosting RIG-I-mediated antiviral immune responses by controlling the ubiquitination of RIG-I and MAVS.

DNA-damage-inducible 1 protein (Ddi1) contains an uncharacteristic ubiquitin-like domain that binds ubiquitin

PubMed Central

Nowicka, Urszula; Zhang, Daoning; Walker, Olivier; Krutauz, Daria; Castañeda, Carlos A.; Chaturvedi, Apurva; Chen, Tony Y.; Reis, Noa; Glickman, Michael H.; Fushman, David

2015-01-01

SUMMARY Ddi1 belongs to a family of shuttle proteins targeting polyubiquitinated substrates for proteasomal degradation. Unlike the other proteasomal shuttles, Rad23 and Dsk2, Ddi1 remains an enigma: its function is not fully understood and structural properties are poorly characterized. We determined the structure and binding properties of the ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains of Ddi1 from Saccharomyces cerevisiae. We found that, while Ddi1UBA forms a characteristic UBA:ubiquitin complex, Ddi1UBL has entirely uncharacteristic binding preferences. Despite having a ubiquitin-like fold, Ddi1UBL does not interact with typical UBL-receptors but, unexpectedly, binds ubiquitin, forming a unique interface mediated by hydrophobic contacts and by salt-bridges between oppositely-charged residues of Ddi1UBL and ubiquitin. In stark contrast with ubiquitin and other UBLs, the β-sheet surface of Ddi1UBL is negatively charged and, therefore, is recognized in a completely different way. The dual functionality of Ddi1UBL, capable of binding both ubiquitin and proteasome, suggests a novel mechanism for Ddi1 as a proteasomal shuttle. PMID:25703377

Crystal Structure of the Ubiquitin-associated (UBA) Domain of p62 and Its Interaction with Ubiquitin*

PubMed Central

Isogai, Shin; Morimoto, Daichi; Arita, Kyohei; Unzai, Satoru; Tenno, Takeshi; Hasegawa, Jun; Sou, Yu-shin; Komatsu, Masaaki; Tanaka, Keiji; Shirakawa, Masahiro; Tochio, Hidehito

2011-01-01

p62/SQSTM1/A170 is a multimodular protein that is found in ubiquitin-positive inclusions associated with neurodegenerative diseases. Recent findings indicate that p62 mediates the interaction between ubiquitinated proteins and autophagosomes, leading these proteins to be degraded via the autophagy-lysosomal pathway. This ubiquitin-mediated selective autophagy is thought to begin with recognition of the ubiquitinated proteins by the C-terminal ubiquitin-associated (UBA) domain of p62. We present here the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form. The p62 UBA domain adopts a novel dimeric structure in crystals, which is distinctive from those of other UBA domains. NMR analyses reveal that in solution the domain exists in equilibrium between the dimer and monomer forms, and binding ubiquitin shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The dimer-to-monomer transition is associated with a structural change of the very C-terminal end of the p62 UBA domain, although the UBA fold itself is essentially maintained. Our data illustrate that dimerization and ubiquitin binding of the p62 UBA domain are incompatible with each other. These observations reveal an autoinhibitory mechanism in the p62 UBA domain and suggest that autoinhibition plays a role in the function of p62. PMID:21715324

Assembly and Function of Heterotypic Ubiquitin Chains in Cell-Cycle and Protein Quality Control.

PubMed

Yau, Richard G; Doerner, Kerstin; Castellanos, Erick R; Haakonsen, Diane L; Werner, Achim; Wang, Nan; Yang, X William; Martinez-Martin, Nadia; Matsumoto, Marissa L; Dixit, Vishva M; Rape, Michael

2017-11-02

Posttranslational modification with ubiquitin chains controls cell fate in all eukaryotes. Depending on the connectivity between subunits, different ubiquitin chain types trigger distinct outputs, as seen with K48- and K63-linked conjugates that drive protein degradation or complex assembly, respectively. Recent biochemical analyses also suggested roles for mixed or branched ubiquitin chains, yet without a method to monitor endogenous conjugates, the physiological significance of heterotypic polymers remained poorly understood. Here, we engineered a bispecific antibody to detect K11/K48-linked chains and identified mitotic regulators, misfolded nascent polypeptides, and pathological Huntingtin variants as their endogenous substrates. We show that K11/K48-linked chains are synthesized and processed by essential ubiquitin ligases and effectors that are mutated across neurodegenerative diseases; accordingly, these conjugates promote rapid proteasomal clearance of aggregation-prone proteins. By revealing key roles of K11/K48-linked chains in cell-cycle and quality control, we establish heterotypic ubiquitin conjugates as important carriers of biological information. Copyright © 2017 Elsevier Inc. All rights reserved.

Ubiquitin enzymes in the regulation of immune responses.

PubMed

Ebner, Petra; Versteeg, Gijs A; Ikeda, Fumiyo

2017-08-01

Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses.

Targeting ubiquitination for cancer therapies.

PubMed

Morrow, John Kenneth; Lin, Hui-Kuan; Sun, Shao-Cong; Zhang, Shuxing

2015-01-01

Ubiquitination, the structured degradation and turnover of cellular proteins, is regulated by the ubiquitin-proteasome system (UPS). Most proteins that are critical for cellular regulations and functions are targets of the process. Ubiquitination is comprised of a sequence of three enzymatic steps, and aberrations in the pathway can lead to tumor development and progression as observed in many cancer types. Recent evidence indicates that targeting the UPS is effective for certain cancer treatment, but many more potential targets might have been previously overlooked. In this review, we will discuss the current state of small molecules that target various elements of ubiquitination. Special attention will be given to novel inhibitors of E3 ubiquitin ligases, especially those in the SCF family.

E3 ubiquitin ligase CHIP interacts with C-type lectin-like receptor CLEC-2 and promotes its ubiquitin-proteasome degradation.

PubMed

Shao, Miaomiao; Li, Lili; Song, Shushu; Wu, Weicheng; Peng, Peike; Yang, Caiting; Zhang, Mingming; Duan, Fangfang; Jia, Dongwei; Zhang, Jie; Wu, Hao; Zhao, Ran; Wang, Lan; Ruan, Yuanyuan; Gu, Jianxin

2016-10-01

C-type lectin-like receptor 2 (CLEC-2) was originally identified as a member of non-classical C-type lectin-like receptors in platelets and immune cells. Activation of CLEC-2 is involved in thrombus formation, lymphatic/blood vessel separation, platelet-mediated tumor metastasis and immune response. Nevertheless, the regulation of CLEC-2 expression is little understood. In this study, we identified that the C terminus of Hsc70-interacting protein (CHIP) interacted with CLEC-2 by mass spectrometry analysis, and CHIP decreased the protein expression of CLEC-2 through lysine-48-linked ubiquitination and proteasomal degradation. Deleted and point mutation also revealed that CHIP controlled CLEC-2 protein expression via both tetratricopeptide repeats (TPR) domain and Ubox domain in a HSP70/90-independent manner. Moreover, reduced CHIP expression was associated with decreased CLEC-2 polyubiquitination and increased CLEC-2 protein levels in PMA-induced differentiation of THP-1 monocytes into macrophages. These results indicate that CLEC-2 is the target substrate of E3 ubiquitin ligase CHIP, and suggest that the CHIP/CLEC-2 axis may play an important role in the modulation of immune response. Copyright © 2016 Elsevier Inc. All rights reserved.

Ubiquitin enzymes in the regulation of immune responses

PubMed Central

Ebner, Petra; Versteeg, Gijs A.; Ikeda, Fumiyo

2017-01-01

Abstract Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses. PMID:28524749

The APC/C Ubiquitin Ligase: From Cell Biology to Tumorigenesis

PubMed Central

Penas, Clara; Ramachandran, Vimal; Ayad, Nagi George

2011-01-01

The ubiquitin proteasome system (UPS) is required for normal cell proliferation, vertebrate development, and cancer cell transformation. The UPS consists of multiple proteins that work in concert to target a protein for degradation via the 26S proteasome. Chains of an 8.5-kDa protein called ubiquitin are attached to substrates, thus allowing recognition by the 26S proteasome. Enzymes called ubiquitin ligases or E3s mediate specific attachment to substrates. Although there are over 600 different ubiquitin ligases, the Skp1–Cullin–F-box (SCF) complexes and the anaphase promoting complex/cyclosome (APC/C) are the most studied. SCF involvement in cancer has been known for some time while APC/C’s cancer role has recently emerged. In this review we will discuss the importance of APC/C to normal cell proliferation and development, underscoring its possible contribution to transformation. We will also examine the hypothesis that modulating a specific interaction of the APC/C may be therapeutically attractive in specific cancer subtypes. Finally, given that the APC/C pathway is relatively new as a cancer target, therapeutic interventions affecting APC/C activity may be beneficial in cancers that are resistant to classical chemotherapy. PMID:22655255

A single ubiquitin is sufficient for cargo protein entry into MVBs in the absence of ESCRT ubiquitination

PubMed Central

Stringer, Daniel K.

2011-01-01

ESCRTs (endosomal sorting complexes required for transport) bind and sequester ubiquitinated membrane proteins and usher them into multivesicular bodies (MVBs). As Ubiquitin (Ub)-binding proteins, ESCRTs themselves become ubiquitinated. However, it is unclear whether this regulates a critical aspect of their function or is a nonspecific consequence of their association with the Ub system. We investigated whether ubiquitination of the ESCRTs was required for their ability to sort cargo into the MVB lumen. Although we found that Rsp5 was the main Ub ligase responsible for ubiquitination of ESCRT-0, elimination of Rsp5 or elimination of the ubiquitinatable lysines within ESCRT-0 did not affect MVB sorting. Moreover, by fusing the catalytic domain of deubiquitinating peptidases onto ESCRTs, we could block ESCRT ubiquitination and the sorting of proteins that undergo Rsp5-dependent ubiquitination. Yet, proteins fused to a single Ub moiety were efficiently delivered to the MVB lumen, which strongly indicates that a single Ub is sufficient in sorting MVBs in the absence of ESCRT ubiquitination. PMID:21242292

How Chemical Synthesis of Ubiquitin Conjugates Helps To Understand Ubiquitin Signal Transduction.

PubMed

Hameed, Dharjath S; Sapmaz, Aysegul; Ovaa, Huib

2017-03-15

Ubiquitin (Ub) is a small post-translational modifier protein involved in a myriad of biochemical processes including DNA damage repair, proteasomal proteolysis, and cell cycle control. Ubiquitin signaling pathways have not been completely deciphered due to the complex nature of the enzymes involved in ubiquitin conjugation and deconjugation. Hence, probes and assay reagents are important to get a better understanding of this pathway. Recently, improvements have been made in synthesis procedures of Ub derivatives. In this perspective, we explain various research reagents available and how chemical synthesis has made an important contribution to Ub research.

Dynamic survey of mitochondria by ubiquitin

PubMed Central

Escobar-Henriques, Mafalda; Langer, Thomas

2014-01-01

Ubiquitin is a post-translational modifier with proteolytic and non-proteolytic roles in many biological processes. At mitochondria, it performs regulatory homeostatic functions and contributes to mitochondrial quality control. Ubiquitin is essential for mitochondrial fusion, regulates mitochondria-ER contacts, and participates in maternal mtDNA inheritance. Under stress, mitochondrial dysfunction induces ubiquitin-dependent responses that involve mitochondrial proteome remodeling and culminate in organelle removal by mitophagy. In addition, many ubiquitin-dependent mechanisms have been shown to regulate innate immune responses and xenophagy. Here, we review the emerging roles of ubiquitin at mitochondria. PMID:24569520

Dynamic ubiquitin signaling in cell cycle regulation

PubMed Central

Gilberto, Samuel

2017-01-01

The cell division cycle is driven by a collection of enzymes that coordinate DNA duplication and separation, ensuring that genomic information is faithfully and perpetually maintained. The activity of the effector proteins that perform and coordinate these biological processes oscillates by regulated expression and/or posttranslational modifications. Ubiquitylation is a cardinal cellular modification and is long known for driving cell cycle transitions. In this review, we emphasize emerging concepts of how ubiquitylation brings the necessary dynamicity and plasticity that underlie the processes of DNA replication and mitosis. New studies, often focusing on the regulation of chromosomal proteins like DNA polymerases or kinetochore kinases, are demonstrating that ubiquitylation is a versatile modification that can be used to fine-tune these cell cycle events, frequently through processes that do not involve proteasomal degradation. Understanding how the increasing variety of identified ubiquitin signals are transduced will allow us to develop a deeper mechanistic perception of how the multiple factors come together to faithfully propagate genomic information. Here, we discuss these and additional conceptual challenges that are currently under study toward understanding how ubiquitin governs cell cycle regulation. PMID:28684425

Dynamic ubiquitin signaling in cell cycle regulation.

PubMed

Gilberto, Samuel; Peter, Matthias

2017-08-07

The cell division cycle is driven by a collection of enzymes that coordinate DNA duplication and separation, ensuring that genomic information is faithfully and perpetually maintained. The activity of the effector proteins that perform and coordinate these biological processes oscillates by regulated expression and/or posttranslational modifications. Ubiquitylation is a cardinal cellular modification and is long known for driving cell cycle transitions. In this review, we emphasize emerging concepts of how ubiquitylation brings the necessary dynamicity and plasticity that underlie the processes of DNA replication and mitosis. New studies, often focusing on the regulation of chromosomal proteins like DNA polymerases or kinetochore kinases, are demonstrating that ubiquitylation is a versatile modification that can be used to fine-tune these cell cycle events, frequently through processes that do not involve proteasomal degradation. Understanding how the increasing variety of identified ubiquitin signals are transduced will allow us to develop a deeper mechanistic perception of how the multiple factors come together to faithfully propagate genomic information. Here, we discuss these and additional conceptual challenges that are currently under study toward understanding how ubiquitin governs cell cycle regulation. © 2017 Gilberto and Peter.

Ubiquitin-dependent endocytosis, trafficking and turnover of neuronal membrane proteins

PubMed Central

Schwarz, Lindsay A.; Patrick, Gentry N.

2011-01-01

Extracellular signaling between cells is often transduced via receptors that reside at the cell membrane. In neurons this receptor-mediated signaling can promote a variety of cellular events such as differentiation, axon outgrowth and guidance, synaptic development and function. Endocytic membrane trafficking of receptors can ensure that the strength and duration of an extracellular signal is properly regulated. The covalent modification of membrane proteins by ubiquitin is a key biological mechanism to control receptor internalization and endocytic sorting to recycling and degradative pathways in many cell types. In this review we highlight recent findings regarding the ubiquitin-dependent trafficking and turnover of receptors in neurons and the implications for neuronal development and function. PMID:21884797

Analyses of Ca2+ dynamics using a ubiquitin-10 promoter-driven Yellow Cameleon 3.6 indicator reveal reliable transgene expression and differences in cytoplasmic Ca2+ responses in Arabidopsis and rice (Oryza sativa) roots.

PubMed

Behera, Smrutisanjita; Wang, Nili; Zhang, Chunxia; Schmitz-Thom, Ina; Strohkamp, Sarah; Schültke, Stefanie; Hashimoto, Kenji; Xiong, Lizhong; Kudla, Jörg

2015-04-01

Ca(2+) signatures are central to developmental processes and adaptive responses in plants. However, high-resolution studies of Ca(2+) dynamics using genetically encoded Ca(2+) indicators (GECIs) such as Yellow Cameleon (YC) proteins have so far not been conducted in important model crops such as rice (Oryza sativa). We conducted a comparative study of 35S and ubiquitin-10 (UBQ10) promoter functionality in Arabidopsis thaliana and O. sativa plants expressing the Ca(2+) indicator Yellow Cameleon 3.6 (YC3.6) under control of the UBQ10 or 35S promoter. Ca(2+) signatures in roots of both species were analyzed during exposure to hyperpolarization/depolarization cycles or in response to application of the amino acid glutamate. We found a superior performance of the UBQ10 promoter with regard to expression pattern, levels and expression stabilities in both species. We observed remarkable differences between the two species in the spatiotemporal parameters of the observed Ca(2+) signatures. Rice appeared in general to respond with a lower maximal signal amplitude but greatly increased signal duration when compared with Arabidopsis. Our results identify important advantages to using the UBQ10 promoter in Arabidopsis and rice and in T-DNA mutant backgrounds. Moreover, the observed differences in Ca(2+) signaling in the two species underscore the need for comparative studies to achieve a comprehensive understanding of Ca(2+) signaling in plants. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

The E3 ubiquitin ligase Trim7 mediates c-Jun/AP-1 activation by Ras signalling

PubMed Central

Chakraborty, Atanu; Diefenbacher, Markus E.; Mylona, Anastasia; Kassel, Olivier; Behrens, Axel

2015-01-01

The c-Jun/AP-1 transcription factor controls key cellular behaviours, including proliferation and apoptosis, in response to JNK and Ras/MAPK signalling. While the JNK pathway has been well characterised, the mechanism of activation by Ras was elusive. Here we identify the uncharacterised ubiquitin ligase Trim7 as a critical component of AP-1 activation via Ras. We found that MSK1 directly phosphorylates Trim7 in response to direct activation by the Ras–Raf–MEK–ERK pathway, and this modification stimulates Trim7 E3 ubiquitin ligase activity. Trim7 mediates Lys63-linked ubiquitination of the AP-1 coactivator RACO-1, leading to RACO-1 protein stabilisation. Consequently, Trim7 depletion reduces RACO-1 levels and AP-1-dependent gene expression. Moreover, transgenic overexpression of Trim7 increases lung tumour burden in a Ras-driven cancer model, and knockdown of Trim7 in established xenografts reduces tumour growth. Thus, phosphorylation-ubiquitination crosstalk between MSK1, Trim7 and RACO-1 completes the long sought-after mechanism linking growth factor signalling and AP-1 activation. PMID:25851810

Rabex-5 ubiquitin ligase activity restricts Ras signaling to establish pathway homeostasis in Drosophila.

PubMed

Yan, Hua; Jahanshahi, Maryam; Horvath, Elizabeth A; Liu, Hsiu-Yu; Pfleger, Cathie M

2010-08-10

The Ras signaling pathway allows cells to translate external cues into diverse biological responses. Depending on context and the threshold reached, Ras signaling can promote growth, proliferation, differentiation, or cell survival. Failure to maintain precise control of Ras can have adverse physiological consequences. Indeed, excess Ras signaling disrupts developmental patterning and causes developmental disorders [1, 2], and in mature tissues, it can lead to cancer [3-5]. We identify Rabex-5 as a new component of Ras signaling crucial for achieving proper pathway outputs in multiple contexts in vivo. We show that Drosophila Rabex-5 restricts Ras signaling to establish organism size, wing vein pattern, and eye versus antennal fate. Rabex-5 has both Rab5 guanine nucleotide exchange factor (GEF) activity that regulates endocytic trafficking [6] and ubiquitin ligase activity [7, 8]. Surprisingly, overexpression studies demonstrate that Rabex-5 ubiquitin ligase activity, not its Rab5 GEF activity, is required to restrict wing vein specification and to suppress the eye phenotypes of oncogenic Ras expression. Furthermore, genetic interaction experiments indicate that Rabex-5 acts at the step of Ras, and tissue culture studies show that Rabex-5 promotes Ras ubiquitination. Together, these findings reveal a new mechanism for attenuating Ras signaling in vivo and suggest an important role for Rabex-5-mediated Ras ubiquitination in pathway homeostasis. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

RING-type E3 ligases: Master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination

PubMed Central

Metzger, Meredith B.; Pruneda, Jonathan N.; Klevit, Rachel E.; Weissman, Allan M.

2013-01-01

RING finger domain and RING finger-like ubiquitin ligases (E3s), such as U-box proteins, constitute the vast majority of known E3s. RING-type E3s function together with ubiquitin-conjugating enzymes (E2s) to mediate ubiquitination and are implicated in numerous cellular processes. In part because of their importance in human physiology and disease, these proteins and their cellular functions represent an intense area of study. Here we review recent advances in RING-type E3 recognition of substrates, their cellular regulation, and their varied architecture. Additionally, recent structural insights into RING-type E3 function, with a focus on important interactions with E2s and ubiquitin, are reviewed. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. PMID:23747565

Ubiquitin acetylation inhibits polyubiquitin chain elongation

PubMed Central

Ohtake, Fumiaki; Saeki, Yasushi; Sakamoto, Kensaku; Ohtake, Kazumasa; Nishikawa, Hiroyuki; Tsuchiya, Hikaru; Ohta, Tomohiko; Tanaka, Keiji; Kanno, Jun

2015-01-01

Ubiquitylation is a versatile post-translational modification (PTM). The diversity of ubiquitylation topologies, which encompasses different chain lengths and linkages, underlies its widespread cellular roles. Here, we show that endogenous ubiquitin is acetylated at lysine (K)-6 (AcK6) or K48. Acetylated ubiquitin does not affect substrate monoubiquitylation, but inhibits K11-, K48-, and K63-linked polyubiquitin chain elongation by several E2 enzymes in vitro. In cells, AcK6-mimetic ubiquitin stabilizes the monoubiquitylation of histone H2B—which we identify as an endogenous substrate of acetylated ubiquitin—and of artificial ubiquitin fusion degradation substrates. These results characterize a mechanism whereby ubiquitin, itself a PTM, is subject to another PTM to modulate mono- and polyubiquitylation, thus adding a new regulatory layer to ubiquitin biology. PMID:25527407

Smurf2 negatively modulates RIG-I-dependent antiviral response by targeting VISA/MAVS for ubiquitination and degradation.

PubMed

Pan, Yu; Li, Rui; Meng, Jun-Ling; Mao, He-Ting; Zhang, Yu; Zhang, Jun

2014-05-15

VISA (also known as MAVS, Cardif, IPS-1) is the essential adaptor protein for virus-induced activation of IFN regulatory factors 3 and 7 and production of type I IFNs. Understanding the regulatory mechanisms for VISA will provide detailed insights into the positive or negative regulation of innate immune responses. In this study, we identified Smad ubiquitin regulatory factor (Smurf) 2, one of the Smad ubiquitin regulator factor proteins, as an important negative regulator of virus-triggered type I IFN signaling, which targets at the VISA level. Overexpression of Smurf2 inhibits virus-induced IFN-β and IFN-stimulated response element activation. The E3 ligase defective mutant Smurf2/C716A loses the ability to suppress virus-induced type I IFN signaling, suggesting that the negative regulation is dependent on the ubiquitin E3 ligase activity of Smurf2. Further studies demonstrated that Smurf2 interacted with VISA and targeted VISA for K48-linked ubiquitination, which promoted the degradation of VISA. Consistently, knockout or knockdown of Smurf2 expression therefore promoted antiviral signaling, which was correlated with the increase in protein stability of VISA. Our findings suggest that Smurf2 is an important nonredundant negative regulator of virus-triggered type I IFN signaling by targeting VISA for K48-linked ubiquitination and degradation.

Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Imura, Yoshiyuki, E-mail: imura@brs.nihon-u.ac.jp; Molho, Melissa; Chuang, Chingkai

Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement bothmore » defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly. - Highlights: • Tombusvirus p33 replication protein interacts with cellular RAD6/Ubc2 E2 enzymes. • Deletion of RAD6 reduces tombusvirus replication in yeast. • Silencing of UBC2 in plants inhibits tombusvirus replication. • Mono- and bi-ubiquitination of p33 replication protein in yeast and in vitro. • Rad6p promotes the recruitment of cellular ESCRT proteins into the tombusvirus replicase.« less

Central catalytic domain of BRAP (RNF52) recognizes the types of ubiquitin chains and utilizes oligo-ubiquitin for ubiquitylation.

PubMed

Shoji, Shisako; Hanada, Kazuharu; Ohsawa, Noboru; Shirouzu, Mikako

2017-09-07

Really interesting new gene (RING)-finger protein 52 (RNF52), an E3 ubiquitin ligase, is found in eukaryotes from yeast to humans. Human RNF52 is known as breast cancer type 1 susceptibility protein (BRCA1)-associated protein 2 (BRAP or BRAP2). The central catalytic domain of BRAP comprises four subdomains: nucleotide-binding α/β plait (NBP), really interesting new gene (RING) zinc finger, ubiquitin-specific protease (UBP)-like zinc finger (ZfUBP), and coiled-coil (CC). This domain architecture is conserved in RNF52 orthologs; however, the domain's function in the ubiquitin system has not been delineated. In the present study, we discovered that the RNF52 domain, comprising NBP-RING-ZfUBP-CC, binds to ubiquitin chains (oligo-ubiquitin) but not to the ubiquitin monomers, and can utilize various ubiquitin chains for ubiquitylation and auto-ubiquitylation. The RNF52 domain preferentially bound to M1- and K63-linked di-ubiquitin chains, weakly to K27-linked chains, but not to K6-, K11-, or K48-linked chains. The binding preferences of the RNF52 domain for ubiquitin-linkage types corresponded to ubiquitin usage in the ubiquitylation reaction, except for K11-, K29-, and K33-linked chains. Additionally, the RNF52 domain directly ligated the intact M1-linked, tri-, and tetra-ubiquitin chains and recognized the structural alterations caused by the phosphomimetic mutation of these ubiquitin chains. Full-length BRAP had nearly the same specificity for the ubiquitin-chain types as the RNF52 domain alone. Mass spectrometry analysis of oligomeric ubiquitylation products, mediated by the RNF52 domain, revealed that the ubiquitin-linkage types and auto-ubiquitylation sites depend on the length of ubiquitin chains. Here, we propose a model for the oligomeric ubiquitylation process, controlled by the RNF52 domain, which is not a sequential assembly process involving monomers. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Central catalytic domain of BRAP (RNF52) recognizes the types of ubiquitin chains and utilizes oligo-ubiquitin for ubiquitylation

PubMed Central

Hanada, Kazuharu; Ohsawa, Noboru

2017-01-01

Really interesting new gene (RING)-finger protein 52 (RNF52), an E3 ubiquitin ligase, is found in eukaryotes from yeast to humans. Human RNF52 is known as breast cancer type 1 susceptibility protein (BRCA1)-associated protein 2 (BRAP or BRAP2). The central catalytic domain of BRAP comprises four subdomains: nucleotide-binding α/β plait (NBP), really interesting new gene (RING) zinc finger, ubiquitin-specific protease (UBP)-like zinc finger (ZfUBP), and coiled-coil (CC). This domain architecture is conserved in RNF52 orthologs; however, the domain's function in the ubiquitin system has not been delineated. In the present study, we discovered that the RNF52 domain, comprising NBP–RING–ZfUBP–CC, binds to ubiquitin chains (oligo-ubiquitin) but not to the ubiquitin monomers, and can utilize various ubiquitin chains for ubiquitylation and auto-ubiquitylation. The RNF52 domain preferentially bound to M1- and K63-linked di-ubiquitin chains, weakly to K27-linked chains, but not to K6-, K11-, or K48-linked chains. The binding preferences of the RNF52 domain for ubiquitin-linkage types corresponded to ubiquitin usage in the ubiquitylation reaction, except for K11-, K29-, and K33-linked chains. Additionally, the RNF52 domain directly ligated the intact M1-linked, tri-, and tetra-ubiquitin chains and recognized the structural alterations caused by the phosphomimetic mutation of these ubiquitin chains. Full-length BRAP had nearly the same specificity for the ubiquitin-chain types as the RNF52 domain alone. Mass spectrometry analysis of oligomeric ubiquitylation products, mediated by the RNF52 domain, revealed that the ubiquitin-linkage types and auto-ubiquitylation sites depend on the length of ubiquitin chains. Here, we propose a model for the oligomeric ubiquitylation process, controlled by the RNF52 domain, which is not a sequential assembly process involving monomers. PMID:28768733

Deciphering the Ubiquitin Code.

PubMed

Dittmar, Gunnar; Selbach, Matthias

2017-03-02

In this issue of Molecular Cell, Zhang et al. (2017) systematically identify proteins interacting with all possible di-ubiquitin linkages, thus providing a catalog of readers of the ubiquitin code. Copyright © 2017 Elsevier Inc. All rights reserved.

Selective autophagy: ubiquitin-mediated recognition and beyond.

PubMed

Kraft, Claudine; Peter, Matthias; Hofmann, Kay

2010-09-01

Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degradation pathways. Whereas the ubiquitin-proteasome system is involved in the rapid degradation of proteins, autophagy pathways can selectively remove protein aggregates and damaged or excess organelles. Proteasome-mediated degradation requires previous ubiquitylation of the cargo, which is then recognized by ubiquitin receptors directing it to 26S proteasomes. Although autophagy has long been viewed as a random cytoplasmic degradation system, the involvement of ubiquitin as a specificity factor for selective autophagy is rapidly emerging. Recent evidence also suggests active crosstalk between proteasome-mediated degradation and selective autophagy. Here, we discuss the molecular mechanisms that link autophagy and the proteasome system, as well as the emerging roles of ubiquitin and ubiquitin-binding proteins in selective autophagy. On the basis of the evolutionary history of autophagic ubiquitin receptors, we propose a common origin for metazoan ubiquitin-dependent autophagy and the cytoplasm-to-vacuole targeting pathway of yeast.

Cyclophilin A-regulated ubiquitination is critical for RIG-I-mediated antiviral immune responses

PubMed Central

Liu, Wei; Li, Jing; Zheng, Weinan; Shang, Yingli; Zhao, Zhendong; Wang, Shanshan; Bi, Yuhai; Zhang, Shuang; Xu, Chongfeng; Duan, Ziyuan; Zhang, Lianfeng; Wang, Yue L; Jiang, Zhengfan; Liu, Wenjun; Sun, Lei

2017-01-01

RIG-I is a key cytosolic pattern recognition receptor that interacts with MAVS to induce type I interferons (IFNs) against RNA virus infection. In this study, we found that cyclophilin A (CypA), a peptidyl-prolyl cis/trans isomerase, functioned as a critical positive regulator of RIG-I-mediated antiviral immune responses. Deficiency of CypA impaired RIG-I-mediated type I IFN production and promoted viral replication in human cells and mice. Upon Sendai virus infection, CypA increased the interaction between RIG-I and its E3 ubiquitin ligase TRIM25, leading to enhanced TRIM25-mediated K63-linked ubiquitination of RIG-I that facilitated recruitment of RIG-I to MAVS. In addition, CypA and TRIM25 competitively interacted with MAVS, thereby inhibiting TRIM25-induced K48-linked ubiquitination of MAVS. Taken together, our findings reveal an essential role of CypA in boosting RIG-I-mediated antiviral immune responses by controlling the ubiquitination of RIG-I and MAVS. DOI: http://dx.doi.org/10.7554/eLife.24425.001 PMID:28594325

PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination

PubMed Central

Barrera, Susana P.; Castrejon-Tellez, Vicente; Trinidad, Margarita; Robles-Escajeda, Elisa; Vargas-Medrano, Javier; Varela-Ramirez, Armando; Miranda, Manuel

2015-01-01

Neurotransmitter transporter ubiquitination is emerging as the main mechanism for endocytosis and sorting of cargo into lysosomes. In this study, we demonstrate PKC-dependent ubiquitination of three different isoforms of the glycine transporter 1 (GlyT1). Incubation of cells expressing transporter with the PKC activator phorbol ester induced a dramatic, time-dependent increase in GlyT1 ubiquitination, followed by accumulation of GlyT1 in EEA1 positive early endosomes. This occurred via a mechanism that was abolished by inhibition of PKC. GlyT1 endocytosis was confirmed in both retinal sections and primary cultures of mouse amacrine neurons. Replacement of only all lysines in the N-and C-termini to arginines prevented ubiquitination and endocytosis, displaying redundancy in the mechanism of ubiquitination. Interestingly, a 40–50% reduction in glycine uptake was detected in phorbol-ester stimulated cells expressing the WT-GlyT1, whereas no significant change was for the mutant protein, demonstrating that endocytosis participates in the reduction of uptake. Consistent with previous findings for the dopamine transporter DAT, ubiquitination of GlyT1 tails functions as sorting signal to deliver transporter into the lysosome and removal of ubiquitination sites dramatically attenuated the rate of GlyT1 degradation. Finally, we showed for the first time that PKC-dependent GlyT1 phosphorylation was not affected by removal of ubiquitination sites, suggesting separate PKC-dependent signaling events for these posttranslational modifications. PMID:26418248

The ubiquitin-specific protease USP15 promotes RIG-I-mediated antiviral signaling by deubiquitylating TRIM25.

PubMed

Pauli, Eva-Katharina; Chan, Ying Kai; Davis, Meredith E; Gableske, Sebastian; Wang, May K; Feister, Katharina F; Gack, Michaela U

2014-01-07

Ubiquitylation is an important mechanism for regulating innate immune responses to viral infections. Attachment of lysine 63 (Lys(63))-linked ubiquitin chains to the RNA sensor retinoic acid-inducible gene-I (RIG-I) by the ubiquitin E3 ligase tripartite motif protein 25 (TRIM25) leads to the activation of RIG-I and stimulates production of the antiviral cytokines interferon-α (IFN-α) and IFN-β. Conversely, Lys(48)-linked ubiquitylation of TRIM25 by the linear ubiquitin assembly complex (LUBAC) stimulates the proteasomal degradation of TRIM25, thereby inhibiting the RIG-I signaling pathway. Here, we report that ubiquitin-specific protease 15 (USP15) deubiquitylates TRIM25, preventing the LUBAC-dependent degradation of TRIM25. Through protein purification and mass spectrometry analysis, we identified USP15 as an interaction partner of TRIM25 in human cells. Knockdown of endogenous USP15 by specific small interfering RNA markedly enhanced the ubiquitylation of TRIM25. In contrast, expression of wild-type USP15, but not its catalytically inactive mutant, reduced the Lys(48)-linked ubiquitylation of TRIM25, leading to its stabilization. Furthermore, ectopic expression of USP15 enhanced the TRIM25- and RIG-I-dependent production of type I IFN and suppressed RNA virus replication. In contrast, depletion of USP15 resulted in decreased IFN production and markedly enhanced viral replication. Together, these data identify USP15 as a critical regulator of the TRIM25- and RIG-I-mediated antiviral immune response, thereby highlighting the intricate regulation of innate immune signaling.

The Ubiquitin-Specific Protease USP15 Promotes RIG-I–Mediated Antiviral Signaling by Deubiquitylating TRIM25

PubMed Central

Pauli, Eva-Katharina; Chan, Ying Kai; Davis, Meredith E.; Gableske, Sebastian; Wang, May K.; Feister, Katharina F.; Gack, Michaela U.

2014-01-01

Ubiquitylation is an important mechanism for regulating innate immune responses to viral infections. Attachment of lysine 63 (Lys63)–linked ubiquitin chains to the RNA sensor retinoic acid–inducible gene-I (RIG-I) by the ubiquitin E3 ligase tripartite motif protein 25 (TRIM25) leads to the activation of RIG-I and stimulates production of the antiviral cytokines interferon-α (IFN-α) and IFN-β. Conversely, Lys48-linked ubiquitylation of TRIM25 by the linear ubiquitin assembly complex (LUBAC) stimulates the proteasomal degradation of TRIM25, thereby inhibiting the RIG-I signaling pathway. Here, we report that ubiquitin-specific protease 15 (USP15) deubiquitylates TRIM25, preventing the LUBAC-dependent degradation of TRIM25. Through protein purification and mass spectrometry analysis, we identified USP15 as an interaction partner of TRIM25 in human cells. Knockdown of endogenous USP15 by specific small interfering RNA markedly enhanced the ubiquitylation of TRIM25. In contrast, expression of wild-type USP15, but not its catalytically inactive mutant, reduced the Lys48-linked ubiquitylation of TRIM25, leading to its stabilization. Furthermore, ectopic expression of USP15 enhanced the TRIM25- and RIG-I–dependent production of type I IFN and suppressed RNA virus replication. In contrast, depletion of USP15 resulted in decreased IFN production and markedly enhanced viral replication. Together, these data identify USP15 as a critical regulator of the TRIM25- and RIG-I–mediated antiviral immune response, thereby highlighting the intricate regulation of innate immune signaling. PMID:24399297

Structural Motifs Involved in Ubiquitin-Mediated Processing of the NF-κB Precursor p105: Roles of the Glycine-Rich Region and a Downstream Ubiquitination Domain

PubMed Central

Orian, Amir; Schwartz, Alan L.; Israël, Alain; Whiteside, Simon; Kahana, Chaim; Ciechanover, Aaron

1999-01-01

The ubiquitin proteolytic system plays a major role in a variety of basic cellular processes. In the majority of these processes, the target proteins are completely degraded. In one exceptional case, generation of the p50 subunit of the transcriptional regulator NF-κB, the precursor protein p105 is processed in a limited manner: the N-terminal domain yields the p50 subunit, whereas the C-terminal domain is degraded. The identity of the mechanisms involved in this unique process have remained elusive. It has been shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is an important processing signal. Here we show that the GRR does not interfere with conjugation of ubiquitin to p105 but probably does interfere with the processing of the ubiquitin-tagged precursor by the 26S proteasome. Structural analysis reveals that a short sequence containing a few Gly residues and a single essential Ala is sufficient to generate p50. Mechanistically, the presence of the GRR appears to stop further degradation of p50 and to stabilize the molecule. It appears that the localization of the GRR within p105 plays an important role in directing processing: transfer of the GRR within p105 or insertion of the GRR into homologous or heterologous proteins is not sufficient to promote processing in most cases, which is probably due to the requirement for an additional specific ubiquitination and/or recognition domain(s). Indeed, we have shown that amino acid residues 441 to 454 are important for processing. In particular, both Lys 441 and Lys 442 appear to serve as major ubiquitination targets, while residues 446 to 454 are independently important for processing and may serve as the ubiquitin ligase recognition motif. PMID:10207090

Ubiquitinated Proteome: Ready for Global?*

PubMed Central

Shi, Yi; Xu, Ping; Qin, Jun

2011-01-01

Ubiquitin (Ub) is a small and highly conserved protein that can covalently modify protein substrates. Ubiquitination is one of the major post-translational modifications that regulate a broad spectrum of cellular functions. The advancement of mass spectrometers as well as the development of new affinity purification tools has greatly expedited proteome-wide analysis of several post-translational modifications (e.g. phosphorylation, glycosylation, and acetylation). In contrast, large-scale profiling of lysine ubiquitination remains a challenge. Most recently, new Ub affinity reagents such as Ub remnant antibody and tandem Ub binding domains have been developed, allowing for relatively large-scale detection of several hundreds of lysine ubiquitination events in human cells. Here we review different strategies for the identification of ubiquitination site and discuss several issues associated with data analysis. We suggest that careful interpretation and orthogonal confirmation of MS spectra is necessary to minimize false positive assignments by automatic searching algorithms. PMID:21339389

Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin

PubMed Central

Im, Eunju; Yoo, Lang; Hyun, Minju; Shin, Woo Hyun

2016-01-01

Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the pars compacta of the substantia nigra and accumulation of ubiquitinated proteins in aggregates called Lewy bodies. Several mutated genes have been found in familial PD patients, including SNCA (α-synuclein), PARK2 (parkin), PINK1, PARK7 (DJ-1), LRRK2 and ATP13A2. Many pathogenic mutations of PARK2, which encodes the ubiquitin E3 ligase parkin, result in loss of function, leading to accumulation of parkin substrates and consequently contributing to dopaminergic cell death. ISG15 is a member of the ubiquitin-like modifier family and is induced by stimulation with type I interferons. Similar to ubiquitin and ubiquitination, covalent conjugation of ISG15 to target proteins (ISGylation) regulates their biochemical properties. In this study, we identified parkin as a novel target of ISGylation specifically mediated by the ISG15-E3 ligase HERC5. In addition, we identified two ISGylation sites, Lys-349 and Lys-369, in the in-between-ring domain of parkin. ISGylation of these sites promotes parkin's ubiquitin E3 ligase activity by suppressing the intramolecular interaction that maintains its autoinhibited conformation and increases its cytoprotective effect. In conclusion, covalent ISG15 conjugation is a novel mode of modulating parkin activity, and alteration in this pathway may be associated with PD pathogenesis. PMID:27534820

Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin.

PubMed

Im, Eunju; Yoo, Lang; Hyun, Minju; Shin, Woo Hyun; Chung, Kwang Chul

2016-08-01

Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the pars compacta of the substantia nigra and accumulation of ubiquitinated proteins in aggregates called Lewy bodies. Several mutated genes have been found in familial PD patients, including SNCA (α-synuclein), PARK2 (parkin), PINK1, PARK7 (DJ-1), LRRK2 and ATP13A2 Many pathogenic mutations of PARK2, which encodes the ubiquitin E3 ligase parkin, result in loss of function, leading to accumulation of parkin substrates and consequently contributing to dopaminergic cell death. ISG15 is a member of the ubiquitin-like modifier family and is induced by stimulation with type I interferons. Similar to ubiquitin and ubiquitination, covalent conjugation of ISG15 to target proteins (ISGylation) regulates their biochemical properties. In this study, we identified parkin as a novel target of ISGylation specifically mediated by the ISG15-E3 ligase HERC5. In addition, we identified two ISGylation sites, Lys-349 and Lys-369, in the in-between-ring domain of parkin. ISGylation of these sites promotes parkin's ubiquitin E3 ligase activity by suppressing the intramolecular interaction that maintains its autoinhibited conformation and increases its cytoprotective effect. In conclusion, covalent ISG15 conjugation is a novel mode of modulating parkin activity, and alteration in this pathway may be associated with PD pathogenesis. © 2016 The Authors.

Mass spectrometry techniques for studying the ubiquitin system.

PubMed

Heap, Rachel E; Gant, Megan S; Lamoliatte, Frederic; Peltier, Julien; Trost, Matthias

2017-10-15

Post-translational control of proteins through covalent attachment of ubiquitin plays important roles in all eukaryotic cell functions. The ubiquitin system in humans consists of 2 E1, 35 E2 and >600 E3 ubiquitin ligases as well as hundreds of deubiquitylases, which reverse ubiquitin attachment. Moreover, there are hundreds of proteins with ubiquitin-binding domains that bind one of the eight possible polyubiquitin chains. Dysfunction of the ubiquitin system is associated with many diseases such as cancer, autoimmunity and neurodegeneration, demonstrating the importance of ubiquitylation. Therefore, enzymes of the ubiquitin system are considered highly attractive drug targets. In recent years, mass spectrometry (MS)-based techniques have become increasingly important in the deciphering of the ubiquitin system. This short review addresses the state-of-the-art MS techniques for the identification of ubiquitylated proteins and their ubiquitylation sites. We also discuss the identification and quantitation of ubiquitin chain topologies and highlight how the activity of enzymes in the ubiquitin pathway can be measured. Finally, we present current MS tools that can be used for drug discovery in the ubiquitin space. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

A ubiquitin carboxyl extension protein secreted from a plant-parasitic nematode Globodera rostochiensis is cleaved in planta to promote plant parasitism.

PubMed

Chronis, Demosthenis; Chen, Shiyan; Lu, Shunwen; Hewezi, Tarek; Carpenter, Sara C D; Loria, Rosemary; Baum, Thomas J; Wang, Xiaohong

2013-04-01

Nematode effector proteins originating from esophageal gland cells play central roles in suppressing plant defenses and in formation of the plant feeding cells that are required for growth and development of cyst nematodes. A gene (GrUBCEP12) encoding a unique ubiquitin carboxyl extension protein (UBCEP) that consists of a signal peptide for secretion, a mono-ubiquitin domain, and a 12 amino acid carboxyl extension protein (CEP12) domain was cloned from the potato cyst nematode Globodera rostochiensis. This GrUBCEP12 gene was expressed exclusively within the nematode's dorsal esophageal gland cell, and was up-regulated in the parasitic second-stage juvenile, correlating with the time when feeding cell formation is initiated. We showed that specific GrUBCEP12 knockdown via RNA interference reduced nematode parasitic success, and that over-expression of the secreted Gr(Δ) (SP) UBCEP12 protein in potato resulted in increased nematode susceptibility, providing direct evidence that this secreted effector is involved in plant parasitism. Using transient expression assays in Nicotiana benthamiana, we found that Gr(Δ) (SP) UBCEP12 is processed into free ubiquitin and a CEP12 peptide (GrCEP12) in planta, and that GrCEP12 suppresses resistance gene-mediated cell death. A target search showed that expression of RPN2a, a gene encoding a subunit of the 26S proteasome, was dramatically suppressed in Gr(Δ) (SP) UBCEP12 but not GrCEP12 over-expression plants when compared with control plants. Together, these results suggest that, when delivered into host plant cells, Gr(Δ) (SP) UBCEP12 becomes two functional units, one acting to suppress plant immunity and the other potentially affecting the host 26S proteasome, to promote feeding cell formation. © 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.

Small ubiquitin-related modifier is secreted and shows cytokine-like activity.

PubMed

Hosono, Hidetaka; Yokosawa, Hideyoshi

2008-05-01

Small ubiquitin-related modifier (SUMO) is a type I ubiquitin-like protein family member and is covalently attached to various target proteins. Through this post-translational modification, SUMO plays important roles in various cellular events. Here, we show that SUMO is secreted from cultured cells in an endoplasmic reticulum (ER)/Golgi-independent manner and that this secretion occurs without covalent binding to target proteins or chain formation. Overexpression experiments using C-terminally truncated mutants of SUMO revealed that the secretion requires the C-terminal sequence. Recombinant SUMO-3 protein was capable of binding to and promoting the proliferation of cultured cells. Thus, we propose that SUMO functions as a cytokine-like molecule extracellularly.

The E3 ubiquitin ligase NEDD4 induces endocytosis and lysosomal sorting of connexin 43 to promote loss of gap junctions.

PubMed

Totland, Max Z; Bergsland, Christian H; Fykerud, Tone A; Knudsen, Lars M; Rasmussen, Nikoline L; Eide, Peter W; Yohannes, Zeremariam; Sørensen, Vigdis; Brech, Andreas; Lothe, Ragnhild A; Leithe, Edward

2017-09-01

Intercellular communication via gap junctions has an important role in controlling cell growth and in maintaining tissue homeostasis. Connexin 43 (Cx43; also known as GJA1) is the most abundantly expressed gap junction channel protein in humans and acts as a tumor suppressor in multiple tissue types. Cx43 is often dysregulated at the post-translational level during cancer development, resulting in loss of gap junctions. However, the molecular basis underlying the aberrant regulation of Cx43 in cancer cells has remained elusive. Here, we demonstrate that the oncogenic E3 ubiquitin ligase NEDD4 regulates the Cx43 protein level in HeLa cells, both under basal conditions and in response to protein kinase C activation. Furthermore, overexpression of NEDD4, but not a catalytically inactive form of NEDD4, was found to result in nearly complete loss of gap junctions and increased lysosomal degradation of Cx43 in both HeLa and C33A cervical carcinoma cells. Collectively, the data provide new insights into the molecular basis underlying the regulation of gap junction size and represent the first evidence that an oncogenic E3 ubiquitin ligase promotes loss of gap junctions and Cx43 degradation in human carcinoma cells. © 2017. Published by The Company of Biologists Ltd.

Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination

PubMed Central

Mortensen, Franziska; Schneider, Daniel; Barbic, Tanja; Sladewska-Marquardt, Anna; Kühnle, Simone; Marx, Andreas; Scheffner, Martin

2015-01-01

Deregulation of the ubiquitin ligase E6 associated protein (E6AP) encoded by the UBE3A gene has been associated with three different clinical pictures. Hijacking of E6AP by the E6 oncoprotein of distinct human papillomaviruses (HPV) contributes to the development of cervical cancer, whereas loss of E6AP expression or function is the cause of Angelman syndrome, a neurodevelopmental disorder, and increased expression of E6AP has been involved in autism spectrum disorders. Although these observations indicate that the activity of E6AP has to be tightly controlled, only little is known about how E6AP is regulated at the posttranslational level. Here, we provide evidence that the hydrophobic patch of ubiquitin comprising Leu-8 and Ile-44 is important for E6AP-mediated ubiquitination, whereas it does not affect the catalytic properties of the isolated catalytic HECT domain of E6AP. Furthermore, we show that the HPV E6 oncoprotein rescues the disability of full-length E6AP to use a respective hydrophobic patch mutant of ubiquitin for ubiquitination and that it stimulates E6AP-mediated ubiquitination of Ring1B, a known substrate of E6AP, in vitro and in cells. Based on these data, we propose that E6AP exists in at least two different states, an active and a less active or latent one, and that the activity of E6AP is controlled by noncovalent interactions with ubiquitin and allosteric activators such as the HPV E6 oncoprotein. PMID:26216987

Ubiquitin in Influenza Virus Entry and Innate Immunity.

PubMed

Rudnicka, Alina; Yamauchi, Yohei

2016-10-24

Viruses are obligatory cellular parasites. Their mission is to enter a host cell, to transfer the viral genome, and to replicate progeny whilst diverting cellular immunity. The role of ubiquitin is to regulate fundamental cellular processes such as endocytosis, protein degradation, and immune signaling. Many viruses including influenza A virus (IAV) usurp ubiquitination and ubiquitin-like modifications to establish infection. In this focused review, we discuss how ubiquitin and unanchored ubiquitin regulate IAV host cell entry, and how histone deacetylase 6 (HDAC6), a cytoplasmic deacetylase with ubiquitin-binding activity, mediates IAV capsid uncoating. We also discuss the roles of ubiquitin in innate immunity and its implications in the IAV life cycle.

Ubiquitin in Influenza Virus Entry and Innate Immunity

PubMed Central

Rudnicka, Alina; Yamauchi, Yohei

2016-01-01

Viruses are obligatory cellular parasites. Their mission is to enter a host cell, to transfer the viral genome, and to replicate progeny whilst diverting cellular immunity. The role of ubiquitin is to regulate fundamental cellular processes such as endocytosis, protein degradation, and immune signaling. Many viruses including influenza A virus (IAV) usurp ubiquitination and ubiquitin-like modifications to establish infection. In this focused review, we discuss how ubiquitin and unanchored ubiquitin regulate IAV host cell entry, and how histone deacetylase 6 (HDAC6), a cytoplasmic deacetylase with ubiquitin-binding activity, mediates IAV capsid uncoating. We also discuss the roles of ubiquitin in innate immunity and its implications in the IAV life cycle. PMID:27783058

Overexpressed ubiquitin ligase Cullin7 in breast cancer promotes cell proliferation and invasion via down-regulating p53

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Hongsheng; Wu, Fenping; Wang, Yan

2014-08-08

Highlights: • Cullin7 is overexpressed in human breast cancer samples. • Cullin7 stimulated proliferation and invasion of breast cancer cells. • Inhibition of p53 contributes to Cullin7-induced proliferation and invasion. - Abstract: Ubiquitin ligase Cullin7 has been identified as an oncogene in some malignant diseases such as choriocarcinoma and neuroblastoma. However, the role of Cullin7 in breast cancer carcinogenesis remains unclear. In this study, we compared Cullin7 protein levels in breast cancer tissues with normal breast tissues and identified significantly higher expression of Cullin7 protein in breast cancer specimens. By overexpressing Cullin7 in breast cancer cells HCC1937, we found thatmore » Cullin7 could promote cell growth and invasion in vitro. In contrast, the cell growth and invasion was inhibited by silencing Cullin7 in breast cancer cell BT474. Moreover, we demonstrated that Cullin7 promoted breast cancer cell proliferation and invasion via down-regulating p53 expression. Thus, our study provided evidence that Cullin7 functions as a novel oncogene in breast cancer and may be a potential therapeutic target for breast cancer management.« less

The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation.

PubMed

Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

2016-07-22

The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation*

PubMed Central

Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

2016-01-01

The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. PMID:27302062

Nogo-B receptor promotes the chemoresistance of human hepatocellular carcinoma via the ubiquitination of p53 protein

PubMed Central

Long, Fei; Liu, Ying; Liu, Zhenzhen; Li, Song; Yang, Xuejun; Sun, Deguang; Wang, Haibo; Liu, Qinlong; Liang, Rui; Li, Yan; Gao, Zhenming; Shao, Shujuan; Miao, Qing Robert; Wang, Liming

2016-01-01

Nogo-B receptor (NgBR), a type I single transmembrane domain receptor is the specific receptor for Nogo-B. Our previous work demonstrated that NgBR is highly expressed in breast cancer cells, where it promotes epithelial mesenchymal transition (EMT), an important step in metastasis. Here, we show that both in vitro and in vivo increased expression of NgBR contributes to the increased chemoresistance of Bel7402/5FU cells, a stable 5-FU (5-Fluorouracil) resistant cell line related Bel7402 cells. NgBR knockdown abrogates S-phase arrest in Bel7402/5FU cells, which correlates with a reduction in G1/S phase checkpoint proteins p53 and p21. In addition, NgBR suppresses p53 protein levels through activation of the PI3K/Akt/MDM2 pathway, which promotes p53 degradation via the ubiquitin proteasome pathway and thus increases the resistance of human hepatocellular cancer cells to 5-FU. Furthermore, we found that NgBR expression is associated with a poor prognosis of human hepatocellular carcinoma (HCC) patients. These results suggest that targeting NgBR in combination with chemotherapeutic drugs, such as 5-FU, could improve the efficacy of current anticancer treatments. PMID:26840457

A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function

PubMed Central

Liu, Yaobin; Huang, Xiangao; He, Xian; Zhou, Yanqing; Jiang, Xiaogang; Chen-Kiang, Selina; Jaffrey, Samie R.; Xu, Guoqiang

2015-01-01

The immunomodulatory drug (IMiD) thalidomide and its structural analogs lenalidomide and pomalidomide are highly effective in treating clinical indications. Thalidomide binds to cereblon (CRBN), a substrate receptor of the cullin-4 really interesting new gene (RING) E3 ligase complex. Here, we examine the effect of thalidomide and its analogs on CRBN ubiquitination and its functions in human cell lines. We find that the ubiquitin modification of CRBN includes K48-linked polyubiquitin chains and that thalidomide blocks the formation of CRBN-ubiquitin conjugates. Furthermore, we show that ubiquitinated CRBN is targeted for proteasomal degradation. Treatment of human myeloma cell lines such as MM1.S, OPM2, and U266 with thalidomide (100 μM) and its structural analog lenalidomide (10 μM) results in stabilization of CRBN and elevation of CRBN protein levels. This in turn leads to the reduced level of CRBN target proteins and enhances the sensitivity of human multiple myeloma cells to IMiDs. Our results reveal a novel mechanism by which thalidomide and its analogs modulate the CRBN function in cells. Through inhibition of CRBN ubiquitination, thalidomide and its analogs allow CRBN to accumulate, leading to the increased cullin-4 RING E3 ligase-mediated degradation of target proteins.—Liu, Y., Huang, X., He, X., Zhou, Y., Jiang, X., Chen-Kiang, S., Jaffrey, S. R., Xu, G. A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function. PMID:26231201

Activation of the Slx5–Slx8 Ubiquitin Ligase by Poly-small Ubiquitin-like Modifier Conjugates*S⃞

PubMed Central

Mullen, Janet R.; Brill, Steven J.

2008-01-01

Protein sumoylation is a regulated process that is important for the health of human and yeast cells. In budding yeast, a subset of sumoylated proteins is targeted for ubiquitination by a conserved heterodimeric ubiquitin (Ub) ligase, Slx5–Slx8, which is needed to suppress the accumulation of high molecular weight small ubiquitin-like modifier (SUMO) conjugates. Structure-function analysis indicates that the Slx5–Slx8 complex contains multiple SUMO-binding domains that are collectively required for in vivo function. To determine the specificity of Slx5–Slx8, we assayed its Ub ligase activity using sumoylated Siz2 as an in vitro substrate. In contrast to unsumoylated or multisumoylated Siz2, substrates containing poly-SUMO conjugates were efficiently ubiquitinated by Slx5–Slx8. Although Siz2 itself was ubiquitinated, the bulk of the Ub was conjugated to SUMO residues. Slx5–Slx8 primarily mono-ubiquitinated the N-terminal SUMO moiety of the chain. These data indicate that the Slx5–Slx8 Ub ligase is stimulated by poly-SUMO conjugates and that it can ubiquitinate a poly-SUMO chain. PMID:18499666

Linear ubiquitin chains: enzymes, mechanisms and biology

PubMed Central

2017-01-01

Ubiquitination is a versatile post-translational modification that regulates a multitude of cellular processes. Its versatility is based on the ability of ubiquitin to form multiple types of polyubiquitin chains, which are recognized by specific ubiquitin receptors to induce the required cellular response. Linear ubiquitin chains are linked through Met 1 and have been established as important players of inflammatory signalling and apoptotic cell death. These chains are generated by a ubiquitin E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) that is thus far the only E3 ligase capable of forming linear ubiquitin chains. The complex consists of three subunits, HOIP, HOIL-1L and SHARPIN, each of which have specific roles in the observed biological functions of LUBAC. Furthermore, LUBAC has been found to be associated with OTULIN and CYLD, deubiquitinases that disassemble linear chains and counterbalance the E3 ligase activity of LUBAC. Gene mutations in HOIP, HOIL-1L and OTULIN are found in human patients who suffer from autoimmune diseases, and HOIL-1L mutations are also found in myopathy patients. In this paper, we discuss the mechanisms of linear ubiquitin chain generation and disassembly by their respective enzymes and review our current understanding of their biological functions and association with human diseases. PMID:28446710

A Discrete Ubiquitin-Mediated Network Regulates the Strength of NOD2 Signaling

PubMed Central

Tigno-Aranjuez, Justine T.; Bai, Xiaodong

2013-01-01

Dysregulation of NOD2 signaling is implicated in the pathology of various inflammatory diseases, including Crohn's disease, asthma, and sarcoidosis, making signaling proteins downstream of NOD2 potential therapeutic targets. Inhibitor-of-apoptosis (IAP) proteins, particularly cIAP1, are essential mediators of NOD2 signaling, and in this work, we describe a molecular mechanism for cIAP1's regulation in the NOD2 signaling pathway. While cIAP1 promotes RIP2's tyrosine phosphorylation and subsequent NOD2 signaling, this positive regulation is countered by another E3 ubiquitin ligase, ITCH, through direct ubiquitination of cIAP1. This ITCH-mediated ubiquitination leads to cIAP1's lysosomal degradation. Pharmacologic inhibition of cIAP1 expression in ITCH−/− macrophages attenuates heightened ITCH−/− macrophage muramyl dipeptide-induced responses. Transcriptome analysis, combined with pharmacologic inhibition of cIAP1, further defines specific pathways within the NOD2 signaling pathway that are targeted by cIAP1. This information provides genetic signatures that may be useful in repurposing cIAP1-targeted therapies to correct NOD2-hyperactive states and identifies a ubiquitin-regulated signaling network centered on ITCH and cIAP1 that controls the strength of NOD2 signaling. PMID:23109427

Ubiquitination of Cdc20 by the APC occurs through an intramolecular mechanism

PubMed Central

Foe, Ian T.; Foster, Scott A.; Cheung, Stephanie K.; DeLuca, Steven Z.; Morgan, David O.; Toczyski, David P.

2012-01-01

SUMMARY Background Cells control progression through late mitosis by regulating Cdc20 and Cdh1, the two mitotic activators of the Anaphase Promoting Complex (APC). The control of Cdc20 protein levels during the cell cycle is not well understood. Results Here, we demonstrate that Cdc20 is degraded in budding yeast by multiple APC-dependent mechanisms. We find that the majority of Cdc20 turnover does not involve a second activator molecule, but instead depends on in cis Cdc20 autoubiquitination while it is bound to its activator-binding site on the APC core. Unlike in trans ubiquitination of Cdc20 substrates, the APC ubiquitinates Cdc20 independent of APC activation by Cdc20’s C-box. Cdc20 turnover by this intramolecular mechanism is cell cycle-regulated, contributing to the decline in Cdc20 levels that occurs after anaphase. Interestingly, high substrate levels in vitro significantly reduce Cdc20 autoubiquitination. Conclusion We show here that Cdc20 fluctuates through the cell cycle via a distinct form of APC-mediated ubiquitination. This in cis autoubiquitination may preferentially occur in early anaphase, following depletion of Cdc20 substrates. This suggests that distinct mechanisms are able to target Cdc20 for ubiquitination at different points during the cell cycle. PMID:22079111

Lack of promoter IV-driven BDNF transcription results in depression-like behavior.

PubMed

Sakata, K; Jin, L; Jha, S

2010-10-01

Transcription of Bdnf is controlled by multiple promoters, in which promoter IV contributes significantly to activity-dependent Bdnf transcription. We have generated promoter IV mutant mice [brain-derived neurotrophic factor (BDNF)-KIV] in which promoter IV-driven expression of BDNF is selectively disrupted by inserting a green fluorescent protein (GFP)-STOP cassette within the Bdnf exon IV locus. BDNF-KIV animals exhibited depression-like behavior as shown by the tail suspension test (TST), sucrose preference test (SPT) and learned helplessness test (LHT). In addition, BDNF-KIV mice showed reduced activity in the open field test (OFT) and reduced food intake in the novelty-suppressed feeding test (NSFT). The mutant mice did not display anxiety-like behavior in the light and dark box test and elevated plus maze tests. Interestingly, the mutant mice showed defective response inhibition in the passive avoidance test (PAT) even though their learning ability was intact when measured with the active avoidance test (AAT). These results suggest that promoter IV-dependent BDNF expression plays a critical role in the control of mood-related behaviors. This is the first study that directly addressed the effects of endogenous promoter-driven expression of BDNF in depression-like behavior. © 2010 The Authors. Genes, Brain and Behavior © 2010 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.

Regulation of HTLV-1 Tax Stability, Cellular Trafficking and NF-κB Activation by the Ubiquitin-Proteasome Pathway

PubMed Central

Lavorgna, Alfonso; Harhaj, Edward William

2014-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATLL) in 3%–5% of infected individuals after a long latent period. HTLV-1 Tax is a trans-activating protein that regulates viral gene expression and also modulates cellular signaling pathways to enhance T-cell proliferation and cell survival. The Tax oncoprotein promotes T-cell transformation, in part via constitutive activation of the NF-κB transcription factor; however, the underlying mechanisms remain unknown. Ubiquitination is a type of post-translational modification that occurs in a three-step enzymatic cascade mediated by E1, E2 and E3 enzymes and regulates protein stability as well as signal transduction, protein trafficking and the DNA damage response. Emerging studies indicate that Tax hijacks the ubiquitin machinery to activate ubiquitin-dependent kinases and downstream NF-κB signaling. Tax interacts with the E2 conjugating enzyme Ubc13 and is conjugated on C-terminal lysine residues with lysine 63-linked polyubiquitin chains. Tax K63-linked polyubiquitination may serve as a platform for signaling complexes since this modification is critical for interactions with NEMO and IKK. In addition to NF-κB signaling, mono- and polyubiquitination of Tax also regulate its subcellular trafficking and stability. Here, we review recent advances in the diverse roles of ubiquitin in Tax function and how Tax usurps the ubiquitin-proteasome pathway to promote oncogenesis. PMID:25341660

Isolation and functional characterization of a cotton ubiquitination-related promoter and 5'UTR that drives high levels of expression in root and flower tissues.

PubMed

Viana, Antonio A B; Fragoso, Rodrigo R; Guimarães, Luciane M; Pontes, Naiara; Oliveira-Neto, Osmundo B; Artico, Sinara; Nardeli, Sarah M; Alves-Ferreira, Marcio; Batista, João A N; Silva, Maria C M; Grossi-de-Sa, Maria F

2011-11-24

Cotton (Gossypium spp.) is an important crop worldwide that provides raw material to 40% of the textile fiber industry. Important traits have been studied aiming the development of genetically modified crops including resistance to insect and diseases, and tolerance to drought, cold and herbicide. Therefore, the characterization of promoters and regulatory regions is also important to achieve high gene expression and/or a specific expression pattern. Commonly, genes involved in ubiquitination pathways are highly and differentially expressed. In this study, we analyzed the expression of a cotton ubiquitin-conjugating enzyme (E2) family member with no previous characterization. Nucleotide analysis revealed high identity with cotton E2 homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the E2 active site, and an intron that is spliced in E2 homologues, but not in GhGDRP85. The GhGDRP85 gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2) and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in Arabidopsis thaliana. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2. uceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2) and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues.

HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency.

PubMed

Guilbert, Solenn M; Lambert, Herman; Rodrigue, Marc-Antoine; Fuchs, Margit; Landry, Jacques; Lavoie, Josée N

2018-02-05

BCL2-associated athanogene (BAG)-3 is viewed as a platform that would physically and functionally link distinct classes of molecular chaperones of the heat shock protein (HSP) family for the stabilization and clearance of damaged proteins. In this study, we show that HSPB8, a member of the small heat shock protein subfamily, cooperates with BAG3 to coordinate the sequestration of harmful proteins and the cellular adaptive response upon proteasome inhibition. Silencing of HSPB8, like depletion of BAG3, inhibited targeting of ubiquitinated proteins to the juxtanuclear aggresome, a mammalian system of spatial quality control. However, aggresome targeting was restored in BAG3-depleted cells by a mutant BAG3 defective in HSPB8 binding, uncoupling HSPB8 function from its binding to BAG3. Depletion of HSPB8 impaired formation of ubiquitinated microaggregates in an early phase and interfered with accurate modifications of the stress sensor p62/sequestosome (SQSTM)-1. This impairment correlated with decreased coupling of BAG3 to p62/SQSTM1 in response to stress, hindering Kelch-like ECH-associated protein (KEAP)-1 sequestration and stabilization of nuclear factor E2-related factor (Nrf)-2, an important arm of the antioxidant defense. Notably, the myopathy-associated mutation of BAG3 (P209L), which lies within the HSPB8-binding motif, deregulated the association between BAG3 and p62/SQSTM1 and the KEAP1-Nrf2 signaling axis. Together, our findings support a so-far-unrecognized role for the HSPB8-BAG3 connection in mounting of an efficient stress response, which may be involved in BAG3-related human diseases.-Guilbert, S. M., Lambert, H., Rodrigue, M.-A., Fuchs, M., Landry, J., Lavoie, J. N. HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency.

PELI1 functions as a dual modulator of necroptosis and apoptosis by regulating ubiquitination of RIPK1 and mRNA levels of c-FLIP.

PubMed

Wang, Huibing; Meng, Huyan; Li, Xingyan; Zhu, Kezhou; Dong, Kangyun; Mookhtiar, Adnan K; Wei, Huiting; Li, Ying; Sun, Shao-Cong; Yuan, Junying

2017-11-07

Apoptosis and necroptosis are two distinct cell death mechanisms that may be activated in cells on stimulation by TNFα. It is still unclear, however, how apoptosis and necroptosis may be differentially regulated. Here we screened for E3 ubiquitin ligases that could mediate necroptosis. We found that deficiency of Pellino 1 (PELI1), an E3 ubiquitin ligase, blocked necroptosis. We show that PELI1 mediates K63 ubiquitination on K115 of RIPK1 in a kinase-dependent manner during necroptosis. Ubiquitination of RIPK1 by PELI1 promotes the formation of necrosome and execution of necroptosis. Although PELI1 is not directly involved in mediating the activation of RIPK1, it is indispensable for promoting the binding of activated RIPK1 with its downstream mediator RIPK3 to promote the activation of RIPK3 and MLKL. Inhibition of RIPK1 kinase activity blocks PELI1-mediated ubiquitination of RIPK1 in necroptosis. However, we show that PELI1 deficiency sensitizes cells to both RIPK1-dependent and RIPK1-independent apoptosis as a result of down-regulated expression of c-FLIP, an inhibitor of caspase-8. Finally, we show that Peli1 -/- mice are sensitized to TNFα-induced apoptosis. Thus, PELI1 is a key modulator of RIPK1 that differentially controls the activation of necroptosis and apoptosis. Published under the PNAS license.

The HIP2~Ubiquitin Conjugate Forms a Non-Compact Monomeric Thioester during Di-Ubiquitin Synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, Benjamin W.; Barber, Kathryn R.; Shilton, Brian H.

2015-03-23

Polyubiquitination is a post-translational event used to control the degradation of damaged or unwanted proteins by modifying the target protein with a chain of ubiquitin molecules. One potential mechanism for the assembly of polyubiquitin chains involves the dimerization of an E2 conjugating enzyme allowing conjugated ubiquitin molecules to be put into close proximity to assist reactivity. HIP2 (UBE2K) and Ubc1 (yeast homolog of UBE2K) are unique E2 conjugating enzymes that each contain a C-terminal UBA domain attached to their catalytic domains, and they have basal E3-independent polyubiquitination activity. Although the isolated enzymes are monomeric, polyubiquitin formation activity assays show thatmore » both can act as ubiquitin donors or ubiquitin acceptors when in the activated thioester conjugate suggesting dimerization of the E2-ubiquitin conjugates. Stable disulfide complexes, analytical ultracentrifugation and small angle x-ray scattering were used to show that the HIP2-Ub and Ubc1-Ub thioester complexes remain predominantly monomeric in solution. Models of the HIP2-Ub complex derived from SAXS data show the complex is not compact but instead forms an open or backbent conformation similar to UbcH5b~Ub or Ubc13~Ub where the UBA domain and covalently attached ubiquitin reside on opposite ends of the catalytic domain. Activity assays showed that full length HIP2 exhibited a five-fold increase in the formation rate of di-ubiquitin compared to a HIP2 lacking the UBA domain. This difference was not observed for Ubc1 and may be attributed to the closer proximity of the UBA domain in HIP2 to the catalytic core than for Ubc1.« less

Specificity and disease in the ubiquitin system

PubMed Central

Chaugule, Viduth K.; Walden, Helen

2016-01-01

Post-translational modification (PTM) of proteins by ubiquitination is an essential cellular regulatory process. Such regulation drives the cell cycle and cell division, signalling and secretory pathways, DNA replication and repair processes and protein quality control and degradation pathways. A huge range of ubiquitin signals can be generated depending on the specificity and catalytic activity of the enzymes required for attachment of ubiquitin to a given target. As a consequence of its importance to eukaryotic life, dysfunction in the ubiquitin system leads to many disease states, including cancers and neurodegeneration. This review takes a retrospective look at our progress in understanding the molecular mechanisms that govern the specificity of ubiquitin conjugation. PMID:26862208

Linear ubiquitin chains: enzymes, mechanisms and biology.

PubMed

Rittinger, Katrin; Ikeda, Fumiyo

2017-04-01

Ubiquitination is a versatile post-translational modification that regulates a multitude of cellular processes. Its versatility is based on the ability of ubiquitin to form multiple types of polyubiquitin chains, which are recognized by specific ubiquitin receptors to induce the required cellular response. Linear ubiquitin chains are linked through Met 1 and have been established as important players of inflammatory signalling and apoptotic cell death. These chains are generated by a ubiquitin E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) that is thus far the only E3 ligase capable of forming linear ubiquitin chains. The complex consists of three subunits, HOIP, HOIL-1L and SHARPIN, each of which have specific roles in the observed biological functions of LUBAC. Furthermore, LUBAC has been found to be associated with OTULIN and CYLD, deubiquitinases that disassemble linear chains and counterbalance the E3 ligase activity of LUBAC. Gene mutations in HOIP, HOIL-1L and OTULIN are found in human patients who suffer from autoimmune diseases, and HOIL-1L mutations are also found in myopathy patients. In this paper, we discuss the mechanisms of linear ubiquitin chain generation and disassembly by their respective enzymes and review our current understanding of their biological functions and association with human diseases. © 2017 The Authors.

Parkin-phosphoubiquitin complex reveals cryptic ubiquitin-binding site required for RBR ligase activity.

PubMed

Kumar, Atul; Chaugule, Viduth K; Condos, Tara E C; Barber, Kathryn R; Johnson, Clare; Toth, Rachel; Sundaramoorthy, Ramasubramanian; Knebel, Axel; Shaw, Gary S; Walden, Helen

2017-05-01

RING-between-RING (RBR) E3 ligases are a class of ubiquitin ligases distinct from RING or HECT E3 ligases. An important RBR ligase is Parkin, mutations in which lead to early-onset hereditary Parkinsonism. Parkin and other RBR ligases share a catalytic RBR module but are usually autoinhibited and activated via distinct mechanisms. Recent insights into Parkin regulation predict large, unknown conformational changes during Parkin activation. However, current data on active RBR ligases reflect the absence of regulatory domains. Therefore, it remains unclear how individual RBR ligases are activated, and whether they share a common mechanism. We now report the crystal structure of a human Parkin-phosphoubiquitin complex, which shows that phosphoubiquitin binding induces movement in the 'in-between RING' (IBR) domain to reveal a cryptic ubiquitin-binding site. Mutation of this site negatively affects Parkin's activity. Furthermore, ubiquitin binding promotes cooperation between Parkin molecules, which suggests a role for interdomain association in the RBR ligase mechanism.

Role of the ubiquitin-proteasome system in brain ischemia: friend or foe?

PubMed

Caldeira, Margarida V; Salazar, Ivan L; Curcio, Michele; Canzoniero, Lorella M T; Duarte, Carlos B

2014-01-01

The ubiquitin-proteasome system (UPS) is a catalytic machinery that targets numerous cellular proteins for degradation, thus being essential to control a wide range of basic cellular processes and cell survival. Degradation of intracellular proteins via the UPS is a tightly regulated process initiated by tagging a target protein with a specific ubiquitin chain. Neurons are particularly vulnerable to any change in protein composition, and therefore the UPS is a key regulator of neuronal physiology. Alterations in UPS activity may induce pathological responses, ultimately leading to neuronal cell death. Brain ischemia triggers a complex series of biochemical and molecular mechanisms, such as an inflammatory response, an exacerbated production of misfolded and oxidized proteins, due to oxidative stress, and the breakdown of cellular integrity mainly mediated by excitotoxic glutamatergic signaling. Brain ischemia also damages protein degradation pathways which, together with the overproduction of damaged proteins and consequent upregulation of ubiquitin-conjugated proteins, contribute to the accumulation of ubiquitin-containing proteinaceous deposits. Despite recent advances, the factors leading to deposition of such aggregates after cerebral ischemic injury remain poorly understood. This review discusses the current knowledge on the role of the UPS in brain function and the molecular mechanisms contributing to UPS dysfunction in brain ischemia with consequent accumulation of ubiquitin-containing proteins. Chemical inhibitors of the proteasome and small molecule inhibitors of deubiquitinating enzymes, which promote the degradation of proteins by the proteasome, were both shown to provide neuroprotection in brain ischemia, and this apparent contradiction is also discussed in this review. Copyright © 2013 Elsevier Ltd. All rights reserved.

Parkin mediates the ubiquitination of VPS35 and modulates retromer-dependent endosomal sorting.

PubMed

Williams, Erin T; Glauser, Liliane; Tsika, Elpida; Jiang, Haisong; Islam, Shariful; Moore, Darren J

2018-06-11

Mutations in a number of genes cause familial forms of Parkinson's disease (PD), including mutations in the vacuolar protein sorting 35 ortholog (VPS35) and parkin genes. In this study, we identify a novel functional interaction between parkin and VPS35. We demonstrate that parkin interacts with and robustly ubiquitinates VPS35 in human neural cells. Familial parkin mutations are impaired in their ability to ubiquitinate VPS35. Parkin mediates the attachment of an atypical poly-ubiquitin chain to VPS35 with three lysine residues identified within the C-terminal region of VPS35 that are covalently modified by ubiquitin. Notably, parkin-mediated VPS35 ubiquitination does not promote the proteasomal degradation of VPS35. Furthermore, parkin does not influence the steady-state levels or turnover of VPS35 in neural cells and VPS35 levels are normal in the brains of parkin knockout mice. These data suggest that ubiquitination of VPS35 by parkin may instead serve a non-degradative cellular function potentially by regulating retromer-dependent sorting. Accordingly, we find that components of the retromer-associated WASH complex are markedly decreased in the brain of parkin knockout mice, suggesting that parkin may modulate WASH complex-dependent retromer sorting. Parkin gene silencing in primary cortical neurons selectively disrupts the vesicular sorting of the autophagy receptor ATG9A, a WASH-dependent retromer cargo. Parkin is not required for dopaminergic neurodegeneration induced by the expression of PD-linked D620N VPS35 in mice, consistent with VPS35 being located downstream of parkin function. Our data reveal a novel functional interaction of parkin with VPS35 that may be important for retromer-mediated endosomal sorting and PD.

Proteasome subunit Rpn13 is a novel ubiquitin receptor

PubMed Central

Husnjak, Koraljka; Elsasser, Suzanne; Zhang, Naixia; Chen, Xiang; Randles, Leah; Shi, Yuan; Hofmann, Kay; Walters, Kylie; Finley, Daniel; Dikic, Ivan

2010-01-01

Proteasomal receptors that recognize ubiquitin chains attached to substrates are key mediators of selective protein degradation in eukaryotes. Here we report the identification of a new ubiquitin receptor, Rpn13/ARM1, a known component of the proteasome. Rpn13 binds ubiquitin via a conserved N-terminal region termed the Pru domain (Pleckstrin-like receptor for ubiquitin), which binds K48-linked diubiquitin with an affinity of ∼90 nM. Like proteasomal ubiquitin receptor Rpn10/S5a, Rpn13 also binds ubiquitin-like domains of the UBL/UBA family of ubiquitin receptors. A synthetic phenotype results in yeast when specific mutations of the ubiquitin binding sites of Rpn10 and Rpn13 are combined, indicating functional linkage between these ubiquitin receptors. Since Rpn13 is also the proteasomal receptor for Uch37, a deubiquitinating enzyme, our findings suggest a coupling of chain recognition and disassembly at the proteasome. PMID:18497817

Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels.

PubMed

Fu, Ssu-Ju; Jeng, Chung-Jiuan; Ma, Chia-Hao; Peng, Yi-Jheng; Lee, Chi-Ming; Fang, Ya-Ching; Lee, Yi-Ching; Tang, Sung-Chun; Hu, Meng-Chun; Tang, Chih-Yung

2017-03-01

Voltage-gated Ca V 2.1 channels comprise a pore-forming α 1A subunit with auxiliary α 2 δ and β subunits. Ca V 2.1 channels play an essential role in regulating synaptic signaling. Mutations in the human gene encoding the Ca V 2.1 subunit are associated with the cerebellar disease episodic ataxia type 2 (EA2). Several EA2-causing mutants exhibit impaired protein stability and exert dominant-negative suppression of Ca V 2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human Ca V 2.1 subunit by identifying RNF138, an E3 ubiquitin ligase, as a novel Ca V 2.1-binding partner. In neurons, RNF138 and Ca V 2.1 coexist in the same protein complex and display notable subcellular colocalization at presynaptic and postsynaptic regions. Overexpression of RNF138 promotes polyubiquitination and accelerates protein turnover of Ca V 2.1. Disrupting endogenous RNF138 function with a mutant (RNF138-H36E) or shRNA infection significantly upregulates the Ca V 2.1 protein level and enhances Ca V 2.1 protein stability. Disrupting endogenous RNF138 function also effectively rescues the defective protein expression of EA2 mutants, as well as fully reversing EA2 mutant-induced excessive proteasomal degradation of Ca V 2.1 WT subunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on Ca V 2.1 WT functional expression, which can be attributed to defective membrane trafficking of Ca V 2.1 WT in the presence of EA2 mutants. We propose that RNF138 plays a critical role in the homeostatic regulation of Ca V 2.1 protein level and functional expression and that RNF138 serves as the primary E3 ubiquitin ligase promoting EA2-associated aberrant degradation of human Ca V 2.1 subunits. SIGNIFICANCE STATEMENT Loss-of-function mutations in the human Ca V 2.1 subunit are linked to episodic ataxia type 2 (EA2), a dominantly inherited disease characterized by

Ubiquitin-like and ubiquitin-associated domain proteins: significance in proteasomal degradation

PubMed Central

Lau, Alan F.

2009-01-01

The ubiquitin–proteasome pathway of protein degradation is one of the major mechanisms that are involved in the maintenance of the proper levels of cellular proteins. The regulation of proteasomal degradation thus ensures proper cell functions. The family of proteins containing ubiquitin-like (UbL) and ubiquitin-associated (UBA) domains has been implicated in proteasomal degradation. UbL–UBA domain containing proteins associate with substrates destined for degradation as well as with subunits of the proteasome, thus regulating the proper turnover of proteins. PMID:19468686

USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A.

PubMed

Uckelmann, Michael; Densham, Ruth M; Baas, Roy; Winterwerp, Herrie H K; Fish, Alexander; Sixma, Titia K; Morris, Joanna R

2018-01-15

BRCA1-BARD1-catalyzed ubiquitination of histone H2A is an important regulator of the DNA damage response, priming chromatin for repair by homologous recombination. However, no specific deubiquitinating enzymes (DUBs) are known to antagonize this function. Here we identify ubiquitin specific protease-48 (USP48) as a H2A DUB, specific for the C-terminal BRCA1 ubiquitination site. Detailed biochemical analysis shows that an auxiliary ubiquitin, an additional ubiquitin that itself does not get cleaved, modulates USP48 activity, which has possible implications for its regulation in vivo. In cells we reveal that USP48 antagonizes BRCA1 E3 ligase function and in BRCA1-proficient cells loss of USP48 results in positioning 53BP1 further from the break site and in extended resection lengths. USP48 repression confers a survival benefit to cells treated with camptothecin and its activity acts to restrain gene conversion and mutagenic single-strand annealing. We propose that USP48 promotes genome stability by antagonizing BRCA1 E3 ligase function.

Ubiquitin is part of the retrovirus budding machinery

NASA Astrophysics Data System (ADS)

Patnaik, Akash; Chau, Vincent; Wills, John W.

2000-11-01

Retroviruses contain relatively large amounts of ubiquitin, but the significance of this finding has been unknown. Here, we show that drugs that are known to reduce the level of free ubiquitin in the cell dramatically reduced the release of Rous sarcoma virus, an avian retrovirus. This effect was suppressed by overexpressing ubiquitin and also by directly fusing ubiquitin to the C terminus of Gag, the viral protein that directs budding and particle release. The block to budding was found to be at the plasma membrane, and electron microscopy revealed that the reduced level of ubiquitin results in a failure of mature virus particles to separate from each other and from the plasma membrane during budding. These data indicate that ubiquitin is actually part of the budding machinery.

Receptor Tyrosine Kinase Ubiquitination and De-Ubiquitination in Signal Transduction and Receptor Trafficking

PubMed Central

Critchley, William R.; Pellet-Many, Caroline; Ringham-Terry, Benjamin; Zachary, Ian C.; Ponnambalam, Sreenivasan

2018-01-01

Receptor tyrosine kinases (RTKs) are membrane-based sensors that enable rapid communication between cells and their environment. Evidence is now emerging that interdependent regulatory mechanisms, such as membrane trafficking, ubiquitination, proteolysis and gene expression, have substantial effects on RTK signal transduction and cellular responses. Different RTKs exhibit both basal and ligand-stimulated ubiquitination, linked to trafficking through different intracellular compartments including the secretory pathway, plasma membrane, endosomes and lysosomes. The ubiquitin ligase superfamily comprising the E1, E2 and E3 enzymes are increasingly implicated in this post-translational modification by adding mono- and polyubiquitin tags to RTKs. Conversely, removal of these ubiquitin tags by proteases called de-ubiquitinases (DUBs) enables RTK recycling for another round of ligand sensing and signal transduction. The endocytosis of basal and activated RTKs from the plasma membrane is closely linked to controlled proteolysis after trafficking and delivery to late endosomes and lysosomes. Proteolytic RTK fragments can also have the capacity to move to compartments such as the nucleus and regulate gene expression. Such mechanistic diversity now provides new opportunities for modulating RTK-regulated cellular responses in health and disease states. PMID:29543760

Ubiquitin-Modifying Enzymes and Regulation of the Inflammasome.

PubMed

Kattah, Michael G; Malynn, Barbara A; Ma, Averil

2017-11-10

Ubiquitin and ubiquitin-modifying enzymes play critical roles in a wide variety of intracellular signaling pathways. Inflammatory signaling cascades downstream of TNF, TLR agonists, antigen receptor cross-linking, and cytokine receptors, all rely on ubiquitination events to direct subsequent immune responses. In the past several years, inflammasome activation and subsequent signal transduction have emerged as an excellent example of how ubiquitin signals control inflammatory responses. Inflammasomes are multiprotein signaling complexes that ultimately lead to caspase activation and release of the interleukin-1 (IL-1) family members, IL-1β and IL-18. Inflammasome activation is critical for the host's defense against pathogens, but dysregulation of inflammasomes may contribute to the pathogenesis of multiple diseases. Ultimately, understanding how various ubiquitin interacting proteins control inflammatory signaling cascades could provide new pathways for therapeutic intervention. Here we review specific ubiquitin-modifying enzymes and ubiquitination events that orchestrate inflammatory responses, with an emphasis on the NLRP3 inflammasome. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pathological Heterogeneity of Frontotemporal Lobar Degeneration with Ubiquitin-Positive Inclusions Delineated by Ubiquitin Immunohistochemistry and Novel Monoclonal Antibodies

PubMed Central

Sampathu, Deepak M.; Neumann, Manuela; Kwong, Linda K.; Chou, Thomas T.; Micsenyi, Matthew; Truax, Adam; Bruce, Jennifer; Grossman, Murray; Trojanowski, John Q.; Lee, Virginia M.-Y.

2006-01-01

Frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) is a common neuropathological subtype of frontotemporal dementia. Although this subtype of frontotemporal dementia is defined by the presence of ubiquitin-positive but tau- and α-synuclein-negative inclusions, it is unclear whether all cases of FTLD-U have the same underlying pathogenesis. Examination of tissue sections from FTLD-U brains stained with anti-ubiquitin antibodies revealed heterogeneity in the morphological characteristics of pathological inclusions among subsets of cases. Three types of FTLD-U were delineated based on morphology and distribution of ubiquitin-positive inclusions. To address the hypothesis that FTLD-U is pathologically heterogeneous, novel monoclonal antibodies (mAbs) were generated by immunization of mice with high molecular mass (Mr > 250 kd) insoluble material prepared by biochemical fractionation of FTLD-U brains. Novel mAbs were identified that immunolabeled all of the ubiquitin-positive inclusions in one subset of FTLD-U cases, whereas other mAbs stained the ubiquitin-positive inclusions in a second subset of cases. These novel mAbs did not stain inclusions in other neurodegenerative disorders, including tauopathies and α-synucleinopathies. Therefore, ubiquitin immunohistochemistry and the immunostaining properties of the novel mAbs generated here suggest that FTLD-U is pathologically he-terogeneous. Identification of the disease proteins recognized by these mAbs will further advance understanding of molecular substrates of FTLD-U neurodegenerative pathways. PMID:17003490

Decoding the patterns of ubiquitin recognition by ubiquitin-associated domains from free energy simulations.

PubMed

Bouvier, Benjamin

2014-01-07

Ubiquitin is a highly conserved, highly represented protein acting as a regulating signal in numerous cellular processes. It leverages a single hydrophobic binding patch to recognize and bind a large variety of protein domains with remarkable specificity, but can also self-assemble into chains of poly-diubiquitin units in which these interfaces are sequestered, profoundly altering the individual monomers' recognition characteristics. Despite numerous studies, the origins of this varied specificity and the competition between substrates for the binding of the ubiquitin interface patch remain under heated debate. This study uses enhanced sampling all-atom molecular dynamics to simulate the unbinding of complexes of mono- or K48-linked diubiquitin bound to several ubiquitin-associated domains, providing insights into the mechanism and free energetics of ubiquitin recognition and binding. The implications for the subtle tradeoff between the stability of the polyubiquitin signal and its easy recognition by target protein assemblies are discussed, as is the enhanced affinity of the latter for long polyubiquitin chains compared to isolated mono- or diubiquitin.

FBXL5 interacts with p150 {sup Glued} and regulates its ubiquitination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang Ning; Liu Jing; Ding Xia

2007-07-20

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. p150 {sup Glued} is the dynactin subunit responsible for binding to dynein and microtubules. The F-box proteins constitute one of the four subunits of ubiquitin protein ligase complex called SCFs (SKP1-cullin-F-box), which governs phosphorylation-dependent ubiquitination and subsequent proteolysis. Our recent study showed that the proteolysis of mitotic kinesin CENP-E is mediated by SCF via a direct Skp1 link [D. Liu, N. Zhang, J. Du, X. Cai, M. Zhu, C. Jin, Z. Dou, C. Feng, Y. Yang, L. Liu, K. Takeyasu, W. Xie, X. Yao,more » Interaction of Skp1 with CENP-E at the midbody is essential for cytokinesis, Biochem. Biophys. Res. Commun. 345 (2006) 394-402]. Here we show that F-box protein FBXL5 interacts with p150 {sup Glued} and orchestrates its turnover via ubiquitination. FBXL5 binds to p150 {sup Glued} in vitro and in vivo. FBXL5 and p150 {sup Glued} co-localize primarily in the cytoplasm with peri-nuclear enrichment in HeLa cells. Overexpression of FBXL5 promotes poly-ubiquitination of p150 {sup Glued} and protein turnover of p150 {sup Glued} . Our findings provide a potential mechanism by which p150 {sup Glued} protein function is regulated by SCFs.« less

Synthetic and semi-synthetic strategies to study ubiquitin signaling.

PubMed

van Tilburg, Gabriëlle Ba; Elhebieshy, Angela F; Ovaa, Huib

2016-06-01

The post-translational modification ubiquitin can be attached to the ɛ-amino group of lysine residues or to a protein's N-terminus as a mono ubiquitin moiety. Via its seven intrinsic lysine residues and its N-terminus, it can also form ubiquitin chains on substrates in many possible ways. To study ubiquitin signals, many synthetic and semi-synthetic routes have been developed for generation of ubiquitin-derived tools and conjugates. The strength of these methods lies in their ability to introduce chemo-selective ligation handles at sites that currently cannot be enzymatically modified. Here, we review the different synthetic and semi-synthetic methods available for ubiquitin conjugate synthesis and their contribution to how they have helped investigating conformational diversity of diubiquitin signals. Next, we discuss how these methods help understanding the ubiquitin conjugation-deconjugation system by recent advances in ubiquitin ligase probes and diubiquitin-based DUB probes. Lastly, we discuss how these methods help studying post-translational modification of ubiquitin itself. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ubiquitination dynamics in the early-branching eukaryote Giardia intestinalis

PubMed Central

Niño, Carlos A; Chaparro, Jenny; Soffientini, Paolo; Polo, Simona; Wasserman, Moises

2013-01-01

Ubiquitination is a highly dynamic and versatile posttranslational modification that regulates protein function, stability, and interactions. To investigate the roles of ubiquitination in a primitive eukaryotic lineage, we utilized the early-branching eukaryote Giardia intestinalis. Using a combination of biochemical, immunofluorescence-based, and proteomics approaches, we assessed the ubiquitination status during the process of differentiation in Giardia. We observed that different types of ubiquitin modifications present specific cellular and temporal distribution throughout the Giardia life cycle from trophozoites to cyst maturation. Ubiquitin signal was detected in the wall of mature cysts, and enzymes implicated in cyst wall biogenesis were identified as substrates for ubiquitination. Interestingly, inhibition of proteasome activity did not affect trophozoite replication and differentiation, while it caused a decrease in cyst viability, arguing for proteasome involvement in cyst wall maturation. Using a proteomics approach, we identified around 200 high-confidence ubiquitinated candidates that vary their ubiquitination status during differentiation. Our results indicate that ubiquitination is critical for several cellular processes in this primitive eukaryote. PMID:23613346

Grouper tshβ Promoter-Driven Transgenic Zebrafish Marks Proximal Kidney Tubule Development

PubMed Central

Wang, Yang; Sun, Zhi-Hui; Zhou, Li; Li, Zhi; Gui, Jian-Fang

2014-01-01

Kidney tubule plays a critical role in recovering or secreting solutes, but the detailed morphogenesis remains unclear. Our previous studies have found that grouper tshβ (gtshβ) is also expressed in kidney, however, the distribution significance is still unknown. To understand the gtshβ role and kidney tubule morphogenesis, here, we have generated a transgenic zebrafish line Tg(gtshβ:GFP) with green fluorescent protein driven by the gtshβ promoter. Similar to the endogenous tshβ in zebrafish or in grouper, the gtshβ promoter-driven GFP is expressed in pituitary and kidney, and the developing details of proximal kidney tubule are marked in the transgenic zebrafish line. The gfp initially transcribes at 16 hours post fertilization (hpf) above the dorsal mesentery, and partially co-localizes with pronephric tubular markers slc20a1a and cdh17. Significantly, the GFP specifically localizes in proximal pronephric segments during embryogenesis and resides at kidney duct epithelium in adult fish. To test whether the gtshβ promoter-driven GFP may serve as a readout signal of the tubular development, we have treated the embryos with retinoic acid signaing (RA) reagents, in which exogenous RA addition results in a distal extension of the proximal segments, while RA inhibition induces a weakness and shortness of the proximal segments. Therefore, this transgenic line provides a useful tool for genetic or chemical analysis of kidney tubule. PMID:24905828

Characterization and Promoter Analysis of a Cotton Ring-Type Ubiquitin Ligase (E3) Gene

USDA-ARS?s Scientific Manuscript database

A cotton fiber cDNA, GhRING1, and its corresponding gene have been cloned and characterized. The GhRING1 gene encodes a RING-type ubiquitin ligase (E3) containing 337 amino acids (aa). The GhRING1 protein contains a RING finger motif with conserved cysteine and histine residues at the C-terminus a...

Correlation between ubiquitination and defects of bull spermatozoa and removal of defective spermatozoa using anti-ubiquitin antibody-coated magnetized beads.

PubMed

Zhang, Jian; Su, Jie; Hu, Shuxiang; Zhang, Jindun; Ding, Rui; Guo, Jitong; Cao, Guifang; Li, Rongfeng; Sun, Qing-Yuan; Li, Xihe

2018-05-01

Ubiquitination is an important cellular process in spermatogenesis and involves the regulation of spermatid differentiation and spermiogenesis. In the current study, the correlation between bull sperm ubiquitination and sperm defects was analyzed, and the feasibility using anti-ubiquitin specific antibody immobilized magnetic beads to remove the spermatozoa with defects was assessed. A total of nine bulls were examined, and the amount of sperm ubiquitination ranged from 55 to 151. Correspondingly, the percentage of sperm deformity ranged from 9.3% to 28.1%. The coefficient of correlation was r = 0.92, indicating a significant correlation between the percentage of sperm deformity and the amount of ubiquitination (P < 0.05). The results from use of fluorescence staining and single-channel flow cytometry indicated there was a significant correlation between the sperm deformity and amount of ubiquitination (r = 0.86, P < 0.05). Results gained by use of the TUNEL and ubiquitination assays by double-channel flow cytometry indicated that the proportion of genetically defective spermatozoa with ubiquitination in Q3 and Q2 quartiles was markedly greater than that of spermatozoa with ubiquitination in Q1 and Q4 quartiles (82.1% compared with 17.9%). All these results confirmed that sperm ubiquitination is associated with genetic DNA defects (P < 0.01). Furthermore, nine semen samples with sperm motility of less than 50% (minimal motility), 50% to 70% (moderate motility) and greater than 70% (greatest motility) were selected for sorting defective spermatozoa using anti-ubiquitin specific antibody-coated magnetic beads. Strikingly, the percentage of sperm deformity significantly decreased from 18.8%, 19.0% and 17.1% to 11.7%, 11.0% and 11.0%, respectively (P < 0.05), suggesting that this method might be a feasible technology to improve the productivity via removal of the defective spermatozoa from bull semen. Copyright © 2018 Elsevier B.V. All rights

An Interaction Landscape of Ubiquitin Signaling.

PubMed

Zhang, Xiaofei; Smits, Arne H; van Tilburg, Gabrielle B A; Jansen, Pascal W T C; Makowski, Matthew M; Ovaa, Huib; Vermeulen, Michiel

2017-03-02

Intracellular signaling via the covalent attachment of different ubiquitin linkages to protein substrates is fundamental to many cellular processes. Although linkage-selective ubiquitin interactors have been studied on a case-by-case basis, proteome-wide analyses have not been conducted yet. Here, we present ubiquitin interactor affinity enrichment-mass spectrometry (UbIA-MS), a quantitative interaction proteomics method that makes use of chemically synthesized diubiquitin to enrich and identify ubiquitin linkage interactors from crude cell lysates. UbIA-MS reveals linkage-selective diubiquitin interactions in multiple cell types. For example, we identify TAB2 and TAB3 as novel K6 diubiquitin interactors and characterize UCHL3 as a K27-linkage selective interactor that regulates K27 polyubiquitin chain formation in cells. Additionally, we show a class of monoubiquitin and K6 diubiquitin interactors whose binding is induced by DNA damage. We expect that our proteome-wide diubiquitin interaction landscape and established workflows will have broad applications in the ongoing efforts to decipher the complex language of ubiquitin signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

A Review on Ubiquitination of Neurotrophin Receptors: Facts and Perspectives

PubMed Central

Sánchez-Sánchez, Julia; Arévalo, Juan Carlos

2017-01-01

Ubiquitination is a reversible post-translational modification involved in a plethora of different physiological functions. Among the substrates that are ubiquitinated, neurotrophin receptors (TrkA, TrkB, TrkC, and p75NTR) have been studied recently. TrkA is the most studied receptor in terms of its ubiquitination, and different E3 ubiquitin ligases and deubiquitinases have been implicated in its ubiquitination, whereas not much is known about the other neurotrophin receptors aside from their ubiquitination. Additional studies are needed that focus on the ubiquitination of TrkB, TrkC, and p75NTR in order to further understand the role of ubiquitination in their physiological and pathological functions. Here we review what is currently known regarding the ubiquitination of neurotrophin receptors and its physiological and pathological relevance. PMID:28335430

A Subset of Ubiquitin-Conjugating Enzymes Is Essential for Plant Immunity.

PubMed

Zhou, Bangjun; Mural, Ravi V; Chen, Xuanyang; Oates, Matt E; Connor, Richard A; Martin, Gregory B; Gough, Julian; Zeng, Lirong

2017-02-01

Of the three classes of enzymes involved in ubiquitination, ubiquitin-conjugating enzymes (E2) have been often incorrectly considered to play merely an auxiliary role in the process, and few E2 enzymes have been investigated in plants. To reveal the role of E2 in plant innate immunity, we identified and cloned 40 tomato genes encoding ubiquitin E2 proteins. Thioester assays indicated that the majority of the genes encode enzymatically active E2. Phylogenetic analysis classified the 40 tomato E2 enzymes into 13 groups, of which members of group III were found to interact and act specifically with AvrPtoB, a Pseudomonas syringae pv tomato effector that uses its ubiquitin ligase (E3) activity to suppress host immunity. Knocking down the expression of group III E2 genes in Nicotiana benthamiana diminished the AvrPtoB-promoted degradation of the Fen kinase and the AvrPtoB suppression of host immunity-associated programmed cell death. Importantly, silencing group III E2 genes also resulted in reduced pattern-triggered immunity (PTI). By contrast, programmed cell death induced by several effector-triggered immunity elicitors was not affected on group III-silenced plants. Functional characterization suggested redundancy among group III members for their role in the suppression of plant immunity by AvrPtoB and in PTI and identified UBIQUITIN-CONJUGATING11 (UBC11), UBC28, UBC29, UBC39, and UBC40 as playing a more significant role in PTI than other group III members. Our work builds a foundation for the further characterization of E2s in plant immunity and reveals that AvrPtoB has evolved a strategy for suppressing host immunity that is difficult for the plant to thwart. © 2017 American Society of Plant Biologists. All Rights Reserved.

USP7 small-molecule inhibitors interfere with ubiquitin binding.

PubMed

Kategaya, Lorna; Di Lello, Paola; Rougé, Lionel; Pastor, Richard; Clark, Kevin R; Drummond, Jason; Kleinheinz, Tracy; Lin, Eva; Upton, John-Paul; Prakash, Sumit; Heideker, Johanna; McCleland, Mark; Ritorto, Maria Stella; Alessi, Dario R; Trost, Matthias; Bainbridge, Travis W; Kwok, Michael C M; Ma, Taylur P; Stiffler, Zachary; Brasher, Bradley; Tang, Yinyan; Jaishankar, Priyadarshini; Hearn, Brian R; Renslo, Adam R; Arkin, Michelle R; Cohen, Frederick; Yu, Kebing; Peale, Frank; Gnad, Florian; Chang, Matthew T; Klijn, Christiaan; Blackwood, Elizabeth; Martin, Scott E; Forrest, William F; Ernst, James A; Ndubaku, Chudi; Wang, Xiaojing; Beresini, Maureen H; Tsui, Vickie; Schwerdtfeger, Carsten; Blake, Robert A; Murray, Jeremy; Maurer, Till; Wertz, Ingrid E

2017-10-26

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy

Ubiquitination of specific mitochondrial matrix proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lehmann, Gilad; Ziv, Tamar; Braten, Ori

2016-06-17

Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinatedmore » proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.« less

A MAPK-Driven Feedback Loop Suppresses Rac Activity to Promote RhoA-Driven Cancer Cell Invasion

PubMed Central

Hetmanski, Joseph H. R.; Zindy, Egor; Schwartz, Jean-Marc; Caswell, Patrick T.

2016-01-01

Cell migration in 3D microenvironments is fundamental to development, homeostasis and the pathobiology of diseases such as cancer. Rab-coupling protein (RCP) dependent co-trafficking of α5β1 and EGFR1 promotes cancer cell invasion into fibronectin (FN) containing extracellular matrix (ECM), by potentiating EGFR1 signalling at the front of invasive cells. This promotes a switch in RhoGTPase signalling to inhibit Rac1 and activate a RhoA-ROCK-Formin homology domain-containing 3 (FHOD3) pathway and generate filopodial actin-spike protrusions which drive invasion. To further understand the signalling network that drives RCP-driven invasive migration, we generated a Boolean logical model based on existing network pathways/models, where each node can be interrogated by computational simulation. The model predicted an unanticipated feedback loop, whereby Raf/MEK/ERK signalling maintains suppression of Rac1 by inhibiting the Rac-activating Sos1-Eps8-Abi1 complex, allowing RhoA activity to predominate in invasive protrusions. MEK inhibition was sufficient to promote lamellipodia formation and oppose filopodial actin-spike formation, and led to activation of Rac and inactivation of RhoA at the leading edge of cells moving in 3D matrix. Furthermore, MEK inhibition abrogated RCP/α5β1/EGFR1-driven invasive migration. However, upon knockdown of Eps8 (to suppress the Sos1-Abi1-Eps8 complex), MEK inhibition had no effect on RhoGTPase activity and did not oppose invasive migration, suggesting that MEK-ERK signalling suppresses the Rac-activating Sos1-Abi1-Eps8 complex to maintain RhoA activity and promote filopodial actin-spike formation and invasive migration. Our study highlights the predictive potential of mathematical modelling approaches, and demonstrates that a simple intervention (MEK-inhibition) could be of therapeutic benefit in preventing invasive migration and metastasis. PMID:27138333

Parkin-phosphoubiquitin complex reveals a cryptic ubiquitin binding site required for RBR ligase activity

PubMed Central

Kumar, Atul; Chaugule, Viduth K; Condos, Tara E C; Barber, Kathryn R; Johnson, Clare; Toth, Rachel; Sundaramoorthy, Ramasubramanian; Knebel, Axel; Shaw, Gary S; Walden, Helen

2017-01-01

RING-BETWEENRING-RING (RBR) E3 ligases are a class of ubiquitin ligases distinct from RING or HECT E3 ligases. An important RBR is Parkin, mutations in which lead to early onset hereditary Parkinsonism. Parkin and other RBRs share a catalytic RBR module, but are usually autoinhibited and activated via distinct mechanisms. Recent insights into Parkin regulation predict large, unknown conformational changes during activation of Parkin. However, current data on active RBRs are in the absence of regulatory domains. Therefore, how individual RBRs are activated, and whether they share a common mechanism remains unclear. We now report the crystal structure of a human Parkin-phosphoubiquitin complex, which shows that phosphoubiquitin binding induces a movement in the IBR domain to reveal a cryptic ubiquitin binding site. Mutation of this site negatively impacts on Parkin’s activity. Furthermore, ubiquitin binding promotes cooperation between Parkin molecules, suggesting a role for interdomain association in RBR ligase mechanism. PMID:28414322

The importance of regulatory ubiquitination in cancer and metastasis

PubMed Central

Gallo, L. H.; Ko, J.; Donoghue, D. J.

2017-01-01

ABSTRACT Ubiquitination serves as a degradation mechanism of proteins, but is involved in additional cellular processes such as activation of NFκB inflammatory response and DNA damage repair. We highlight the E2 ubiquitin conjugating enzymes, E3 ubiquitin ligases and Deubiquitinases that support the metastasis of a plethora of cancers. E3 ubiquitin ligases also modulate pluripotent cancer stem cells attributed to chemotherapy resistance. We further describe mutations in E3 ubiquitin ligases that support tumor proliferation and adaptation to hypoxia. Thus, this review describes how tumors exploit members of the vast ubiquitin signaling pathways to support aberrant oncogenic signaling for survival and metastasis. PMID:28166483

Decoding the Ubiquitin-Mediated Pathway of Arthropod Disease Vectors

PubMed Central

Choy, Anthony; Severo, Maiara S.; Sun, Ruobai; Girke, Thomas; Gillespie, Joseph J.; Pedra, Joao H. F.

2013-01-01

Protein regulation by ubiquitin has been extensively described in model organisms. However, characterization of the ubiquitin machinery in disease vectors remains mostly unknown. This fundamental gap in knowledge presents a concern because new therapeutics are needed to control vector-borne diseases, and targeting the ubiquitin machinery as a means for disease intervention has been already adopted in the clinic. In this study, we employed a bioinformatics approach to uncover the ubiquitin-mediated pathway in the genomes of Anopheles gambiae, Aedes aegypti, Culex quinquefasciatus, Ixodes scapularis, Pediculus humanus and Rhodnius prolixus. We observed that (1) disease vectors encode a lower percentage of ubiquitin-related genes when compared to Drosophila melanogaster, Mus musculus and Homo sapiens but not Saccharomyces cerevisiae; (2) overall, there are more proteins categorized as E3 ubiquitin ligases when compared to E2-conjugating or E1-activating enzymes; (3) the ubiquitin machinery within the three mosquito genomes is highly similar; (4) ubiquitin genes are more than doubled in the Chagas disease vector (R. prolixus) when compared to other arthropod vectors; (5) the deer tick I. scapularis and the body louse (P. humanus) genomes carry low numbers of E1-activating enzymes and HECT-type E3 ubiquitin ligases; (6) R. prolixus have low numbers of RING-type E3 ubiquitin ligases; and (7) C. quinquefasciatus present elevated numbers of predicted F-box E3 ubiquitin ligases, JAB and UCH deubiquitinases. Taken together, these findings provide novel opportunities to study the interaction between a pathogen and an arthropod vector. PMID:24205097

Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsumoto, Hotaru; Saitoh, Hisato, E-mail: hisa@kumamoto-u.ac.jp; Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto

2016-07-29

We have previously reported the co-localization of O-propargyl-puromycin (OP-Puro) with SUMO-2/3 and ubiquitin at promyelocytic leukemia-nuclear bodies (PML-NBs) in the presence of the proteasome inhibitor MG132, implying a role for the ubiquitin family in sequestering OP-puromycylated immature polypeptides to the nucleus during impaired proteasome activity. Here, we found that as expected puromycin induced SUMO-1/2/3 accumulation with ubiquitin at multiple nuclear foci in HeLa cells when co-exposed to MG132. Co-administration of puromycin and MG132 also facilitated redistribution of PML and the SUMO-targeted ubiquitin ligase RNF4 concurrently with SUMO-2/3. As removal of the drugs from the medium led to disappearance of themore » SUMO-2/3-ubiquitin nuclear foci, our findings indicated that nuclear assembly/disassembly of SUMO-2/3 and ubiquitin was pharmacologically manipulable, supporting our previous observation on OP-Puro, which predicted the ubiquitin family function in sequestrating aberrant proteins to the nucleus. -- Highlights: •Puromycin exhibits the O-propargyl-puromycin effect. •Puromycin induces SUMO redistribution upon proteasome inhibition. •Ubiquitin and RNF4 accumulate at PML-nuclear bodies with SUMO-2/3. •The ubiquitin family may function in nuclear sequestration of immature proteins.« less

Isolation and functional characterization of a cotton ubiquitination-related promoter and 5'UTR that drives high levels of expression in root and flower tissues

PubMed Central

2011-01-01

Background Cotton (Gossypium spp.) is an important crop worldwide that provides raw material to 40% of the textile fiber industry. Important traits have been studied aiming the development of genetically modified crops including resistance to insect and diseases, and tolerance to drought, cold and herbicide. Therefore, the characterization of promoters and regulatory regions is also important to achieve high gene expression and/or a specific expression pattern. Commonly, genes involved in ubiquitination pathways are highly and differentially expressed. In this study, we analyzed the expression of a cotton ubiquitin-conjugating enzyme (E2) family member with no previous characterization. Results Nucleotide analysis revealed high identity with cotton E2 homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the E2 active site, and an intron that is spliced in E2 homologues, but not in GhGDRP85. The GhGDRP85 gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2) and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in Arabidopsis thaliana. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2. Conclusions uceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2) and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues. PMID:22115195

[Ubiquitin-proteasome system and sperm DNA repair: An update].

PubMed

Zhang, Guo-Wei; Cai, Hong-Cai; Shang, Xue-Jun

2016-09-01

The ubiquitin-proteasome system (UPS) is a proteasome system widely present in the human body, which is composed of ubiquitin (Ub), ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2), ubiquitin protein ligases (E3), 26S proteasome, and deubiquitinating enzymes (DUBs) and involved in cell cycle regulation, immune response, signal transduction, DNA repair as well as protein degradation. Sperm DNA is vulnerable to interference or damage in the progression of chromosome association and homologous recombination. Recent studies show that UPS participates in DNA repair in spermatogenesis by modulating DNA repair enzymes via ubiquitination, assisting in the identification of DNA damage sites, raising damage repair-related proteins, initiating the DNA repair pathway, maintaining chromosome stability, and ensuring the normal process of spermatogenesis.

The ubiquitin-proteasome system is necessary for long-term synaptic depression in Aplysia.

PubMed

Fioravante, Diasinou; Liu, Rong-Yu; Byrne, John H

2008-10-08

The neuropeptide Phe-Met-Arg-Phe-NH(2) (FMRFa) can induce transcription-dependent long-term synaptic depression (LTD) in Aplysia sensorimotor synapses. We investigated the role of the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to presynaptic inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by CREB2, which is generally regarded as a transcription repressor. Binding of CREB2 to the promoter region of ap-uch was accompanied by histone hyperacetylation, suggesting that CREB2 cannot only inhibit but also promote gene expression. CREB2 was phosphorylated after FMRFa, and blocking phospho-CREB2 blocked LTD. In addition to changes in the expression of ap-uch, the synaptic vesicle-associated protein synapsin was downregulated in LTD in a proteasome-dependent manner. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions.

Enhanced ubiquitination of cytoskeletal proteins in pressure overloaded myocardium is accompanied by changes in specific E3 ligases.

PubMed

Balasubramanian, Sundaravadivel; Mani, Santhoshkumar; Shiraishi, Hirokazu; Johnston, Rebecca K; Yamane, Kentaro; Willey, Christopher D; Cooper, George; Tuxworth, William J; Kuppuswamy, Dhandapani

2006-10-01

Ubiquitin conjugation of proteins is critical for cell homeostasis and contributes to both cell survival and death. Here we studied ubiquitination of proteins in pressure overloaded (PO) myocardium in the context of cardiomyocyte survival. Analysis using a feline right ventricular pressure overload (RVPO) model revealed a robust and transient increase in ubiquitination of proteins present in the Triton X-100-insoluble fraction in 24 to 48 h PO myocardium, and confocal micrographs indicate this increase in ubiquitination occurs subsarcolemmaly near the intercalated disc area of cardiomyocytes. The ubiquitination was accompanied by changes in E3 ligases including Cbl, E6AP, Mdm2 and cIAP in the same period of PO, although atrophy-related E3 ligases, MuRF1 and MuRF3 were unaltered. Furthermore, Cbl displayed a substantial increase in both levels of expression and tyrosine phosphorylation in 48 h PO myocardium. Confocal studies revealed enrichment of Cbl at the intercalated discs of 48 h PO cardiomyocytes, as evidenced by its colocalization with N-cadherin. Although apoptosis was observed in 48 h PO myocardium by TUNEL staining, cardiomyocytes showing ubiquitin staining were not positive for TUNEL staining. Furthermore, 48 h PO resulted in the phosphorylation of inhibitor of nuclear factor kappa B (IkappaB), suggesting its ubiquitin-mediated degradation and the nuclear localization of NFkappaB for the expression of specific cell survival factors such as cIAPs. Together these data indicate that increased levels of E3 ligases that regulate cell homeostasis and promote cell survival could ubiquitinate multiple cytoskeletal protein targets and that these events that occur during the early phase of PO may contribute to both cardiomyocyte survival and hypertrophy.

A unique deubiquitinase that deconjugates phosphoribosyl-linked protein ubiquitination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, Jiazhang; Yu, Kaiwen; Fei, Xiaowen

Ubiquitination regulates many aspects of host immunity and thus is a common target for infectious agents. Recent studies revealed that members of the SidE effector family of the bacterial pathogen Legionella pneumophila attacked several small GTPases associated with the endoplasmic reticulum by a novel ubiquitination mechanism that does not require the E1 and E2 enzymes of the host ubiquitination machinery. Following ubiquitin activation by ADP- ribosylation via a mono-ADP-ribosylation motif, ADP-ribosylated ubiquitin is cleaved by a phosphodiesterasedomainwithinSdeA,whichisconcomitantwiththelinkof phosphoribosylated ubiquitin to serine residues in the substrate. Here we demonstrate that the activity of SidEs is regulated by SidJ, another effector encodedmore » by a gene situated in the locus coding for three members of the SidE family (SdeC, SdeB and SdeA). SidJ functions to remove ubiquitin from SidEs-modified substrates by cleaving the phosphodiester bond that links phosphoribosylated ubiquitin to protein substrates. Further, the deubiquitinase activity of SidJ is essential for its role in L. pneumophila infection. Finally, the activity of SidJ is required for efficiently reducing the abundance of ubiquitinated Rab33b in infected cells within a few hours after bacterial uptake. Our results establish SidJ as a deubiquitinase that functions to impose temporal regulation of the activity of the SidE effectors. The identification of SidJ may shed light on future study of signaling cascades mediated by this unique ubiquitination that also potentially regulates cellular processes in eukaryotic cells.« less

Role of Growth Arrest and DNA Damage–inducible α in Akt Phosphorylation and Ubiquitination after Mechanical Stress-induced Vascular Injury

PubMed Central

Mitra, Sumegha; Sammani, Saad; Wang, Ting; Boone, David L.; Meyer, Nuala J.; Dudek, Steven M.; Moreno-Vinasco, Liliana; Garcia, Joe G. N.

2011-01-01

Rationale: The stress-induced growth arrest and DNA damage–inducible α (GADD45a) gene is up-regulated by mechanical stress with GADD45a knockout (GADD45a−/−) mice demonstrating both increased susceptibility to ventilator-induced lung injury (VILI) and reduced levels of the cell survival and vascular permeability signaling effector (Akt). However, the functional role of GADD45a in the pathogenesis of VILI is unknown. Objectives: We sought to define the role of GADD45a in the regulation of Akt activation induced by mechanical stress. Methods: VILI-challenged GADD45a−/− mice were administered a constitutively active Akt1 vector and injury was assessed by bronchoalveolar lavage cell counts and protein levels. Human pulmonary artery endothelial cells (EC) were exposed to 18% cyclic stretch (CS) under conditions of GADD45a silencing and used for immunoprecipitation, Western blotting or immunofluoresence. EC were also transfected with mutant ubiquitin vectors to characterize site-specific Akt ubiquitination. DNA methylation was measured using methyl-specific polymerase chain reaction assay. Measurements and Main Results: Studies exploring the linkage of GADD45a with mechanical stress and Akt regulation revealed VILI-challenged GADD45a−/− mice to have significantly reduced lung injury on overexpression of Akt1 transgene. Increased mechanical stress with 18% CS in EC induced Akt phosphorylation via E3 ligase tumor necrosis factor receptor–associated factor 6 (TRAF6)–mediated Akt K63 ubiquitination resulting in Akt trafficking and activation at the membrane. GADD45a is essential to this process because GADD45a-silenced endothelial cells and GADD45a−/− mice exhibited increased Akt K48 ubiquitination leading to proteasomal degradation. These events involve loss of ubiquitin carboxyl terminal hydrolase 1 (UCHL1), a deubiquitinating enzyme that normally removes K48 polyubiquitin chains bound to Akt thus promoting Akt K63 ubiquitination. Loss of GADD45a

Structural determinants of ubiquitin-CXC chemokine receptor 4 interaction.

PubMed

Saini, Vikas; Marchese, Adriano; Tang, Wei-Jen; Majetschak, Matthias

2011-12-23

Ubiquitin, a post-translational protein modifier inside the cell, functions as a CXC chemokine receptor (CXCR) 4 agonist outside the cell. However, the structural determinants of the interaction between extracellular ubiquitin and CXCR4 remain unknown. Utilizing C-terminal truncated ubiquitin and ubiquitin mutants, in which surface residues that are known to interact with ubiquitin binding domains in interacting proteins are mutated (Phe-4, Leu-8, Ile-44, Asp-58, Val-70), we provide evidence that the ubiquitin-CXCR4 interaction follows a two-site binding mechanism in which the hydrophobic surfaces surrounding Phe-4 and Val-70 are important for receptor binding, whereas the flexible C terminus facilitates receptor activation. Based on these findings and the available crystal structures, we then modeled the ubiquitin-CXCR4 interface with the RosettaDock software followed by small manual adjustments, which were guided by charge complementarity and anticipation of a conformational switch of CXCR4 upon activation. This model suggests three residues of CXCR4 (Phe-29, Phe-189, Lys-271) as potential interaction sites. Binding studies with HEK293 cells overexpressing wild type and CXCR4 after site-directed mutagenesis confirm that these residues are important for ubiquitin binding but that they do not contribute to the binding of stromal cell-derived factor 1α. Our findings suggest that the structural determinants of the CXCR4 agonist activity of ubiquitin mimic the typical structure-function relationship of chemokines. Furthermore, we provide evidence for separate and specific ligand binding sites on CXCR4. As exogenous ubiquitin has been shown to possess therapeutic potential, our findings are expected to facilitate the structure-based design of new compounds with ubiquitin-mimetic actions on CXCR4.

Mitochondrial Ubiquitin Ligase in Cardiovascular Disorders.

PubMed

Yu, Tao; Zhang, Yinfeng; Li, Pei-Feng

2017-01-01

Mitochondrial dynamics play a critical role in cellular responses and physiological process. However, their dysregulation leads to a functional degradation, which results in a diverse array of common disorders, including cardiovascular disease. In this background, the mitochondrial ubiquitin ligase has been attracting substantial research interest in recent years. Mitochondrial ubiquitin ligase is localized in the mitochondrial outer membrane, where it plays an essential role in the regulation of mitochondrial dynamics and apoptosis. In this chapter, we provide a comprehensive overview of the functions of mitochondrial ubiquitin ligases identified hitherto, with a special focus on cardiovascular disorders.

Atomic-level description of ubiquitin folding

PubMed Central

Piana, Stefano; Lindorff-Larsen, Kresten; Shaw, David E.

2013-01-01

Equilibrium molecular dynamics simulations, in which proteins spontaneously and repeatedly fold and unfold, have recently been used to help elucidate the mechanistic principles that underlie the folding of fast-folding proteins. The extent to which the conclusions drawn from the analysis of such proteins, which fold on the microsecond timescale, apply to the millisecond or slower folding of naturally occurring proteins is, however, unclear. As a first attempt to address this outstanding issue, we examine here the folding of ubiquitin, a 76-residue-long protein found in all eukaryotes that is known experimentally to fold on a millisecond timescale. Ubiquitin folding has been the subject of many experimental studies, but its slow folding rate has made it difficult to observe and characterize the folding process through all-atom molecular dynamics simulations. Here we determine the mechanism, thermodynamics, and kinetics of ubiquitin folding through equilibrium atomistic simulations. The picture emerging from the simulations is in agreement with a view of ubiquitin folding suggested from previous experiments. Our findings related to the folding of ubiquitin are also consistent, for the most part, with the folding principles derived from the simulation of fast-folding proteins, suggesting that these principles may be applicable to a wider range of proteins. PMID:23503848

Histone ubiquitination: a tagging tail unfolds?

PubMed

Jason, Laure J M; Moore, Susan C; Lewis, John D; Lindsey, George; Ausió, Juan

2002-02-01

Despite the fact that histone H2A ubiquitination affects about 10-15% of this histone in most eukaryotic cells, histone ubiquitination is among one of the less-well-characterized post-translational histone modifications. Nevertheless, some important observations have been made in recent years. Whilst several enzymes had been known to ubiquitinate histones in vitro, recent studies in yeast have led to the unequivocal identification of the enzyme responsible for this post-translational modification in this organism. A strong functional co-relation to meiosis and spermiogenesis has also now been well documented, although its participation in other functional aspects of chromatin metabolism, such as transcription or DNA repair, still remains rather speculative and controversial. Because of its nature, histone ubiquitination represents the most bulky structural change to histones and as such it would be expected to exert an important effect on chromatin structure. Past and recent structural studies, however, indicate a surprising lack of effect of (H2A/H2B) ubiquitination on nucleosome architecture and of uH2A on chromatin folding. These results suggest that this modification may serve as a signal for recognition by functionally relevant trans-acting factors and/or operate synergistically in conjunction with other post-translational modifications such as for instance acetylation. Copyright 2002 Wiley Periodicals, Inc.

Rines/RNF180, a novel RING finger gene-encoded product, is a membrane-bound ubiquitin ligase.

PubMed

Ogawa, Miyuki; Mizugishi, Kiyomi; Ishiguro, Akira; Koyabu, Yoshio; Imai, Yuzuru; Takahashi, Ryosuke; Mikoshiba, Katsuhiko; Aruga, Jun

2008-04-01

We identified and characterized a novel RING finger gene, Rines/RNF180, which is well conserved among vertebrates. Putative Rines gene product (Rines) contains a RING finger domain, a basic coiled-coil domain, a novel conserved domain (DSPRC) and a C-terminal hydrophobic region that is predicted to be a transmembrane domain. N-terminally epitope tagged-Rines (Nt-Rines) was detected in the endoplasmic reticulum membrane/nuclear envelope in cultured mammalian cells. Nt-Rines was not extracted by high salt or alkaline buffers and was degraded in intact endoplasmic reticulum treated with proteinase K, indicating that Nt-Rines is an integral membrane protein with most of its N-terminal regions in the cytoplasm. Rines was expressed in brain, kidney, testis and uterus of adult mice, and in developing lens and brain, particularly in the ventricular layer of the cerebral cortex at embryonic stages. In cultured cells, Nt-Rines can bind another protein and promoted its degradation. The degradation was inhibited by proteasomal inhibitors. In addition, Nt-Rines itself was heavily ubiquitinated and degraded by proteasome. The involvement of Rines in the ubiquitin-proteasome pathway was further supported by its binding to the UbcH6 ubiquitin-conjugating enzyme and by its trans-ubiquitination enhancing activities. These results suggest that Rines is a membrane-bound E3 ubiquitin ligase.

Ubiquitin over-expression phenotypes and ubiquitin gene molecular misreading during aging in Drosophila melanogaster

PubMed Central

Hoe, Nicholas; Huang, Chung M.; Landis, Gary; Verhage, Marian; Ford, Daniel; Yang, Junsheng; van Leeuwen, Fred W.; Tower, John

2011-01-01

Molecular Misreading (MM) is the inaccurate conversion of genomic information into aberrant proteins. For example, when RNA polymerase II transcribes a GAGAG motif it synthesizes at low frequency RNA with a two-base deletion. If the deletion occurs in a coding region, translation will result in production of misframed proteins. During mammalian aging, misframed versions of human amyloid precursor protein (hApp) and ubiquitin (hUbb) accumulate in the aggregates characteristic of neurodegenerative diseases, suggesting dysfunctional degradation or clearance. Here cDNA clones encoding wild-type hUbb and the frame-shifted version hUbb+1 were expressed in transgenic Drosophila using the doxycycline-regulated system. Misframed proteins were abundantly produced, both from the transgenes and from endogenous Drosophila ubiquitin-encoding genes, and their abundance increased during aging in whole-fly extracts. Over-expression of wild-type hUbb, but not hUbb+1, was toxic during fly development. In contrast, when over-expressed specifically in adult flies, hUbb+1 caused small decreases in life span, whereas hUbb was associated with small increases, preferentially in males. The data suggest that MM occurs in Drosophila and that the resultant misframed proteins accumulate with age. MM of the ubiquitin gene can produce alternative ubiquitin gene products with different and sometimes opposing phenotypic effects. PMID:21415465

Evaluation of Selected Binding Domains for the Analysis of Ubiquitinated Proteomes

NASA Astrophysics Data System (ADS)

Nakayasu, Ernesto S.; Ansong, Charles; Brown, Joseph N.; Yang, Feng; Lopez-Ferrer, Daniel; Qian, Wei-Jun; Smith, Richard D.; Adkins, Joshua N.

2013-08-01

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono- and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ~200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle.

Evaluation of selected binding domains for the analysis of ubiquitinated proteomes

PubMed Central

Nakayasu, Ernesto S.; Ansong, Charles; Brown, Joseph N.; Yang, Feng; Lopez-Ferrer, Daniel; Qian, Wei-Jun; Smith, Richard D.; Adkins, Joshua N.

2013-01-01

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising, but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ∼200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle. PMID:23649778

Phosphorylated ubiquitin chain is the genuine Parkin receptor

PubMed Central

Okatsu, Kei; Koyano, Fumika; Kimura, Mayumi; Kosako, Hidetaka; Saeki, Yasushi

2015-01-01

PINK1 selectively recruits Parkin to depolarized mitochondria for quarantine and removal of damaged mitochondria via ubiquitylation. Dysfunction of this process predisposes development of familial recessive Parkinson’s disease. Although various models for the recruitment process have been proposed, none of them adequately explain the accumulated data, and thus the molecular basis for PINK1 recruitment of Parkin remains to be fully elucidated. In this study, we show that a linear ubiquitin chain of phosphomimetic tetra-ubiquitin(S65D) recruits Parkin to energized mitochondria in the absence of PINK1, whereas a wild-type tetra-ubiquitin chain does not. Under more physiologically relevant conditions, a lysosomal phosphorylated polyubiquitin chain recruited phosphomimetic Parkin to the lysosome. A cellular ubiquitin replacement system confirmed that ubiquitin phosphorylation is indeed essential for Parkin translocation. Furthermore, physical interactions between phosphomimetic Parkin and phosphorylated polyubiquitin chain were detected by immunoprecipitation from cells and in vitro reconstitution using recombinant proteins. We thus propose that the phosphorylated ubiquitin chain functions as the genuine Parkin receptor for recruitment to depolarized mitochondria. PMID:25847540

The paracaspase MALT1 cleaves HOIL1 reducing linear ubiquitination by LUBAC to dampen lymphocyte NF-κB signalling

PubMed Central

Klein, Theo; Fung, Shan-Yu; Renner, Florian; Blank, Michael A.; Dufour, Antoine; Kang, Sohyeong; Bolger-Munro, Madison; Scurll, Joshua M.; Priatel, John J.; Schweigler, Patrick; Melkko, Samu; Gold, Michael R.; Viner, Rosa I.; Régnier, Catherine H.; Turvey, Stuart E.; Overall, Christopher M.

2015-01-01

Antigen receptor signalling activates the canonical NF-κB pathway via the CARD11/BCL10/MALT1 (CBM) signalosome involving key, yet ill-defined roles for linear ubiquitination. The paracaspase MALT1 cleaves and removes negative checkpoint proteins, amplifying lymphocyte responses in NF-κB activation and in B-cell lymphoma subtypes. To identify new human MALT1 substrates, we compare B cells from the only known living MALT1mut/mut patient with healthy MALT1+/mut family members using 10-plex Tandem Mass Tag TAILS N-terminal peptide proteomics. We identify HOIL1 of the linear ubiquitin chain assembly complex as a novel MALT1 substrate. We show linear ubiquitination at B-cell receptor microclusters and signalosomes. Late in the NF-κB activation cycle HOIL1 cleavage transiently reduces linear ubiquitination, including of NEMO and RIP1, dampening NF-κB activation and preventing reactivation. By regulating linear ubiquitination, MALT1 is both a positive and negative pleiotropic regulator of the human canonical NF-κB pathway—first promoting activation via the CBM—then triggering HOIL1-dependent negative-feedback termination, preventing reactivation. PMID:26525107

Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition.

PubMed

Matsumoto, Hotaru; Saitoh, Hisato

2016-07-29

We have previously reported the co-localization of O-propargyl-puromycin (OP-Puro) with SUMO-2/3 and ubiquitin at promyelocytic leukemia-nuclear bodies (PML-NBs) in the presence of the proteasome inhibitor MG132, implying a role for the ubiquitin family in sequestering OP-puromycylated immature polypeptides to the nucleus during impaired proteasome activity. Here, we found that as expected puromycin induced SUMO-1/2/3 accumulation with ubiquitin at multiple nuclear foci in HeLa cells when co-exposed to MG132. Co-administration of puromycin and MG132 also facilitated redistribution of PML and the SUMO-targeted ubiquitin ligase RNF4 concurrently with SUMO-2/3. As removal of the drugs from the medium led to disappearance of the SUMO-2/3-ubiquitin nuclear foci, our findings indicated that nuclear assembly/disassembly of SUMO-2/3 and ubiquitin was pharmacologically manipulable, supporting our previous observation on OP-Puro, which predicted the ubiquitin family function in sequestrating aberrant proteins to the nucleus. Copyright © 2016 Elsevier Inc. All rights reserved.

An AAA Motor-Driven Mechanical Switch in Rpn11 Controls Deubiquitination at the 26S Proteasome.

PubMed

Worden, Evan J; Dong, Ken C; Martin, Andreas

2017-09-07

Poly-ubiquitin chains direct protein substrates to the 26S proteasome, where they are removed by the deubiquitinase Rpn11 during ATP-dependent substrate degradation. Rapid deubiquitination is required for efficient degradation but must be restricted to committed substrates that are engaged with the ATPase motor to prevent premature ubiquitin chain removal and substrate escape. Here we reveal the ubiquitin-bound structure of Rpn11 from S. cerevisiae and the mechanisms for mechanochemical coupling of substrate degradation and deubiquitination. Ubiquitin binding induces a conformational switch of Rpn11's Insert-1 loop from an inactive closed state to an active β hairpin. This switch is rate-limiting for deubiquitination and strongly accelerated by mechanical substrate translocation into the AAA+ motor. Deubiquitination by Rpn11 and ubiquitin unfolding by the ATPases are in direct competition. The AAA+ motor-driven acceleration of Rpn11 is therefore important to ensure that poly-ubiquitin chains are removed only from committed substrates and fast enough to prevent their co-degradation. Copyright © 2017 Elsevier Inc. All rights reserved.

Downregulation of Ubiquitin-conjugating Enzyme UBE2D3 Promotes Telomere Maintenance and Radioresistance of Eca-109 Human Esophageal Carcinoma Cells

PubMed Central

Yang, Hui; Wu, Lin; Ke, Shaobo; Wang, Wenbo; Yang, Lei; Gao, Xiaojia; Fang, Hongyan; Yu, Haijun; Zhong, Yahua; Xie, Conghua; Zhou, Fuxiang; Zhou, Yunfeng

2016-01-01

Ubiquitin-conjugating enzyme UBE2D3 is an important member of the ubiquitin-proteasome pathways. Our previous study showed that the expression of UBE2D3 was negatively related to human telomerase reverse transcriptase (hTERT) and radioresistance in human breast cancer cells. However, in esophageal carcinoma, the exact effects and mechanisms of UBE2D3 in radioresistance remain unclear. This study shows that UBE2D3 knockdown was associated with significant increases in radioresistance to X-rays, telomerase activity, telomere length, and telomere shelterins. UBE2D3 knockdown-mediated radioresistance was related to a decrease in the spontaneous and ionizing radiation-induced apoptosis, resulting from a decrease in the Bax/Bcl-2 ratio. Furthermore, UBE2D3 downregulation was associated with increased G1-S phase transition and prolonged IR-induced G2/M arrest through over expression of cyclin D1, decrease of CDC25A expression and promotion of the ATM/ATR-Chk1-CDC25C pathway. Moreover, UBE2D3 downregulation reduced spontaneous DNA double-strand breaks and accelerated the repair of DNA damage induced by IR. The current data thus demonstrate that UBE2D3 downregulation enhances radioresistance by increased telomere homeostasis and prolonged IR-induced G2/M arrest, but decreases the IR-induced apoptosis and the number of DNA damage foci. These results suggest that UBE2D3 might be a potential molecular target to improve radiotherapy effects in esophageal carcinoma. PMID:27326259

ABA-dependent inhibition of the ubiquitin proteasome system during germination at high temperature in Arabidopsis.

PubMed

Chiu, Rex Shun; Pan, Shiyue; Zhao, Rongmin; Gazzarrini, Sonia

2016-12-01

During germination, endogenous and environmental factors trigger changes in the transcriptome, translatome and proteome to break dormancy. In Arabidopsis thaliana, the ubiquitin proteasome system (UPS) degrades proteins that promote dormancy to allow germination. While research on the UPS has focused on the identification of proteasomal substrates, little information is known about the regulation of its activity. Here we characterized the activity of the UPS during dormancy release and maintenance by monitoring protein ubiquitination and degradation of two proteasomal substrates: Suc-LLVY-AMC, a well characterized synthetic substrate, and FUSCA3 (FUS3), a dormancy-promoting transcription factor degraded by the 26S proteasome. Our data indicate that proteasome activity and protein ubiquitination increase during imbibition at optimal temperature (21°C), and are required for seed germination. However, abscisic acid (ABA) and supraoptimal temperature (32°C) inhibit germination by dampening both protein ubiquitination and proteasome activity. Inhibition of UPS function by high temperature is reduced by the ABA biosynthesis inhibitor, fluridone, and in ABA biosynthetic mutants, suggesting that it is ABA dependent. Accordingly, inhibition of FUS3 degradation at 32°C is also dependent on ABA. Native gels show that inhibition of proteasome activity is caused by interference with the 26S/30S ratio as well as free 19S and 20S levels, impacting the proteasome degradation cycle. Transfer experiments show that ABA-mediated inhibition of proteasome activity at 21°C is restricted to the first 2 days of germination, a time window corresponding to seed sensitivity to environmental and ABA-mediated growth inhibition. Our data show that ABA and high temperature inhibit germination under unfavourable growth conditions by repressing the UPS. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Dengue Virus Genome Uncoating Requires Ubiquitination

PubMed Central

Byk, Laura A.; Iglesias, Néstor G.; De Maio, Federico A.; Gebhard, Leopoldo G.; Rossi, Mario

2016-01-01

ABSTRACT The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle. Flaviviruses are mainly arthropod-borne viruses, including emerging and reemerging pathogens such as dengue, Zika, and West Nile viruses. Currently, dengue virus is one of the most significant human viral pathogens transmitted by mosquitoes and is responsible for about 390 million infections every year around the world. Here, we examined for the first time molecular aspects of dengue virus genome uncoating. We followed the fate of the capsid protein and RNA genome early during infection and found that capsid is degraded after viral internalization by the host ubiquitin-proteasome system. However, proteasome activity and capsid degradation were not necessary to free the genome for initial viral translation. Unexpectedly, genome uncoating was blocked by inhibiting ubiquitination. Using different assays to bypass entry and evaluate the first rounds of viral translation, a narrow window of time during infection that requires ubiquitination but not proteasome activity was identified. In this regard, ubiquitin E1-activating enzyme inhibition was sufficient to stabilize the incoming viral genome in the cytoplasm of infected cells, causing its retention in either endosomes or nucleocapsids. Our data support a model in which dengue virus genome uncoating requires a nondegradative ubiquitination step, providing new insights into this crucial but understudied viral process. PMID:27353759

Lysine Ubiquitination and Acetylation of Human Cardiac 20S Proteasomes

PubMed Central

Lau, Edward; Choi, Howard JH; Ng, Dominic CM; Meyer, David; Fang, Caiyun; Li, Haomin; Wang, Ding; Zelaya, Ivette M; Yates, John R; Lam, Maggie PY

2016-01-01

Purpose Altered proteasome functions are associated with multiple cardiomyopathies. While the proteasome targets poly-ubiquitinated proteins for destruction, it itself is modifiable by ubiquitination. We aim to identify the exact ubiquitination sites on cardiac proteasomes and examine whether they are also subject to acetylations. Experimental design Assembled cardiac 20S proteasome complexes were purified from five human hearts with ischemic cardiomyopathy, then analyzed by high-resolution MS to identify ubiquitination and acetylation sites. We developed a library search strategy that may be used to complement database search in identifying PTM in different samples. Results We identified 63 ubiquitinated lysines from intact human cardiac 20S proteasomes. In parallel, 65 acetylated residues were also discovered, 39 of which shared with ubiquitination sites. Conclusion and clinical relevance This is the most comprehensive characterization of cardiac proteasome ubiquitination to-date. There are significant overlaps between the discovered ubiquitination and acetylation sites, permitting potential crosstalk in regulating proteasome functions. The information presented here will aid future therapeutic strategies aimed at regulating the functions of cardiac proteasomes. PMID:24957502

Plant Virus Infection and the Ubiquitin Proteasome Machinery: Arms Race along the Endoplasmic Reticulum.

PubMed

Verchot, Jeanmarie

2016-11-19

The endoplasmic reticulum (ER) is central to plant virus replication, translation, maturation, and egress. Ubiquitin modification of ER associated cellular and viral proteins, alongside the actions of the 26S proteasome, are vital for the regulation of infection. Viruses can arrogate ER associated ubiquitination as well as cytosolic ubiquitin ligases with the purpose of directing the ubiquitin proteasome system (UPS) to new targets. Such targets include necessary modification of viral proteins which may stabilize certain complexes, or modification of Argonaute to suppress gene silencing. The UPS machinery also contributes to the regulation of effector triggered immunity pattern recognition receptor immunity. Combining the results of unrelated studies, many positive strand RNA plant viruses appear to interact with cytosolic Ub-ligases to provide novel avenues for controlling the deleterious consequences of disease. Viral interactions with the UPS serve to regulate virus infection in a manner that promotes replication and movement, but also modulates the levels of RNA accumulation to ensure successful biotrophic interactions. In other instances, the UPS plays a central role in cellular immunity. These opposing roles are made evident by contrasting studies where knockout mutations in the UPS can either hamper viruses or lead to more aggressive diseases. Understanding how viruses manipulate ER associated post-translational machineries to better manage virus-host interactions will provide new targets for crop improvement.

Tuning BRCA1 and BARD1 activity to investigate RING ubiquitin ligase mechanisms.

PubMed

Stewart, Mikaela D; Duncan, Emily D; Coronado, Ernesto; DaRosa, Paul A; Pruneda, Jonathan N; Brzovic, Peter S; Klevit, Rachel E

2017-03-01

The tumor-suppressor protein BRCA1 works with BARD1 to catalyze the transfer of ubiquitin onto protein substrates. The N-terminal regions of BRCA1 and BARD1 that contain their RING domains are responsible for dimerization and ubiquitin ligase activity. This activity is a common feature among hundreds of human RING domain-containing proteins. RING domains bind and activate E2 ubiquitin-conjugating enzymes to promote ubiquitin transfer to substrates. We show that the identity of residues at specific positions in the RING domain can tune activity levels up or down. We report substitutions that create a structurally intact BRCA1/BARD1 heterodimer that is inactive in vitro with all E2 enzymes. Other substitutions in BRCA1 or BARD1 RING domains result in hyperactivity, revealing that both proteins have evolved attenuated activity. Loss of attenuation results in decreased product specificity, providing a rationale for why nature has tuned BRCA1 activity. The ability to tune BRCA1 provides powerful tools for understanding its biological functions and provides a basis to assess mechanisms for rescuing the activity of cancer-associated variations. Beyond the applicability to BRCA1, we show the identity of residues at tuning positions that can be used to predict and modulate the activity of an unrelated RING E3 ligase. These findings provide valuable insights into understanding the mechanism and function of RING E3 ligases like BRCA1. © 2017 The Protein Society.

Crystal Structure of a Ube2S-Ubiquitin Conjugate

PubMed Central

Lorenz, Sonja; Bhattacharyya, Moitrayee; Feiler, Christian; Rape, Michael; Kuriyan, John

2016-01-01

Protein ubiquitination occurs through the sequential formation and reorganization of specific protein-protein interfaces. Ubiquitin-conjugating (E2) enzymes, such as Ube2S, catalyze the formation of an isopeptide linkage between the C-terminus of a “donor” ubiquitin and a primary amino group of an “acceptor” ubiquitin molecule. This reaction involves an intermediate, in which the C-terminus of the donor ubiquitin is thioester-bound to the active site cysteine of the E2 and a functionally important interface is formed between the two proteins. A docked model of a Ube2S-donor ubiquitin complex was generated previously, based on chemical shift mapping by NMR, and predicted contacts were validated in functional studies. We now present the crystal structure of a covalent Ube2S-ubiquitin complex. The structure contains an interface between Ube2S and ubiquitin in trans that resembles the earlier model in general terms, but differs in detail. The crystallographic interface is more hydrophobic than the earlier model and is stable in molecular dynamics (MD) simulations. Remarkably, the docked Ube2S-donor complex converges readily to the configuration seen in the crystal structure in 3 out of 8 MD trajectories. Since the crystallographic interface is fully consistent with mutational effects, this indicates that the structure provides an energetically favorable representation of the functionally critical Ube2S-donor interface. PMID:26828794

Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked Ubiquitin Signaling.

PubMed

Michel, Martin A; Swatek, Kirby N; Hospenthal, Manuela K; Komander, David

2017-10-05

Several ubiquitin chain types have remained unstudied, mainly because tools and techniques to detect these posttranslational modifications are scarce. Linkage-specific antibodies have shaped our understanding of the roles and dynamics of polyubiquitin signals but are available for only five out of eight linkage types. We here characterize K6- and K33-linkage-specific "affimer" reagents as high-affinity ubiquitin interactors. Crystal structures of affimers bound to their cognate chain types reveal mechanisms of specificity and a K11 cross-reactivity in the K33 affimer. Structure-guided improvements yield superior affinity reagents suitable for western blotting, confocal fluorescence microscopy and pull-down applications. This allowed us to identify RNF144A and RNF144B as E3 ligases that assemble K6-, K11-, and K48-linked polyubiquitin in vitro. A protocol to enrich K6-ubiquitinated proteins from cells identifies HUWE1 as a main E3 ligase for this chain type, and we show that mitofusin-2 is modified with K6-linked polyubiquitin in a HUWE1-dependent manner. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

HSF-1 activates the ubiquitin proteasome system to promote non-apoptotic developmental cell death in C. elegans.

PubMed

Kinet, Maxime J; Malin, Jennifer A; Abraham, Mary C; Blum, Elyse S; Silverman, Melanie R; Lu, Yun; Shaham, Shai

2016-03-08

Apoptosis is a prominent metazoan cell death form. Yet, mutations in apoptosis regulators cause only minor defects in vertebrate development, suggesting that another developmental cell death mechanism exists. While some non-apoptotic programs have been molecularly characterized, none appear to control developmental cell culling. Linker-cell-type death (LCD) is a morphologically conserved non-apoptotic cell death process operating in Caenorhabditis elegans and vertebrate development, and is therefore a compelling candidate process complementing apoptosis. However, the details of LCD execution are not known. Here we delineate a molecular-genetic pathway governing LCD in C. elegans. Redundant activities of antagonistic Wnt signals, a temporal control pathway, and mitogen-activated protein kinase kinase signaling control heat shock factor 1 (HSF-1), a conserved stress-activated transcription factor. Rather than protecting cells, HSF-1 promotes their demise by activating components of the ubiquitin proteasome system, including the E2 ligase LET-70/UBE2D2 functioning with E3 components CUL-3, RBX-1, BTBD-2, and SIAH-1. Our studies uncover design similarities between LCD and developmental apoptosis, and provide testable predictions for analyzing LCD in vertebrates.

The roles of ubiquitin modifying enzymes in neoplastic disease.

PubMed

Kumari, Nishi; Jaynes, Patrick William; Saei, Azad; Iyengar, Prasanna Vasudevan; Richard, John Lalith Charles; Eichhorn, Pieter Johan Adam

2017-12-01

The initial experiments performed by Rose, Hershko, and Ciechanover describing the identification of a specific degradation signal in short-lived proteins paved the way to the discovery of the ubiquitin mediated regulation of numerous physiological functions required for cellular homeostasis. Since their discovery of ubiquitin and ubiquitin function over 30years ago it has become wholly apparent that ubiquitin and their respective ubiquitin modifying enzymes are key players in tumorigenesis. The human genome encodes approximately 600 putative E3 ligases and 80 deubiquitinating enzymes and in the majority of cases these enzymes exhibit specificity in sustaining either pro-tumorigenic or tumour repressive responses. In this review, we highlight the known oncogenic and tumour suppressive effects of ubiquitin modifying enzymes in cancer relevant pathways with specific focus on PI3K, MAPK, TGFβ, WNT, and YAP pathways. Moreover, we discuss the capacity of targeting DUBs as a novel anticancer therapeutic strategy. Copyright © 2017 Elsevier B.V. All rights reserved.

Mesenchymal stem cell-based HSP70 promoter-driven VEGFA induction by resveratrol promotes angiogenesis in a mouse model.

PubMed

Chen, Young-Bin; Lan, Ying-Wei; Hung, Tsai-Hsien; Chen, Lih-Geeng; Choo, Kong-Bung; Cheng, Winston T K; Lee, Hsuan-Shu; Chong, Kowit-Yu

2015-07-01

Several studies of stem cell-based gene therapy have indicated that long-lasting regeneration following vessel ischemia may be stimulated through VEGFA gene therapy and/or MSC transplantation for reduction of ischemic injury in limb ischemia and heart failure. The therapeutic potential of MSC transplantation can be further improved by genetically modifying MSCs with genes which enhance angiogenesis following ischemic injury. In the present study, we aimed to develop an approach in MSC-based therapy for repair and mitigation of ischemic injury and regeneration of damaged tissues in ischemic disease. HSP70 promoter-driven VEGFA expression was induced by resveratrol (RSV) in MSCs, and in combination with known RSV biological functions, the protective effects of our approach were investigated by using ex vivo aortic ring coculture system and a 3D scaffolds in vivo model. Results of this investigation demonstrated that HSP promoter-driven VEGFA expression in MSC increased approximately 2-fold over the background VEGFA levels upon HSP70 promoter induction by RSV. Exposure of HUVEC cells to medium containing MSC in which VEGFA had been induced by cis-RSV enhanced tube formation in the treated HUVEC cells. RSV-treated MSC cells differentiated into endothelial-like phenotypes, exhibiting markedly elevated expression of endothelial cell markers. These MSCs also induced aortic ring sprouting, characteristic of neovascular formation from pre-existing vessels, and additionally promoted neovascularization at the MSC transplantation site in a mouse model. These observations support a hypothesis that VEGFA expression induced by cis-RSV acting on the HSP70 promoter in transplanted MSC augments the angiogenic effects of stem cell gene therapy. The use of an inducible system also vastly reduces possible clinical risks associated with constitutive VEGFA expression.

A Subset of Ubiquitin-Conjugating Enzymes Is Essential for Plant Immunity1[OPEN

PubMed Central

Connor, Richard A.

2017-01-01

Of the three classes of enzymes involved in ubiquitination, ubiquitin-conjugating enzymes (E2) have been often incorrectly considered to play merely an auxiliary role in the process, and few E2 enzymes have been investigated in plants. To reveal the role of E2 in plant innate immunity, we identified and cloned 40 tomato genes encoding ubiquitin E2 proteins. Thioester assays indicated that the majority of the genes encode enzymatically active E2. Phylogenetic analysis classified the 40 tomato E2 enzymes into 13 groups, of which members of group III were found to interact and act specifically with AvrPtoB, a Pseudomonas syringae pv tomato effector that uses its ubiquitin ligase (E3) activity to suppress host immunity. Knocking down the expression of group III E2 genes in Nicotiana benthamiana diminished the AvrPtoB-promoted degradation of the Fen kinase and the AvrPtoB suppression of host immunity-associated programmed cell death. Importantly, silencing group III E2 genes also resulted in reduced pattern-triggered immunity (PTI). By contrast, programmed cell death induced by several effector-triggered immunity elicitors was not affected on group III-silenced plants. Functional characterization suggested redundancy among group III members for their role in the suppression of plant immunity by AvrPtoB and in PTI and identified UBIQUITIN-CONJUGATING11 (UBC11), UBC28, UBC29, UBC39, and UBC40 as playing a more significant role in PTI than other group III members. Our work builds a foundation for the further characterization of E2s in plant immunity and reveals that AvrPtoB has evolved a strategy for suppressing host immunity that is difficult for the plant to thwart. PMID:27909045

Ubiquitin Utilizes an Acidic Surface Patch to Alter Chromatin Structure

PubMed Central

Debelouchina, Galia T.; Gerecht, Karola; Muir, Tom W.

2016-01-01

Ubiquitylation of histone H2B, associated with gene activation, leads to chromatin decompaction through an unknown mechanism. We used a hydrogen-deuterium exchange strategy coupled with nuclear magnetic resonance spectroscopy to map the ubiquitin surface responsible for its structural effects on chromatin. Our studies revealed that a previously uncharacterized acidic patch on ubiquitin comprising residues Glu16 and Glu18 is essential for decompaction. These residues mediate promiscuous electrostatic interactions with the basic histone proteins, potentially positioning the ubiquitin moiety as a dynamic “wedge” that prevents the intimate association of neighboring nucleosomes. Using two independent cross-linking strategies and an oligomerization assay, we also showed that ubiquitin-ubiquitin contacts occur in the chromatin environment and are important for the solubilization of the chromatin polymers. Our work highlights a novel, chromatin-related aspect of the “ubiquitin code”, and sheds light on how the information rich ubiquitin modification can orchestrate different biochemical outcomes using different surface features. PMID:27870837

N-myc downstream-regulated gene 1 promotes oxaliplatin-triggered apoptosis in colorectal cancer cells via enhancing the ubiquitination of Bcl-2.

PubMed

Yang, Xiao; Zhu, Fan; Yu, Chaoran; Lu, Jiaoyang; Zhang, Luyang; Lv, Yanfeng; Sun, Jing; Zheng, Minhua

2017-07-18

N-myc downstream-regulated gene1 (NDRG1) has been identified as a potent tumor suppressor gene. The molecular mechanisms of anti-tumor activity of NDRG1 involve its suppressive effects on a variety of tumorigenic signaling pathways. The purpose of this study was to investigate the role of NDRG1 in the apoptosis of colorectal cancer (CRC) cells. We first collected the clinical data of locally advanced rectal cancer (LARC) patients receiving oxaliplatin-based neoadjuvant chemotherapy in our medical center. Correlation analysis revealed that NDRG1 positively associated with the downstaging rates and prognosis of patients. Then, the effects of over-expression and depletion of NDRG1 gene on apoptosis of colorectal cancer were tested in vitro and in vivo. NDRG1 over-expression promoted apoptosis in colorectal cancer cells whereas depletion of NDRG1 resulted in resistance to oxaliplatin treatment. Furthermore, we observed that Bcl-2, a major anti-apoptotic protein, was regulated by NDRG1 at post-transcriptional level. By binding Protein kinase Cα (PKCα), a classical regulating factor of Bcl-2, NDRG1 enhanced the ubiquitination and degradation of Bcl-2, thus promoting apoptosis in CRC cells. In addition, NDRG1 inhibited tumor growth and promoted apoptosis in mouse xenograft model. In conclusion,NDRG1 promotes oxaliplatin-triggered apoptosis in colorectal cancer. Therefore, colorectal cancer patients can be stratified by the expression level of NDRG1. NDRG1-positive patients may benefit from oxaliplatin-containing chemotherapy regimens whereas those with negative NDRG1 expression should avoid the usage of this cytotoxic drug.

Cloning of ubiquitin-activating enzyme and ubiquitin-conjugating enzyme genes from Gracilaria lemaneiformis and their activity under heat shock.

PubMed

Li, Guang-Qi; Zang, Xiao-Nan; Zhang, Xue-Cheng; Lu, Ning; Ding, Yan; Gong, Le; Chen, Wen-Chao

2014-03-15

To study the response of Gracilaria lemaneiformis to heat stress, two key enzymes - ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2) - of the Ubiquitin/26S proteasome pathway (UPP) were studied in three strains of G. lemaneiformis-wild type, heat-tolerant cultivar 981 and heat-tolerant cultivar 07-2. The full length DNA sequence of E1 contained only one exon. The open reading frame (ORF) sequence was 981 nucleotides encoding 326 amino acids, which contained conserved ATP binding sites (LYDRQIRLWGLE, ELAKNVLLAGV, LKEMN, VVCAI) and the ubiquitin-activating domains (VVCAI…LMTEAC, VFLDLGDEYSYQ, AIVGGMWGRE). The gene sequence of E2 contained four exons and three introns. The sum of the four exons gave an open reading frame sequence of 444 nucleotides encoding 147 amino acids, which contained a conserved ubiquitin-activating domain (GSICLDIL), ubiquitin-conjugating domains (RIYHPNIN, KVLLSICSLL, DDPLV) and ubiquitin-ligase (E3) recognition sites (KRI, YPF, WSP). Real-time-PCR analysis of transcription levels of E1 and E2 under heat shock conditions (28°C and 32°C) showed that in wild type, transcriptions of E1 and E2 were up-regulated at 28°C, while at 32°C, transcriptions of the two enzymes were below the normal level. In cultivar 981 and cultivar 07-2 of G. lemaneiformis, the transcription levels of the two enzymes were up-regulated at 32°C, and transcription level of cultivar 07-2 was even higher than that of cultivar 981. These results suggest that the UPP plays an important role in high temperature resistance of G. lemaneiformis and the bioactivity of UPP is directly related to the heat-resistant ability of G. lemaneiformis. Copyright © 2013 Elsevier B.V. All rights reserved.

A New Scheme to Characterize and Identify Protein Ubiquitination Sites.

PubMed

Nguyen, Van-Nui; Huang, Kai-Yao; Huang, Chien-Hsun; Lai, K Robert; Lee, Tzong-Yi

2017-01-01

Protein ubiquitination, involving the conjugation of ubiquitin on lysine residue, serves as an important modulator of many cellular functions in eukaryotes. Recent advancements in proteomic technology have stimulated increasing interest in identifying ubiquitination sites. However, most computational tools for predicting ubiquitination sites are focused on small-scale data. With an increasing number of experimentally verified ubiquitination sites, we were motivated to design a predictive model for identifying lysine ubiquitination sites for large-scale proteome dataset. This work assessed not only single features, such as amino acid composition (AAC), amino acid pair composition (AAPC) and evolutionary information, but also the effectiveness of incorporating two or more features into a hybrid approach to model construction. The support vector machine (SVM) was applied to generate the prediction models for ubiquitination site identification. Evaluation by five-fold cross-validation showed that the SVM models learned from the combination of hybrid features delivered a better prediction performance. Additionally, a motif discovery tool, MDDLogo, was adopted to characterize the potential substrate motifs of ubiquitination sites. The SVM models integrating the MDDLogo-identified substrate motifs could yield an average accuracy of 68.70 percent. Furthermore, the independent testing result showed that the MDDLogo-clustered SVM models could provide a promising accuracy (78.50 percent) and perform better than other prediction tools. Two cases have demonstrated the effective prediction of ubiquitination sites with corresponding substrate motifs.

Hydrogen peroxide stimulates ubiquitin-conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes

NASA Technical Reports Server (NTRS)

Li, Yi-Ping; Chen, Yuling; Li, Andrew S.; Reid, Michael B.

2003-01-01

Reactive oxygen species (ROS) are thought to promote muscle atrophy in chronic wasting diseases, but the underlying mechanism has not been determined. Here we show that H2O2 stimulates ubiquitin conjugation to muscle proteins through transcriptional regulation of the enzymes (E2 and E3 proteins) that conjugate ubiquitin to muscle proteins. Incubation of C2C12 myotubes with 100 microM H2O2 increased the rate of 125I-labeled ubiquitin conjugation to muscle proteins in whole cell extracts. This response required at least 4-h exposure to H2O2 and persisted for at least 24 h. Preincubating myotubes with cycloheximide or actinomycin D blocked H2O2 stimulation of ubiquitin-conjugating activity, suggesting that gene transcription is required. Northern blot analyses revealed that H2O2 upregulates expression of specific E3 and E2 proteins that are thought to regulate muscle catabolism, including atrogin1/MAFbx, MuRF1, and E214k. These results suggest that ROS stimulate protein catabolism in skeletal muscle by upregulating the ubiquitin conjugation system.

Ubiquitin and Proteasomes in Transcription

PubMed Central

Geng, Fuqiang; Wenzel, Sabine; Tansey, William P.

2013-01-01

Regulation of gene transcription is vitally important for the maintenance of normal cellular homeostasis. Failure to correctly regulate gene expression, or to deal with problems that arise during the transcription process, can lead to cellular catastrophe and disease. One of the ways cells cope with the challenges of transcription is by making extensive use of the proteolytic and nonproteolytic activities of the ubiquitin-proteasome system (UPS). Here, we review recent evidence showing deep mechanistic connections between the transcription and ubiquitin-proteasome systems. Our goal is to leave the reader with a sense that just about every step in transcription—from transcription initiation through to export of mRNA from the nucleus—is influenced by the UPS and that all major arms of the system—from the first step in ubiquitin (Ub) conjugation through to the proteasome—are recruited into transcriptional processes to provide regulation, directionality, and deconstructive power. PMID:22404630

Ubiquitin-mediated modulation of the cytoplasmic viral RNA sensor RIG-I.

PubMed

Oshiumi, Hiroyuki; Matsumoto, Misako; Seya, Tsukasa

2012-01-01

RIG-I-like receptors, including RIG-I, MDA5 and LGP2, recognize cytoplasmic viral RNA. The RIG-I protein consists of N-terminal CARDs, central RNA helicase and C-terminal domains. RIG-I activation is regulated by ubiquitination. Three ubiquitin ligases target the RIG-I protein. TRIM25 and Riplet ubiquitin ligases are positive regulators of RIG-I and deliver the K63-linked polyubiquitin moiety to RIG-I CARDs and the C-terminal domain. RNF125, another ubiquitin ligase, is a negative regulator of RIG-I and mediates K48-linked polyubiquitination of RIG-I, leading to the degradation of the RIG-I protein by proteasomes. The K63-linked polyubiquitin chains of RIG-I are removed by a deubiquitin enzyme, CYLD. Thus, CYLD is a negative regulator of RIG-I. Furthermore, TRIM25 itself is regulated by ubiquitination. HOIP and HOIL proteins are ubiquitin ligases and are also known as linear ubiquitin assembly complexes (LUBACs). The TRIM25 protein is ubiquitinated by LUBAC and then degraded by proteasomes. The splice variant of RIG-I encodes a protein that lacks the first CARD of RIG-I, and the variant RIG-I protein is not ubiquitinated by TRIM25. Therefore, ubiquitin is the key regulator of the cytoplasmic viral RNA sensor RIG-I.

The elusive structural role of ubiquitinated histones.

PubMed

Moore, Susan C; Jason, Laure; Ausió, Juan

2002-01-01

It is increasingly apparent that histone posttranslational modifications are important in chromatin structure and dynamics. However, histone ubiquitination has received little attention. Histones H1, H3, H2A, and H2B can be ubiquitinated in vivo, but the most prevalent are uH2A and uH2B. The size of this modification suggests some sort of structural impact. Physiological observations suggest that ubiquitinated histones may have multiple functions and structural effects. Ubiquitinated histones have been correlated with transcriptionally active DNA, implying that it may prevent chromatin folding or help maintain an open conformation. Also, in some organisms during spermiogenesis, a process involving extensive chromatin remodeling, uH2A levels increase just prior to histone replacement by protamines. Determination of chromatin's structural changes resulting from histone ubiquitination is therefore important. Recent work using reconstituted nucleosomes and chromatin fibers containing uH2A indicate that in the absence of linker histones, ubiquitination has little structural impact. DNase I digests and analytical ultracentrifugation of reconstituted ubiquitinated nucleosomes show no structural differences. Solubility assays using reconstituted chromatin fibers in the presence of divalent ions demonstrate that uH2A fibers are slightly more prone to aggregation than controls, and analytical ultracentrifugation results with different MgCl2 and NaCl concentrations determined that chromatin folding is not affected by this modification. Additional work to assess possible synergistic affects with histone acetylation also precludes any structural implications. Protamine displacement experiments concluded that the presence of uH2A does not significantly affect the ability of the protamines to displace histones. In addition, uH2A does not interfere with histone H1 binding to the nucleosome. While work with uH2B remains insufficient to come to any definitive conclusions about its

Combined Infrared Multiphoton Dissociation with Ultraviolet Photodissociation for Ubiquitin Characterization

NASA Astrophysics Data System (ADS)

Halim, Mohammad A.; Girod, Marion; MacAleese, Luke; Lemoine, Jérôme; Antoine, Rodolphe; Dugourd, Philippe

2016-09-01

Herein we report the successful implementation of the consecutive and simultaneous photodissociation with high (213 nm) and low (10.6 μm) energy photons (HiLoPD, high-low photodissociation) on ubiquitin in a quadrupole-Orbitrap mass spectrometer. Absorption of high-energy UV photon is dispersed over the whole protein and stimulates extensive C-Cα backbone fragmentation, whereas low-energy IR photon gradually increases the internal energy and thus preferentially dissociates the most labile amide (C-N) bonds. We noticed that simultaneous irradiation of UV and IR lasers on intact ubiquitin in a single MS/MS experiment provides a rich and well-balanced fragmentation array of a/x, b/y, and z ions. Moreover, secondary fragmentation from a/x and z ions leads to the formation of satellite side-chain ions (d, v, and w) and can help to distinguish isomeric residues in a protein. Implementation of high-low photodissociation in a high-resolution mass spectrometer may offer considerable benefits to promote a comprehensive portrait of protein characterization.

Fbw7 promotes ubiquitin-dependent degradation of c-Myb: involvement of GSK3-mediated phosphorylation of Thr-572 in mouse c-Myb.

PubMed

Kitagawa, K; Hiramatsu, Y; Uchida, C; Isobe, T; Hattori, T; Oda, T; Shibata, K; Nakamura, S; Kikuchi, A; Kitagawa, M

2009-06-25

Expression of oncoprotein c-Myb oscillates during hematopoiesis and hematological malignancies. Its quantity is not only regulated through transcriptional control but also through the ubiquitin-proteasome pathway, accompanied by phosphorylation, although the mechanisms are poorly understood. In this report, we tried to identify an E3 ubiquitin ligase, which targets c-Myb for ubiquitin-dependent degradation. We found that an F-box protein, Fbw7, interacted with c-Myb, which is mutated in numerous cancers. Fbw7 facilitated ubiquitylation and degradation of c-Myb in intact cells. Moreover, depletion of Fbw7 by RNA interference delayed turnover and increased the abundance of c-Myb in myeloid leukemia cells concomitantly, and suppressed the transcriptional level of gamma-globin, which receives transcriptional repression from c-Myb. In addition, we analysed sites required for both ubiquitylation and degradation of c-Myb. We found that Thr-572 is critical for Fbw7-mediated ubiquitylation in mouse c-Myb using site-directed mutagenesis. Fbw7 recognized the phosphorylation of Thr-572, which was mediated by glycogen synthase kinase 3 (GSK3). In consequence, the c-Myb protein was markedly stabilized by the substitution of Thr-572 to Ala. These observations suggest that SCF(Fbw7) ubiquitin ligase regulates phosphorylation-dependent degradation of c-Myb protein.

Lysine ubiquitination and acetylation of human cardiac 20S proteasomes.

PubMed

Zong, Nobel; Ping, Peipei; Lau, Edward; Choi, Howard Jh; Ng, Dominic Cm; Meyer, David; Fang, Caiyun; Li, Haomin; Wang, Ding; Zelaya, Ivette M; Yates, John R; Lam, Maggie Py

2014-08-01

Altered proteasome functions are associated with multiple cardiomyopathies. While the proteasome targets polyubiquitinated proteins for destruction, it itself is modifiable by ubiquitination. We aim to identify the exact ubiquitination sites on cardiac proteasomes and examine whether they are also subject to acetylations. Assembled cardiac 20S proteasome complexes were purified from five human hearts with ischemic cardiomyopathy, then analyzed by high-resolution MS to identify ubiquitination and acetylation sites. We developed a library search strategy that may be used to complement database search in identifying PTM in different samples. We identified 63 ubiquitinated lysines from intact human cardiac 20S proteasomes. In parallel, 65 acetylated residues were also discovered, 39 of which shared with ubiquitination sites. This is the most comprehensive characterization of cardiac proteasome ubiquitination to date. There are significant overlaps between the discovered ubiquitination and acetylation sites, permitting potential crosstalk in regulating proteasome functions. The information presented here will aid future therapeutic strategies aimed at regulating the functions of cardiac proteasomes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

TNF-alpha increases ubiquitin-conjugating activity in skeletal muscle by up-regulating UbcH2/E220k

NASA Technical Reports Server (NTRS)

Li, Yi-Ping; Lecker, Stewart H.; Chen, Yuling; Waddell, Ian D.; Goldberg, Alfred L.; Reid, Michael B.

2003-01-01

In some inflammatory diseases, TNF-alpha is thought to stimulate muscle catabolism via an NF-kappaB-dependent process that increases ubiquitin conjugation to muscle proteins. The transcriptional mechanism of this response has not been determined. Here we studied the potential role of UbcH2, a ubiquitin carrier protein and homologue of murine E220k. We find that UbcH2 is constitutively expressed by human skeletal and cardiac muscles, murine limb muscle, and cultured myotubes. TNF-alpha stimulates UbcH2 expression in mouse limb muscles in vivo and in cultured myotubes. The UbcH2 promoter region contains a functional NF-kappaB binding site; NF-kappaB binding to this sequence is increased by TNF-alpha stimulation. A dominant negative inhibitor of NF-kappaB activation blocks both UbcH2 up-regulation and the increase in ubiquitin-conjugating activity stimulated by TNF-alpha. In extracts from TNF-alpha-treated myotubes, ubiquitin-conjugating activity is limited by UbcH2 availability; activity is inhibited by an antiserum to UbcH2 or a dominant negative mutant of UbcH2 and is enhanced by wild-type UbcH2. Thus, UbcH2 up-regulation is a novel response to TNF-alpha/NF-kappaB signaling in skeletal muscle that appears to be essential for the increased ubiquitin conjugation induced by this cytokine.

Chlamydia trachomatis Is Resistant to Inclusion Ubiquitination and Associated Host Defense in Gamma Interferon-Primed Human Epithelial Cells.

PubMed

Haldar, Arun K; Piro, Anthony S; Finethy, Ryan; Espenschied, Scott T; Brown, Hannah E; Giebel, Amanda M; Frickel, Eva-Maria; Nelson, David E; Coers, Jörn

2016-12-13

-Chlamydia host resistance pathway active in human epithelial cells. This defense program promotes the deposition of the small antimicrobial protein ubiquitin on vacuoles containing Chlamydia We show that this ubiquitin-based resistance pathway of human cells is highly effective against a Chlamydia species adapted to rodents but ineffective against human-adapted C. trachomatis This observation indicates that C. trachomatis evolved strategies to avoid entrapment within ubiquitin-labeled vacuoles as part of its adaptation to the human innate immune system. Copyright © 2016 Haldar et al.

K48-linked KLF4 ubiquitination by E3 ligase Mule controls T-cell proliferation and cell cycle progression.

PubMed

Hao, Zhenyue; Sheng, Yi; Duncan, Gordon S; Li, Wanda Y; Dominguez, Carmen; Sylvester, Jennifer; Su, Yu-Wen; Lin, Gloria H Y; Snow, Bryan E; Brenner, Dirk; You-Ten, Annick; Haight, Jillian; Inoue, Satoshi; Wakeham, Andrew; Elford, Alisha; Hamilton, Sara; Liang, Yi; Zúñiga-Pflücker, Juan C; He, Housheng Hansen; Ohashi, Pamela S; Mak, Tak W

2017-01-13

T-cell proliferation is regulated by ubiquitination but the underlying molecular mechanism remains obscure. Here we report that Lys-48-linked ubiquitination of the transcription factor KLF4 mediated by the E3 ligase Mule promotes T-cell entry into S phase. Mule is elevated in T cells upon TCR engagement, and Mule deficiency in T cells blocks proliferation because KLF4 accumulates and drives upregulation of its transcriptional targets E2F2 and the cyclin-dependent kinase inhibitors p21 and p27. T-cell-specific Mule knockout (TMKO) mice develop exacerbated experimental autoimmune encephalomyelitis (EAE), show impaired generation of antigen-specific CD8 + T cells with reduced cytokine production, and fail to clear LCMV infections. Thus, Mule-mediated ubiquitination of the novel substrate KLF4 regulates T-cell proliferation, autoimmunity and antiviral immune responses in vivo.

Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide

PubMed Central

Fischer, Eric S.; Böhm, Kerstin; Lydeard, John R.; Yang, Haidi; Stadler, Michael B.; Cavadini, Simone; Nagel, Jane; Serluca, Fabrizio; Acker, Vincent; Lingaraju, Gondichatnahalli M.; Tichkule, Ritesh B.; Schebesta, Michael; Forrester, William C.; Schirle, Markus; Hassiepen, Ulrich; Ottl, Johannes; Hild, Marc; Beckwith, Rohan E. J.; Harper, J. Wade; Jenkins, Jeremy L.; Thomä, Nicolas H.

2015-01-01

In the 1950s the drug thalidomide administered as a sedative to pregnant women led to the birth of thousands of children with multiple defects. Despite its teratogenicity, thalidomide and its derivatives lenalidomide and pomalidomide (together known as Immunomodulatory Drugs: IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-dysplasia. IMiDs target the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase and promote the ubiquitination of Ikaros/Aiolos transcription factors by CRL4CRBN. Here we present the crystal structure of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes CRBN as a CRL4CRBN substrate receptor, which enantioselectively binds IMiDs. Through an unbiased screen we identify the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN. Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN when recruiting Ikaros/Aiolos for degradation. This dual activity implies that small molecules can principally modulate a ligase to up- or down-regulate the ubiquitination of proteins. PMID:25043012

Cdk5 regulates PSD-95 ubiquitination in neurons

PubMed Central

Bianchetta, Michael J.; Lam, TuKiet T.; Jones, Stephen N.; Morabito, Maria A.

2011-01-01

The kinase Cdk5 and its activator p35 have been implicated in drug addiction, neurodegenerative diseases such as Alzheimer’s, learning and memory, and synapse maturation and plasticity. However the molecular mechanisms by which Cdk5 regulates synaptic plasticity are still unclear. PSD-95 is a major postsynaptic scaffolding protein of glutamatergic synapses that regulates synaptic strength and plasticity. PSD-95 is ubiquitinated by the Ubiquitin E3 Ligase Mdm2, and rapid and transient PSD-95 ubiquitination has been implicated in NMDA receptor-induced AMPA receptor endocytosis. Here we demonstrate that genetic or pharmacological reduction of Cdk5 activity increases the interaction of Mdm2 with PSD-95 and enhances PSD-95 ubiquitination without affecting PSD-95 protein levels in vivo in mice, suggesting a non-proteolytic function of ubiquitinated PSD-95 at synapses. We show that PSD-95 ubiquitination correlates with increased interaction with β-adaptin, a subunit of the clathrin adaptor protein complex AP-2. This interaction is increased by genetic reduction of Cdk5 activity or NMDA receptor stimulation and is dependent on Mdm2. Together these results support a function for Cdk5 in regulating PSD-95 ubiqutination and its interaction with AP-2 and suggest a mechanism by which PSD-95 may regulate NMDA receptor-induced AMPA receptor endocytosis. PMID:21849563

High Performance Liquid Chromatography Resolution of Ubiquitin Pathway Enzymes from Wheat Germ 1

PubMed Central

Sullivan, Michael L.; Callis, Judy; Vierstra, Richard D.

1990-01-01

The highly conserved protein ubiquitin is involved in several cellular processes in eukaryotes as a result of its covalent ligation to a variety of target proteins. Here, we describe the purification of several enzymatic activities involved in ubiquitin-protein conjugate formation and disassembly from wheat germ (Triticum vulgare) by a combination of ubiquitin affinity chromatography and anion-exchange high performance liquid chromatography. Using this procedure, ubiquitin activating enzyme (E1), several distinct ubiquitin carrier proteins (E2s) with molecular masses of 16, 20, 23, 23.5, and 25 kilodaltons, and a ubiquitin-protein hydrolase (isopeptidase) were isolated. Purified E1 formed a thiol ester linkage with 125I-ubiquitin in an ATP-dependent manner and transferred bound ubiquitin to the various purified E2s. The ubiquitin protein hydrolase fraction was sensitive to hemin, and in an ATP-independent reaction, was capable of removing the ubiquitin moiety from both ubiquitin 125I-lysozyme conjugates (ε-amino or isopeptide linkage) and the ubiquitin 52-amino acid extension protein fusion (α-amino or peptide linkage). Using this procedure, wheat germ represents an inexpensive source from which enzymes involved in the ubiquitin pathway may be isolated. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:16667769

Distinct promoter activation mechanisms modulate noise-driven HIV gene expression

NASA Astrophysics Data System (ADS)

Chavali, Arvind K.; Wong, Victor C.; Miller-Jensen, Kathryn

2015-12-01

Latent human immunodeficiency virus (HIV) infections occur when the virus occupies a transcriptionally silent but reversible state, presenting a major obstacle to cure. There is experimental evidence that random fluctuations in gene expression, when coupled to the strong positive feedback encoded by the HIV genetic circuit, act as a ‘molecular switch’ controlling cell fate, i.e., viral replication versus latency. Here, we implemented a stochastic computational modeling approach to explore how different promoter activation mechanisms in the presence of positive feedback would affect noise-driven activation from latency. We modeled the HIV promoter as existing in one, two, or three states that are representative of increasingly complex mechanisms of promoter repression underlying latency. We demonstrate that two-state and three-state models are associated with greater variability in noisy activation behaviors, and we find that Fano factor (defined as variance over mean) proves to be a useful noise metric to compare variability across model structures and parameter values. Finally, we show how three-state promoter models can be used to qualitatively describe complex reactivation phenotypes in response to therapeutic perturbations that we observe experimentally. Ultimately, our analysis suggests that multi-state models more accurately reflect observed heterogeneous reactivation and may be better suited to evaluate how noise affects viral clearance.

Ubiquitination in the antiviral immune response.

PubMed

Davis, Meredith E; Gack, Michaela U

2015-05-01

Ubiquitination has long been known to regulate fundamental cellular processes through the induction of proteasomal degradation of target proteins. More recently, 'atypical' non-degradative types of polyubiquitin chains have been appreciated as important regulatory moieties by modulating the activity or subcellular localization of key signaling proteins. Intriguingly, many of these non-degradative types of ubiquitination regulate the innate sensing pathways initiated by pattern recognition receptors (PRRs), ultimately coordinating an effective antiviral immune response. Here we discuss recent advances in understanding the functional roles of degradative and atypical types of ubiquitination in innate immunity to viral infections, with a specific focus on the signaling pathways triggered by RIG-I-like receptors, Toll-like receptors, and the intracellular viral DNA sensor cGAS. Copyright © 2015 Elsevier Inc. All rights reserved.

Ubiquitination as an efficient molecular strategy employed in salmonella infection

USDA-ARS?s Scientific Manuscript database

The ubiquitin modification has various functions in the host innate immune system in response to the bacterial infection. To counteract the host immunity, Salmonella can specifically target ubiquitin pathways by its effector proteins. In this review, we describe the multiple facets of ubiquitin func...

Quantitative proteomics and terminomics to elucidate the role of ubiquitination and proteolysis in adaptive immunity.

PubMed

Klein, Theo; Viner, Rosa I; Overall, Christopher M

2016-10-28

Adaptive immunity is the specialized defence mechanism in vertebrates that evolved to eliminate pathogens. Specialized lymphocytes recognize specific protein epitopes through antigen receptors to mount potent immune responses, many of which are initiated by nuclear factor-kappa B activation and gene transcription. Most, if not all, pathways in adaptive immunity are further regulated by post-translational modification (PTM) of signalling proteins, e.g. phosphorylation, citrullination, ubiquitination and proteolytic processing. The importance of PTMs is reflected by genetic or acquired defects in these pathways that lead to a dysfunctional immune response. Here we discuss the state of the art in targeted proteomics and systems biology approaches to dissect the PTM landscape specifically regarding ubiquitination and proteolysis in B- and T-cell activation. Recent advances have occurred in methods for specific enrichment and targeted quantitation. Together with improved instrument sensitivity, these advances enable the accurate analysis of often rare PTM events that are opaque to conventional proteomics approaches, now rendering in-depth analysis and pathway dissection possible. We discuss published approaches, including as a case study the profiling of the N-terminome of lymphocytes of a rare patient with a genetic defect in the paracaspase protease MALT1, a key regulator protease in antigen-driven signalling, which was manifested by elevated linear ubiquitination.This article is part of the themed issue 'Quantitative mass spectrometry'. © 2016 The Authors.

PAK4 promotes kinase-independent stabilization of RhoU to modulate cell adhesion

PubMed Central

Dart, Anna E.; Box, Gary M.; Court, William; Gale, Madeline E.; Brown, John P.; Pinder, Sarah E.; Eccles, Suzanne A.

2015-01-01

P21-activated kinase 4 (PAK4) is a Cdc42 effector protein thought to regulate cell adhesion disassembly in a kinase-dependent manner. We found that PAK4 expression is significantly higher in high-grade human breast cancer patient samples, whereas depletion of PAK4 modifies cell adhesion dynamics of breast cancer cells. Surprisingly, systematic analysis of PAK4 functionality revealed that PAK4-driven adhesion turnover is neither dependent on Cdc42 binding nor kinase activity. Rather, reduced expression of PAK4 leads to a concomitant loss of RhoU expression. We report that RhoU is targeted for ubiquitination by the Rab40A–Cullin 5 complex and demonstrate that PAK4 protects RhoU from ubiquitination in a kinase-independent manner. Overexpression of RhoU rescues the PAK4 depletion phenotype, whereas loss of RhoU expression reduces cell adhesion turnover and migration. These data support a new kinase-independent mechanism for PAK4 function, where an important role of PAK4 in cellular adhesions is to stabilize RhoU protein levels. Thus, PAK4 and RhoU cooperate to drive adhesion turnover and promote cell migration. PMID:26598620

The Ubiquitin-Proteasome Pathway and Synaptic Plasticity

ERIC Educational Resources Information Center

Hegde, Ashok N.

2010-01-01

Proteolysis by the ubiquitin-proteasome pathway (UPP) has emerged as a new molecular mechanism that controls wide-ranging functions in the nervous system, including fine-tuning of synaptic connections during development and synaptic plasticity in the adult organism. In the UPP, attachment of a small protein, ubiquitin, tags the substrates for…

Analysis of Structural Features Contributing to Weak Affinities of Ubiquitin/Protein Interactions.

PubMed

Cohen, Ariel; Rosenthal, Eran; Shifman, Julia M

2017-11-10

Ubiquitin is a small protein that enables one of the most common post-translational modifications, where the whole ubiquitin molecule is attached to various target proteins, forming mono- or polyubiquitin conjugations. As a prototypical multispecific protein, ubiquitin interacts non-covalently with a variety of proteins in the cell, including ubiquitin-modifying enzymes and ubiquitin receptors that recognize signals from ubiquitin-conjugated substrates. To enable recognition of multiple targets and to support fast dissociation from the ubiquitin modifying enzymes, ubiquitin/protein interactions are characterized with low affinities, frequently in the higher μM and lower mM range. To determine how structure encodes low binding affinity of ubiquitin/protein complexes, we analyzed structures of more than a hundred such complexes compiled in the Ubiquitin Structural Relational Database. We calculated various structure-based features of ubiquitin/protein binding interfaces and compared them to the same features of general protein-protein interactions (PPIs) with various functions and generally higher affinities. Our analysis shows that ubiquitin/protein binding interfaces on average do not differ in size and shape complementarity from interfaces of higher-affinity PPIs. However, they contain fewer favorable hydrogen bonds and more unfavorable hydrophobic/charge interactions. We further analyzed how binding interfaces change upon affinity maturation of ubiquitin toward its target proteins. We demonstrate that while different features are improved in different experiments, the majority of the evolved complexes exhibit better shape complementarity and hydrogen bond pattern compared to wild-type complexes. Our analysis helps to understand how low-affinity PPIs have evolved and how they could be converted into high-affinity PPIs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Manipulation of ubiquitin/SUMO pathways in human herpesviruses infection.

PubMed

Gan, Jin; Qiao, Niu; Strahan, Roxanne; Zhu, Caixia; Liu, Lei; Verma, Subhash C; Wei, Fang; Cai, Qiliang

2016-11-01

Post-translational modification of proteins with ubiquitin/small ubiquitin-like modifier (SUMO) molecules triggers multiple signaling pathways that are critical for many aspects of cellular physiology. Given that viruses hijack the biosynthetic and degradative systems of their host, it is not surprising that viruses encode proteins to manipulate the host's cellular machinery for ubiquitin/SUMO modification at multiple levels. Infection with a herpesvirus, among the most ubiquitous human DNA viruses, has been linked to many human diseases, including cancers. The interplay between human herpesviruses and the ubiquitylation/SUMOylation modification system has been extensively investigated in the past decade. In this review, we present an overview of recent advances to address how the ubiquitin/SUMO-modified system alters the latency and lytic replication of herpesvirus and how herpesviruses usurp the ubiquitin/SUMO pathways against the host's intrinsic and innate immune response to favor their pathogenesis. Copyright © 2016 John Wiley & Sons, Ltd.

E2 enzyme inhibition by stabilization of a low affinity interface with ubiquitin

PubMed Central

St-Cyr, Daniel J.; Ziemba, Amy; Garg, Pankaj; Plamondon, Serge; Auer, Manfred; Sidhu, Sachdev; Marinier, Anne; Kleiger, Gary; Tyers, Mike; Sicheri, Frank

2014-01-01

Weak protein interactions between ubiquitin and the ubiquitin-proteasome system (UPS) enzymes that mediate its covalent attachment to substrates serve to position ubiquitin for optimal catalytic transfer. We show that a small molecule inhibitor of the E2 ubiquitin conjugating enzyme Cdc34A, called CC0651, acts by trapping a weak interaction between ubiquitin and the E2 donor ubiquitin binding site. A structure of the ternary CC0651-Cdc34A-ubiquitin complex reveals that the inhibitor engages a composite binding pocket formed from Cdc34A and ubiquitin. CC0651 also suppresses the spontaneous hydrolysis rate of the Cdc34A-ubiquitin thioester, without overtly affecting the interaction between Cdc34A and the RING domain subunit of the E3 enzyme. Stabilization of the numerous other weak interactions between ubiquitin and UPS enzymes by small molecules may be a feasible strategy to selectively inhibit different UPS activities. PMID:24316736

Tandem UIMs confer Lys48 ubiquitin chain substrate preference to deubiquitinase USP25

PubMed Central

Kawaguchi, Kohei; Uo, Kazune; Tanaka, Toshiaki; Komada, Masayuki

2017-01-01

Ubiquitin-specific protease (USP) 25, belonging to the USP family of deubiquitinases, harbors two tandem ubiquitin-interacting motifs (UIMs), a ~20-amino-acid α-helical stretch that binds to ubiquitin. However, the role of the UIMs in USP25 remains unclear. Here we show that the tandem UIM region binds to Lys48-, but not Lys63-, linked ubiquitin chains, where the two UIMs played a critical and cooperative role. Purified USP25 exhibited higher ubiquitin isopeptidase activity to Lys48-, than to Lys63-, linked ubiquitin chains. Mutations that disrupted the ubiquitin-binding ability of the tandem UIMs resulted in a reduced ubiquitin isopeptidase activity of USP25, suggesting a role for the UIMs in exerting the full catalytic activity of USP25. Moreover, when mutations that convert the binding preference from Lys48- to Lys63-linked ubiquitin chains were introduced into the tandem UIM region, the USP25 mutants acquired elevated and reduced isopeptidase activity toward Lys63- and Lys48-linked ubiquitin chains, respectively. These results suggested that the binding preference of the tandem UIMs toward Lys48-linked ubiquitin chains contributes not only to the full catalytic activity but also to the ubiquitin chain substrate preference of USP25, possibly by selectively holding the Lys48-linked ubiquitin chain substrates in the proximity of the catalytic core. PMID:28327663

Ubiquitination independent of E1 and E2 enzymes by bacterial effectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, Jiazhang; Sheedlo, Michael J.; Yu, Kaiwen

Signaling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalyzed by the E1, E2 and E3 three-enzyme cascade 1, which links the C terminus of ubiquitin via an isopeptide bond mostly to the ε-amino group of a lysine of the substrate. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents 2. For example, many bacterial pathogens exploit ubiquitin signaling using virulence factors that function as E3 ligases, deubiquitinases 3 or asmore » enzymes that directly attack ubiquitin 4. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a niche permissive for its replication in phagocytes 5. Here we demonstrate that members of the SidE effector family (SidEs) of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum (ER). Moreover, we show that these proteins are capable of catalyzing ubiquitination without the need for the E1 and E2 enzymes. The E1/E2-independent ubiquitination catalyzed by these enzymes requires NAD but not ATP and Mg2+. A putative mono ADP-ribosyltransferase (mART) motif critical for the ubiquitination activity is also essential for the role of SidEs in intracellular bacterial replication in a protozoan host. These results establish that ubiquitination can be catalyzed by a single enzyme.« less

The ubiquitin-proteasome system is required for African swine fever replication.

PubMed

Barrado-Gil, Lucía; Galindo, Inmaculada; Martínez-Alonso, Diego; Viedma, Sergio; Alonso, Covadonga

2017-01-01

Several viruses manipulate the ubiquitin-proteasome system (UPS) to initiate a productive infection. Determined viral proteins are able to change the host's ubiquitin machinery and some viruses even encode their own ubiquitinating or deubiquitinating enzymes. African swine fever virus (ASFV) encodes a gene homologous to the E2 ubiquitin conjugating (UBC) enzyme. The viral ubiquitin-conjugating enzyme (UBCv1) is expressed throughout ASFV infection and accumulates at late times post infection. UBCv is also present in the viral particle suggesting that the ubiquitin-proteasome pathway could play an important role at early ASFV infection. We determined that inhibition of the final stage of the ubiquitin-proteasome pathway blocked a post-internalization step in ASFV replication in Vero cells. Under proteasome inhibition, ASF viral genome replication, late gene expression and viral production were severely reduced. Also, ASFV enhanced proteasome activity at late times and the accumulation of polyubiquitinated proteins surrounding viral factories. Core-associated and/or viral proteins involved in DNA replication may be targets for the ubiquitin-proteasome pathway that could possibly assist virus uncoating at final core breakdown and viral DNA release. At later steps, polyubiquitinated proteins at viral factories could exert regulatory roles in cell signaling.

Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae.

PubMed Central

Gilon, T; Chomsky, O; Kulka, R G

1998-01-01

Combinations of different ubiquitin-conjugating (Ubc) enzymes and other factors constitute subsidiary pathways of the ubiquitin system, each of which ubiquitinates a specific subset of proteins. There is evidence that certain sequence elements or structural motifs of target proteins are degradation signals which mark them for ubiquitination by a particular branch of the ubiquitin system and for subsequent degradation. Our aim was to devise a way of searching systematically for degradation signals and to determine to which ubiquitin system subpathways they direct the proteins. We have constructed two reporter gene libraries based on the lacZ or URA3 genes which, in Saccharomyces cerevisiae, express fusion proteins with a wide variety of C-terminal extensions. From these, we have isolated clones producing unstable fusion proteins which are stabilized in various ubc mutants. Among these are 10 clones whose products are stabilized in ubc6, ubc7 or ubc6ubc7 double mutants. The C-terminal extensions of these clones, which vary in length from 16 to 50 amino acid residues, are presumed to contain degradation signals channeling proteins for degradation via the UBC6 and/or UBC7 subpathways of the ubiquitin system. Some of these C-terminal tails share similar sequence motifs, and a feature common to almost all of these sequences is a highly hydrophobic region such as is usually located inside globular proteins or inserted into membranes. PMID:9582269

Proteomic snapshot of the EGF-induced ubiquitin network

PubMed Central

Argenzio, Elisabetta; Bange, Tanja; Oldrini, Barbara; Bianchi, Fabrizio; Peesari, Raghunath; Mari, Sara; Di Fiore, Pier Paolo; Mann, Matthias; Polo, Simona

2011-01-01

The activity, localization and fate of many cellular proteins are regulated through ubiquitination, a process whereby one or more ubiquitin (Ub) monomers or chains are covalently attached to target proteins. While Ub-conjugated and Ub-associated proteomes have been described, we lack a high-resolution picture of the dynamics of ubiquitination in response to signaling. In this study, we describe the epidermal growth factor (EGF)-regulated Ubiproteome, as obtained by two complementary purification strategies coupled to quantitative proteomics. Our results unveil the complex impact of growth factor signaling on Ub-based intracellular networks to levels that extend well beyond what might have been expected. In addition to endocytic proteins, the EGF-regulated Ubiproteome includes a large number of signaling proteins, ubiquitinating and deubiquitinating enzymes, transporters and proteins involved in translation and transcription. The Ub-based signaling network appears to intersect both housekeeping and regulatory circuitries of cellular physiology. Finally, as proof of principle of the biological relevance of the EGF-Ubiproteome, we demonstrated that EphA2 is a novel, downstream ubiquitinated target of epidermal growth factor receptor (EGFR), critically involved in EGFR biological responses. PMID:21245847

Differential ubiquitination in NETs regulates macrophage responses in systemic lupus erythematosus.

PubMed

Barrera-Vargas, Ana; Gómez-Martín, Diana; Carmona-Rivera, Carmelo; Merayo-Chalico, Javier; Torres-Ruiz, Jiram; Manna, Zerai; Hasni, Sarfaraz; Alcocer-Varela, Jorge; Kaplan, Mariana J

2018-06-01

To assess if ubiquitinated proteins potentially present in neutrophil extracellular traps (NETs) can modify cellular responses and induce inflammatory mechanisms in patients with systemic lupus erythematosus (SLE) and healthy subjects. We studied 74 subjects with SLE and 77 healthy controls. Neutrophils and low-density granulocytes were isolated, and NETs were induced. Ubiquitin content was quantified in NETs by western blot analysis, ELISA and immunofluorescence microscopy, while ubiquitination of NET proteins was assessed by immunoprecipitation. Monocyte-derived macrophages from SLE and controls were isolated and stimulated with NETs or ubiquitin. Calcium flux and cytokine synthesis were measured following these stimuli. NETs contain ubiquitinated proteins, with a lower expression of polyubiquitinated proteins in subjects with SLE than in controls. Myeloperoxidase (MPO) is present in ubiquitinated form in NETs. Patients with SLE develop antiubiquitinated MPO antibodies, and titres positively correlate with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score (P<0.01), and negatively correlate with complement components (P<0.01). Stimulation of monocyte-derived macrophages with NETs or with ubiquitin led to enhanced calcium flux. In addition, stimulation with NETs led to enhanced cytokine (tumour necrosis factor-α and interleukin-10) production in macrophages from patients with SLE when compared with controls, which was hampered by inhibition of NET internalisation by macrophages. This is the first study to find ubiquitinated proteins in NETs, and evidence for adaptive immune responses directed towards ubiquitinated NET proteins in SLE. The distinct differences in ubiquitin species profile in NETs compared with healthy controls may contribute to dampened anti-inflammatory responses observed in SLE. These results also support a role for extracellular ubiquitin in inflammation in SLE. © Article author(s) (or their employer(s) unless otherwise stated

Regulation of transcriptional activators by DNA-binding domain ubiquitination

PubMed Central

Landré, Vivien; Revi, Bhindu; Mir, Maria Gil; Verma, Chandra; Hupp, Ted R; Gilbert, Nick; Ball, Kathryn L

2017-01-01

Ubiquitin is a key component of the regulatory network that maintains gene expression in eukaryotes, yet the molecular mechanism(s) by which non-degradative ubiquitination modulates transcriptional activator (TA) function is unknown. Here endogenous p53, a stress-activated transcription factor required to maintain health, is stably monoubiquitinated, following pathway activation by IR or Nutlin-3 and localized to the nucleus where it becomes tightly associated with chromatin. Comparative structure–function analysis and in silico modelling demonstrate a direct role for DNA-binding domain (DBD) monoubiquitination in TA activation. When attached to the DBD of either p53, or a second TA IRF-1, ubiquitin is orientated towards, and makes contact with, the DNA. The contact is made between a predominantly cationic surface on ubiquitin and the anionic DNA. Our data demonstrate an unexpected role for ubiquitin in the mechanism of TA-activity enhancement and provides insight into a new level of transcriptional regulation. PMID:28362432

Regulation of E2s: A Role for Additional Ubiquitin Binding Sites?

PubMed

Middleton, Adam J; Wright, Joshua D; Day, Catherine L

2017-11-10

Attachment of ubiquitin to proteins relies on a sophisticated enzyme cascade that is tightly regulated. The machinery of ubiquitylation responds to a range of signals, which remarkably includes ubiquitin itself. Thus, ubiquitin is not only the central player in the ubiquitylation cascade but also a key regulator. The ubiquitin E3 ligases provide specificity to the cascade and often bind the substrate, while the ubiquitin-conjugating enzymes (E2s) have a pivotal role in determining chain linkage and length. Interaction of ubiquitin with the E2 is important for activity, but the weak nature of these contacts has made them hard to identify and study. By reviewing available crystal structures, we identify putative ubiquitin binding sites on E2s, which may enhance E2 processivity and the assembly of chains of a defined linkage. The implications of these new sites are discussed in the context of known E2-ubiquitin interactions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Promotion of cooperation by payoff-driven migration

NASA Astrophysics Data System (ADS)

Chen, Ya-Shan; Yang, Han-Xin; Guo, Wen-Zhong

2016-05-01

Migration plays an important role in the evolution of cooperation. Previous studies concerning mobile population often assumed that all agents move with the identical velocity. In this paper, we propose a payoff-driven migration in which the velocity of an agent depends on his/her payoff. A parameter α is introduced to adjust the influence of payoff, when α = 0 means that agents all move with the identical velocity while α > 0 means that the lower the payoff is, the faster the moving speed is, and vice versa. For the prisoner's dilemma game, we find that in comparison with the case that agents all move with the same speed, cooperation could be promoted strongly when payoff-dependent velocity is considered. Remarkably, the cooperation level is not a monotonic function of α, and there exists an optimal value of α which can lead to the maximum cooperation level. For the snowdrift game, the cooperation level increases with α.

Low nuclear body formation and tax SUMOylation do not prevent NF-kappaB promoter activation.

PubMed

Bonnet, Amandine; Randrianarison-Huetz, Voahangy; Nzounza, Patrycja; Nedelec, Martine; Chazal, Maxime; Waast, Laetitia; Pene, Sabrina; Bazarbachi, Ali; Mahieux, Renaud; Bénit, Laurence; Pique, Claudine

2012-09-25

The Tax protein encoded by Human T-lymphotropic virus type 1 (HTLV-1) is a powerful activator of the NF-κB pathway, a property critical for HTLV-1-induced immortalization of CD4⁺ T lymphocytes. Tax permanently stimulates this pathway at a cytoplasmic level by activating the IκB kinase (IKK) complex and at a nuclear level by enhancing the binding of the NF-κB factor RelA to its cognate promoters and by forming nuclear bodies, believed to represent transcriptionally active structures. In previous studies, we reported that Tax ubiquitination and SUMOylation play a critical role in Tax localization and NF-κB activation. Indeed, analysis of lysine Tax mutants fused or not to ubiquitin or SUMO led us to propose a two-step model in which Tax ubiquitination first intervenes to activate IKK while Tax SUMOylation is subsequently required for promoter activation within Tax nuclear bodies. However, recent studies showing that ubiquitin or SUMO can modulate Tax activities in either the nucleus or the cytoplasm and that SUMOylated Tax can serve as substrate for ubiquitination suggested that Tax ubiquitination and SUMOylation may mediate redundant rather than successive functions. In this study, we analyzed the properties of a new Tax mutant that is properly ubiquitinated, but defective for both nuclear body formation and SUMOylation. We report that reducing Tax SUMOylation and nuclear body formation do not alter the ability of Tax to activate IKK, induce RelA nuclear translocation, and trigger gene expression from a NF-κB promoter. Importantly, potent NF-κB promoter activation by Tax despite low SUMOylation and nuclear body formation is also observed in T cells, including CD4⁺ primary T lymphocytes. Moreover, we show that Tax nuclear bodies are hardly observed in HTLV-1-infected T cells. Finally, we provide direct evidence that the degree of NF-κB activation by Tax correlates with the level of Tax ubiquitination, but not SUMOylation. These data reveal that the

Low nuclear body formation and tax SUMOylation do not prevent NF-kappaB promoter activation

PubMed Central

2012-01-01

Background The Tax protein encoded by Human T-lymphotropic virus type 1 (HTLV-1) is a powerful activator of the NF-κB pathway, a property critical for HTLV-1-induced immortalization of CD4+ T lymphocytes. Tax permanently stimulates this pathway at a cytoplasmic level by activating the IκB kinase (IKK) complex and at a nuclear level by enhancing the binding of the NF-κB factor RelA to its cognate promoters and by forming nuclear bodies, believed to represent transcriptionally active structures. In previous studies, we reported that Tax ubiquitination and SUMOylation play a critical role in Tax localization and NF-κB activation. Indeed, analysis of lysine Tax mutants fused or not to ubiquitin or SUMO led us to propose a two-step model in which Tax ubiquitination first intervenes to activate IKK while Tax SUMOylation is subsequently required for promoter activation within Tax nuclear bodies. However, recent studies showing that ubiquitin or SUMO can modulate Tax activities in either the nucleus or the cytoplasm and that SUMOylated Tax can serve as substrate for ubiquitination suggested that Tax ubiquitination and SUMOylation may mediate redundant rather than successive functions. Results In this study, we analyzed the properties of a new Tax mutant that is properly ubiquitinated, but defective for both nuclear body formation and SUMOylation. We report that reducing Tax SUMOylation and nuclear body formation do not alter the ability of Tax to activate IKK, induce RelA nuclear translocation, and trigger gene expression from a NF-κB promoter. Importantly, potent NF-κB promoter activation by Tax despite low SUMOylation and nuclear body formation is also observed in T cells, including CD4+ primary T lymphocytes. Moreover, we show that Tax nuclear bodies are hardly observed in HTLV-1-infected T cells. Finally, we provide direct evidence that the degree of NF-κB activation by Tax correlates with the level of Tax ubiquitination, but not SUMOylation. Conclusions These

Molecular dynamics simulations of human E3 ubiquitin ligase Parkin

PubMed Central

Qiu, Shi; Zhu, Shun; Xu, Shan; Han, Yanyan; Liu, Wen; Zuo, Ji

2017-01-01

Human E3 ubiquitin protein ligase parkin (Parkin) mediates mitophagy to maintain mitochondrial homeostasis. Parkin mutations are common genetic causes of early onset familial Parkinson's disease. The molecular mechanism of Parkin activation has been widely studied with emerging evidence suggesting an essential role of the phosphorylated (phospho)-ubiquitin interaction. However, the underlying mechanism of the phospho-ubiquitin interaction remains elusive. In the present study, replica exchange molecular dynamics simulations were performed to examine the conformational dynamics of Parkin in monomer and phospho-ubiquitin-bound states. In the Parkin monomer state, high structural flexibilities were observed in the majority of regions of Parkin particularly in the loop domain between the ubiquitin-like (UBL) and really interesting new gene (RING)0 domain. Binding of phospho-ubiquitin stabilizes the RING1/RING in between RING interface but destabilizes the RING1-UBL interface. Furthermore, using steered molecular dynamics simulations of Parkin mutations, it was demonstrated that salt bridge interactions contribute significantly to the interdomain interactions between the RING1 and UBL domain. Taken together, the results of the present study revealed the conformational dynamics of human full-length Parkin in monomer and phospho-ubiquitin-bound states, providing insights into designing potential therapeutics against Parkinson's disease. PMID:28765939

Receptor for advanced glycation end products is targeted by FBXO10 for ubiquitination and degradation.

PubMed

Evankovich, John; Lear, Travis; Mckelvey, Alison; Dunn, Sarah; Londino, James; Liu, Yuan; Chen, Bill B; Mallampalli, Rama K

2017-09-01

The receptor for advanced glycation end products (RAGE) is a highly expressed cell membrane receptor serving to anchor lung epithelia to matrix components, and it also amplifies inflammatory signaling during acute lung injury. However, mechanisms that regulate its protein concentrations in cells remain largely unknown. Here we show that RAGE exhibits an extended life span in lung epithelia ( t ½ 6 h), is monoubiquitinated at K374, and is degraded in lysosomes. The RAGE ligand ODN2006, a synthetic oligodeoxynucleotide resembling pathogenic hypomethylated CpG DNA, promotes rapid lysosomal RAGE degradation through activation of protein kinase Cζ (PKCζ), which phosphorylates RAGE. PKCζ overexpression enhances RAGE degradation, while PKCζ knockdown stabilizes RAGE protein levels and prevents ODN2006-mediated degradation. We identify that RAGE is targeted by the ubiquitin E3 ligase subunit F-box protein O10 (FBXO10), which associates with RAGE to mediate its ubiquitination and degradation. FBXO10 depletion in cells stabilizes RAGE and is required for ODN2006-mediated degradation. These data suggest that modulation of regulators involved in ubiquitin-mediated disposal of RAGE might serve as unique molecular inputs directing RAGE cellular concentrations and downstream responses, which are critical in an array of inflammatory disorders, including acute lung injury.-Evankovich, J., Lear, T., Mckelvey, A., Dunn, S., Londino, J., Liu, Y., Chen, B. B., Mallampalli, R. K. Receptor for advanced glycation end products is targeted by FBXO10 for ubiquitination and degradation. © FASEB.

Newborn mouse lens proteome and its alteration by lysine 6 mutant ubiquitin

USDA-ARS?s Scientific Manuscript database

Ubiquitin is a tag that often initiates degradation of proteins by the proteasome in the ubiquitin proteasome system. Targeted expression of K6W mutant ubiquitin (K6W-Ub) in the lens results in defects in lens development and cataract formation, suggesting critical functions for ubiquitin in lens. T...

Dengue Virus Genome Uncoating Requires Ubiquitination.

PubMed

Byk, Laura A; Iglesias, Néstor G; De Maio, Federico A; Gebhard, Leopoldo G; Rossi, Mario; Gamarnik, Andrea V

2016-06-28

The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle. Flaviviruses are mainly arthropod-borne viruses, including emerging and reemerging pathogens such as dengue, Zika, and West Nile viruses. Currently, dengue virus is one of the most significant human viral pathogens transmitted by mosquitoes and is responsible for about 390 million infections every year around the world. Here, we examined for the first time molecular aspects of dengue virus genome uncoating. We followed the fate of the capsid protein and RNA genome early during infection and found that capsid is degraded after viral internalization by the host ubiquitin-proteasome system. However, proteasome activity and capsid degradation were not necessary to free the genome for initial viral translation. Unexpectedly, genome uncoating was blocked by inhibiting ubiquitination. Using different assays to bypass entry and evaluate the first rounds of viral translation, a narrow window of time during infection that requires ubiquitination but not proteasome activity was identified. In this regard, ubiquitin E1-activating enzyme inhibition was sufficient to stabilize the incoming viral genome in the cytoplasm of infected cells, causing its retention in either endosomes or nucleocapsids. Our data support a model in which dengue virus genome uncoating requires a nondegradative ubiquitination step, providing new insights into this crucial but understudied viral process. Dengue is the most significant arthropod-borne viral infection in humans. Although the number of cases increases every year, there are no approved therapeutics available for the treatment of dengue infection, and many basic aspects of the viral biology remain elusive. After entry, the viral membrane must fuse with the endosomal membrane to deliver the viral genome into the cytoplasm for translation and replication. A great deal of information has been obtained in the last decade

Genome-wide analysis of Glycine soja ubiquitin (UBQ) genes and functional analysis of GsUBQ10 in response to alkaline stress.

PubMed

Chen, Chao; Chen, Ranran; Wu, Shengyang; Zhu, Dan; Sun, Xiaoli; Liu, Beidong; Li, Qiang; Zhu, Yanming

2018-03-26

Ubiquitin is a highly conserved protein with multiple essential regulation functions through the ubiquitin-proteasome system. Even though its functions in the ubiquitin-mediated protein degradation pathway were very well characterized. The functions of ubiquitin genes in regulating alkaline stress response are not fully established. In this study, we identified 12 potential UBQ genes in Glycine soja genome, and analyzed their evolutionary relationship, conserved domains and promoter cis-elements. We also explored the expression profiles of G. soja UBQ genes under alkaline stress, based on the transcriptome sequencing. We found that the expression of GsUBQ10 was significantly induced by alkaline stress, and function of GsUBQ10 was characterized using overexpression transgenic alfalfa (Medicago sativa). Our results suggested that GsUBQ10 transgenic lines significantly improved the alkaline tolerance in alfalfa. The GsUBQ10 transgenic lines showed lower relative membrane permeability, lower malon dialdehyde content and higher catalase activity than in the wild-type plants. This indicates that GsUBQ10 is involved in regulating the reactive oxygen species accumulation under alkaline stress. Taken together, we identified an ubiquitin gene GsUBQ10 from G. soja, which plays a positive role in responses to alkaline stress in alfalfa. This article is protected by copyright. All rights reserved.

Molecular basis of ubiquitin recognition by the autophagy receptor CALCOCO2

PubMed Central

Xie, Xingqiao; Li, Faxiang; Wang, Yuanyuan; Wang, Yingli; Lin, Zhijie; Cheng, Xiaofang; Liu, Jianping; Chen, Changbin; Pan, Lifeng

2015-01-01

The autophagy receptor CALCOCO2/NDP52 functions as a bridging adaptor and plays an essential role in the selective autophagic degradation of invading pathogens by specifically recognizing ubiquitin-coated intracellular pathogens and subsequently targeting them to the autophagic machinery; thereby it is required for innate immune defense against a range of infectious pathogens in mammals. However, the mechanistic basis underlying CALCOCO2-mediated specific recognition of ubiqutinated pathogens is still unknown. Here, using biochemical and structural analyses, we demonstrated that the cargo-binding region of CALCOCO2 contains a dynamic unconventional zinc finger as well as a C2H2-type zinc-finger, and only the C2H2-type zinc finger specifically recognizes mono-ubiquitin or poly-ubiquitin chains. In addition to elucidating the specific ubiquitin recognition mechanism of CALCOCO2, the structure of the CALCOCO2 C2H2-type zinc finger in complex with mono-ubiquitin also uncovers a unique zinc finger-binding mode for ubiquitin. Our findings provide mechanistic insight into how CALCOCO2 targets ubiquitin-decorated pathogens for autophagic degradations. PMID:26506893

Structure of PINK1 in complex with its substrate ubiquitin

PubMed Central

Schubert, Alexander F.; Gladkova, Christina; Pardon, Els; Wagstaff, Jane L.; Freund, Stefan M.V.; Steyaert, Jan; Maslen, Sarah L.; Komander, David

2018-01-01

Autosomal recessive juvenile Parkinsonism (AR-JP) is caused by mutations in a number of PARK genes, in particular in the E3 ubiquitin ligase Parkin (PARK2), and in its upstream protein kinase PINK1 (PARK6). PINK1 phosphorylates ubiquitin and the Parkin ubiquitin-like domain on structurally protected Ser65 to trigger mitophagy. We here report a crystal structure of a nanobody stabilised complex between Pediculus humanus corporis (Ph)PINK1 bound to ubiquitin in the ‘C-terminally retracted’ (Ub-CR) conformation. The structure reveals many peculiarities of PINK1, including the architecture of the C-terminal region, and reveals how the PINK1 N-lobe binds ubiquitin via a unique insertion. The flexible Ser65-loop in the Ub-CR conformation reaches the activation segment, facilitating placement of Ser65 in a phosphate accepting position. The structure also explains how autophosphorylation in the N-lobe stabilises structurally and functionally important insertions, and reveals the molecular basis for AR-JP causing mutations, some of which disrupt ubiquitin binding. PMID:29160309

Structure of PINK1 in complex with its substrate ubiquitin.

PubMed

Schubert, Alexander F; Gladkova, Christina; Pardon, Els; Wagstaff, Jane L; Freund, Stefan M V; Steyaert, Jan; Maslen, Sarah L; Komander, David

2017-12-07

Autosomal-recessive juvenile Parkinsonism (AR-JP) is caused by mutations in a number of PARK genes, in particular the genes encoding the E3 ubiquitin ligase Parkin (PARK2, also known as PRKN) and its upstream protein kinase PINK1 (also known as PARK6). PINK1 phosphorylates both ubiquitin and the ubiquitin-like domain of Parkin on structurally protected Ser65 residues, triggering mitophagy. Here we report a crystal structure of a nanobody-stabilized complex containing Pediculus humanus corporis (Ph)PINK1 bound to ubiquitin in the 'C-terminally retracted' (Ub-CR) conformation. The structure reveals many peculiarities of PINK1, including the architecture of the C-terminal region, and reveals how the N lobe of PINK1 binds ubiquitin via a unique insertion. The flexible Ser65 loop in the Ub-CR conformation contacts the activation segment, facilitating placement of Ser65 in a phosphate-accepting position. The structure also explains how autophosphorylation in the N lobe stabilizes structurally and functionally important insertions, and reveals the molecular basis of AR-JP-causing mutations, some of which disrupt ubiquitin binding.

Loss-of-function of a ubiquitin-related modifier promotes the mobilization of the active MITE mPing.

PubMed

Tsukiyama, Takuji; Teramoto, Shota; Yasuda, Kanako; Horibata, Akira; Mori, Nanako; Okumoto, Yutaka; Teraishi, Masayoshi; Saito, Hiroki; Onishi, Akiko; Tamura, Kanako; Tanisaka, Takatoshi

2013-05-01

Miniature inverted-repeat transposable elements (MITEs) are widespread in both prokaryotic and eukaryotic genomes, where their copy numbers can attain several thousands. Little is known, however, about the genetic factor(s) affecting their transpositions. Here, we show that disruption of a gene encoding ubiquitin-like protein markedly enhances the transposition activity of a MITE mPing in intact rice plants without any exogenous stresses. We found that the transposition activity of mPing is far higher in the lines harboring a non-functional allele at the Rurm1 (Rice ubiquitin-related modifier-1) locus than in the wild-type line. Although the alteration of cytosine methylation pattern triggers the activation of transposable elements under exogenous stress conditions, the methylation degrees in the whole genome, the mPing-body region, and the mPing-flanking regions of the non-functional Rurm1 line were unchanged. This study provides experimental evidence for one of the models of genome shock theory that genetic accidents within cells enhance the transposition activities of transposable elements.

Increased gibberellin contents contribute to accelerated growth and development of transgenic tobacco overexpressing a wheat ubiquitin gene.

PubMed

Wang, Guo-Kun; Zhang, Meng; Gong, Jiang-Feng; Guo, Qi-Fang; Feng, Ya-Nan; Wang, Wei

2012-12-01

Overexpressing TaUb2 promoted stem growth and resulted in early flowering in transgenic tobacco plants. Ubiquitin are involved in the production, metabolism and proper function of gibberellin. The ubiquitin-26S proteasome system (UPS), in which ubiquitin (Ub) functions as a marker, is a post-translational regulatory system that plays a prominent role in various biological processes. To investigate the impact of different Ub levels on plant growth and development, transgenic tobacco (Nicotiana tabacum L.) plants were engineered to express an Ub gene (TaUb2) from wheat (Triticum aestivum L.) under the control of cauliflower mosaic virus 35S promoter. Transgenic tobacco plants overexpressing TaUb2 demonstrated an accelerated growth rate at early stage and an early flowering phenotype in development. The preceding expression of MADS-box genes also corresponded to the accelerated developmental phenotypes of the transgenic tobacco plants compared to that of wild-type (WT). Total gibberellin (GA) and active GA contents in transgenic tobacco plants were higher than those in WT at the corresponding developmental stages, and some GA metabolism genes were upregulated. Treatment with GA(3) conferred a similarly accelerated grown rate in WT plants to that of transgenic tobacco plants, while growth was inhibited when transgenic tobacco plants were treated with a GA biosynthesis inhibitor. Thus, the results suggest that Ub are involved in the production, metabolism and proper function of GA, which is important in the regulation of plant growth and development.

The ubiquitin-proteasome system in spongiform degenerative disorders

PubMed Central

Whatley, Brandi R.; Li, Lian; Chin, Lih-Shen

2008-01-01

Summary Spongiform degeneration is characterized by vacuolation in nervous tissue accompanied by neuronal death and gliosis. Although spongiform degeneration is a hallmark of prion diseases, this pathology is also present in the brains of patients suffering from Alzheimer's disease, diffuse Lewy body disease, human immunodeficiency virus (HIV) infection, and Canavan's spongiform leukodystrophy. The shared outcome of spongiform degeneration in these diverse diseases suggests that common cellular mechanisms must underlie the processes of spongiform change and neurodegeneration in the central nervous system. Immunohistochemical analysis of brain tissues reveals increased ubiquitin immunoreactivity in and around areas of spongiform change, suggesting the involvement of ubiquitin-proteasome system dysfunction in the pathogenesis of spongiform neurodegeneration. The link between aberrant ubiquitination and spongiform neurodegeneration has been strengthened by the discovery that a null mutation in the E3 ubiquitin-protein ligase mahogunin ring finger-1 (Mgrn1) causes an autosomal recessively inherited form of spongiform neurodegeneration in animals. Recent studies have begun to suggest that abnormal ubiquitination may alter intracellular signaling and cell functions via proteasome-dependent and proteasome-independent mechanisms, leading to spongiform degeneration and neuronal cell death. Further elucidation of the pathogenic pathways involved in spongiform neurodegeneration should facilitate the development of novel rational therapies for treating prion diseases, HIV infection, and other spongiform degenerative disorders. PMID:18790052

PINK1 autophosphorylation is required for ubiquitin recognition.

PubMed

Rasool, Shafqat; Soya, Naoto; Truong, Luc; Croteau, Nathalie; Lukacs, Gergely L; Trempe, Jean-François

2018-04-01

Mutations in PINK1 cause autosomal recessive Parkinson's disease (PD), a neurodegenerative movement disorder. PINK1 is a kinase that acts as a sensor of mitochondrial damage and initiates Parkin-mediated clearance of the damaged organelle. PINK1 phosphorylates Ser65 in both ubiquitin and the ubiquitin-like (Ubl) domain of Parkin, which stimulates its E3 ligase activity. Autophosphorylation of PINK1 is required for Parkin activation, but how this modulates the ubiquitin kinase activity is unclear. Here, we show that autophosphorylation of Tribolium castaneum PINK1 is required for substrate recognition. Using enzyme kinetics and NMR spectroscopy, we reveal that PINK1 binds the Parkin Ubl with a 10-fold higher affinity than ubiquitin via a conserved interface that is also implicated in RING1 and SH3 binding. The interaction requires phosphorylation at Ser205, an invariant PINK1 residue (Ser228 in human). Using mass spectrometry, we demonstrate that PINK1 rapidly autophosphorylates in trans at Ser205. Small-angle X-ray scattering and hydrogen-deuterium exchange experiments provide insights into the structure of the PINK1 catalytic domain. Our findings suggest that multiple PINK1 molecules autophosphorylate first prior to binding and phosphorylating ubiquitin and Parkin. © 2018 The Authors.

Ubiquitin-protein ligases in muscle wasting: multiple parallel pathways?

NASA Technical Reports Server (NTRS)

Lecker, Stewart H.; Goldberg, A. L. (Principal Investigator)

2003-01-01

PURPOSE OF REVIEW: Studies in a wide variety of animal models of muscle wasting have led to the concept that increased protein breakdown via the ubiquitin-proteasome pathway is responsible for the loss of muscle mass seen as muscle atrophy. The complexity of the ubiquitination apparatus has hampered our understanding of how this pathway is activated in atrophying muscles and which ubiquitin-conjugating enzymes in muscle are responsible. RECENT FINDINGS: Recent experiments have shown that two newly identified ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MURF-1, are critical in the development of muscle atrophy. Other in-vitro studies also implicated E2(14k) and E3alpha, of the N-end rule pathway, as playing an important role in the process. SUMMARY: It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as inhibitors of, these E3s.

Ubiquitin-dependent and independent roles of SUMO in proteostasis.

PubMed

Liebelt, Frauke; Vertegaal, Alfred C O

2016-08-01

Cellular proteomes are continuously undergoing alterations as a result of new production of proteins, protein folding, and degradation of proteins. The proper equilibrium of these processes is known as proteostasis, implying that proteomes are in homeostasis. Stress conditions can affect proteostasis due to the accumulation of misfolded proteins as a result of overloading the degradation machinery. Proteostasis is affected in neurodegenerative diseases like Alzheimer's disease, Parkinson's disease, and multiple polyglutamine disorders including Huntington's disease. Owing to a lack of proteostasis, neuronal cells build up toxic protein aggregates in these diseases. Here, we review the role of the ubiquitin-like posttranslational modification SUMO in proteostasis. SUMO alone contributes to protein homeostasis by influencing protein signaling or solubility. However, the main contribution of SUMO to proteostasis is the ability to cooperate with, complement, and balance the ubiquitin-proteasome system at multiple levels. We discuss the identification of enzymes involved in the interplay between SUMO and ubiquitin, exploring the complexity of this crosstalk which regulates proteostasis. These enzymes include SUMO-targeted ubiquitin ligases and ubiquitin proteases counteracting these ligases. Additionally, we review the role of SUMO in brain-related diseases, where SUMO is primarily investigated because of its role during formation of aggregates, either independently or in cooperation with ubiquitin. Detailed understanding of the role of SUMO in these diseases could lead to novel treatment options. Copyright © 2016 the American Physiological Society.

Immune defects caused by mutations in the ubiquitin system.

PubMed

Etzioni, Amos; Ciechanover, Aaron; Pikarsky, Eli

2017-03-01

The importance of the ubiquitin system in health and disease has been widely recognized in recent decades, with better understanding of the various components of the system and their function. Ubiquitination, which is essential to almost all biological processes in eukaryotes, was also found to play an important role in innate and adaptive immune responses. Thus it is not surprising that mutations in genes coding for components of the ubiquitin system cause immune dysregulation. The first defect in the system was described 30 years ago and is due to mutations in the nuclear factor κB (NF-κB) essential modulator, a key regulator of the NF-κB pathway. With use of novel sequencing techniques, many additional mutations in different genes involved in ubiquitination and related to immune system function were identified. This can be clearly illustrated in mutations in the different activation pathways of NF-κB, which result in aberrations in production of various proinflammatory cytokines. The inherited diseases typically manifest with immunodeficiency, autoimmunity, or autoinflammation. In this perspective we provide a short description of the ubiquitin system, with specific emphasis given to its role in the immune system. The various immunodeficiency conditions identified thus far in association with defective ubiquitination are discussed in more detail. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Substrate specificity of the ubiquitin and Ubl proteases

PubMed Central

Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark

2016-01-01

Conjugation and deconjugation of ubiquitin and ubiquitin-like proteins (Ubls) to cellular proteins are highly regulated processes integral to cellular homeostasis. Most often, the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide (isopeptide) linkage. Deubiquitinating enzymes (DUBs) and Ubl-specific proteases (ULPs) comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates. How DUBs and ULPs distinguish among different modifiers, or different polymeric forms of these modifiers, remains poorly understood. The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors, ranging from the topography of specific substrate features, as in different polyubiquitin chain types, to structural elements unique to each enzyme. Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs. We also discuss the unexpected specificities of non-eukaryotic proteases in these families. PMID:27012468

Role of SKP1-CUL1-F-Box-Protein (SCF) E3 Ubiquitin Ligases in Skin Cancer

PubMed Central

Xie, Chuan-Ming; Wei, Wenyi; Sun, Yi

2013-01-01

Many biological processes such as cell proliferation, differentiation, and cell death depend precisely on the timely synthesis and degradation of key regulatory proteins. While protein synthesis can be regulated at multiple levels, protein degradation is mainly controlled by the ubiquitin—proteasome system (UPS), which consists of two distinct steps: (1) ubiquitylation of targeted protein by E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase, and (2) subsequent degradation by the 26S proteasome. Among all E3 ubiquitin ligases, the SCF (SKP1-CUL1-F-box protein) E3 ligases are the largest family and are responsible for the turnover of many key regulatory proteins. Aberrant regulation of SCF E3 ligases is associated with various human diseases, such as cancers, including skin cancer. In this review, we provide a comprehensive overview of all currently published data to define a promoting role of SCF E3 ligases in the development of skin cancer. The future directions in this area of research are also discussed with an ultimate goal to develop small molecule inhibitors of SCF E3 ligases as a novel approach for the treatment of human skin cancer. Furthermore, altered components or substrates of SCF E3 ligases may also be developed as the biomarkers for early diagnosis or predicting prognosis. PMID:23522382

The effect of acetaminophen on ubiquitin homeostasis in Saccharomyces cerevisiae.

PubMed

Huseinovic, Angelina; van Leeuwen, Jolanda S; van Welsem, Tibor; Stulemeijer, Iris; van Leeuwen, Fred; Vermeulen, Nico P E; Kooter, Jan M; Vos, J Chris

2017-01-01

Acetaminophen (APAP), although considered a safe drug, is one of the major causes of acute liver failure by overdose, and therapeutic chronic use can cause serious health problems. Although the reactive APAP metabolite N-acetyl-p-benzoquinoneimine (NAPQI) is clearly linked to liver toxicity, toxicity of APAP is also found without drug metabolism of APAP to NAPQI. To get more insight into mechanisms of APAP toxicity, a genome-wide screen in Saccharomyces cerevisiae for APAP-resistant deletion strains was performed. In this screen we identified genes related to the DNA damage response. Next, we investigated the link between genotype and APAP-induced toxicity or resistance by performing a more detailed screen with a library containing mutants of 1522 genes related to nuclear processes, like DNA repair and chromatin remodelling. We identified 233 strains that had an altered growth rate relative to wild type, of which 107 showed increased resistance to APAP and 126 showed increased sensitivity. Gene Ontology analysis identified ubiquitin homeostasis, regulation of transcription of RNA polymerase II genes, and the mitochondria-to-nucleus signalling pathway to be associated with APAP resistance, while histone exchange and modification, and vesicular transport were connected to APAP sensitivity. Indeed, we observed a link between ubiquitin levels and APAP resistance, whereby ubiquitin deficiency conferred resistance to APAP toxicity while ubiquitin overexpression resulted in sensitivity. The toxicity profile of various chemicals, APAP, and its positional isomer AMAP on a series of deletion strains with ubiquitin deficiency showed a unique resistance pattern for APAP. Furthermore, exposure to APAP increased the level of free ubiquitin and influenced the ubiquitination of proteins. Together, these results uncover a role for ubiquitin homeostasis in APAP-induced toxicity.

The Linear ubiquitin chain assembly complex acts as a liver tumor suppressor and inhibits hepatocyte apoptosis and hepatitis.

PubMed

Shimizu, Yutaka; Peltzer, Nieves; Sevko, Alexandra; Lafont, Elodie; Sarr, Aida; Draberova, Helena; Walczak, Henning

2017-06-01

Linear ubiquitination is a key posttranslational modification that regulates immune signaling and cell death pathways, notably tumor necrosis factor receptor 1 (TNFR1) signaling. The only known enzyme complex capable of forming linear ubiquitin chains under native conditions to date is the linear ubiquitin chain assembly complex, of which the catalytic core component is heme-oxidized iron regulatory protein 2 ubiquitin ligase-1-interacting protein (HOIP). To understand the underlying mechanisms of maintenance of liver homeostasis and the role of linear ubiquitination specifically in liver parenchymal cells, we investigated the physiological role of HOIP in the liver parenchyma. To do so, we created mice harboring liver parenchymal cell-specific deletion of HOIP (Hoip Δhep mice) by crossing Hoip-floxed mice with albumin-Cre mice. HOIP deficiency in liver parenchymal cells triggered tumorigenesis at 18 months of age preceded by spontaneous hepatocyte apoptosis and liver inflammation within the first month of life. In line with the emergence of inflammation, Hoip Δhep mice displayed enhanced liver regeneration and DNA damage. In addition, consistent with increased apoptosis, HOIP-deficient hepatocytes showed enhanced caspase activation and endogenous formation of a death-inducing signaling complex which activated caspase-8. Unexpectedly, exacerbated caspase activation and apoptosis were not dependent on TNFR1, whereas ensuing liver inflammation and tumorigenesis were promoted by TNFR1 signaling. The linear ubiquitin chain assembly complex serves as a previously undescribed tumor suppressor in the liver, restraining TNFR1-independent apoptosis in hepatocytes which, in its absence, is causative of TNFR1-mediated inflammation, resulting in hepatocarcinogenesis. (Hepatology 2017;65:1963-1978). © 2017 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.

Effects of exogenous ubiquitin in a polytrauma model with blunt chest trauma

PubMed Central

Baker, Todd A.; Romero, Jacqueline; Bach, Harold H.; Strom, Joel A.; Gamelli, Richard L.; Majetschak, Matthias

2013-01-01

Objective To determine whether treatment with the CXC chemokine receptor (CXCR) 4 agonist ubiquitin results in beneficial effects in a polytrauma model consisting of bilateral femur fractures plus blunt chest trauma (Injury Severity Score 18-25). Design Treatment study. Setting Research Laboratory. Subjects Seventeen Yorkshire pigs. Interventions Intravenous (i.v.) injection of 1.5 mg/kg ubiquitin or albumin (=control) at 60 min after polytrauma. Measurements and Main Results Anesthetized, mechanically ventilated pigs underwent polytrauma, followed by a simulated 60 min shock phase. At the end of the shock phase ubiquitin or albumin were administered and animals were resuscitated to a mean arterial blood pressure of 70 mmHg until t = 420 min. After i.v. ubiquitin, ubiquitin plasma concentrations increased sixteen-fold to 2870 ± 1015 ng/mL at t = 90 min and decreased with t1/2 = 60 min. Endogenous plasma ubiquitin increased two-fold in the albumin group with peak levels of 359 ± 210 ng/mL. Plasma levels of the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α were unchanged in both groups. Ubiquitin treatment reduced arterial lactate levels and prevented a continuous decrease in arterial oxygenation, which occurred in the albumin group during resuscitation. Wet weight to dry weight ratios of the lung contralateral from the injury, heart, spleen and jejunum were lower with ubiquitin. With ubiquitin treatment, tissue levels of IL-8, IL-10, TNFα and SDF-1α were reduced in the injured lung and of IL-8 in the contralateral lung, respectively. Conclusions Administration of exogenous ubiquitin modulates the local inflammatory response, improves resuscitation, reduces fluid shifts into tissues and preserves arterial oxygenation after blunt polytrauma with lung injury. This study further supports the notion that ubiquitin is a promising protein therapeutic and implies CXCR4 as a drug target after polytrauma. PMID:22622399

Tetraspanin 6 (TSPAN6) Negatively Regulates Retinoic Acid-inducible Gene I-like Receptor-mediated Immune Signaling in a Ubiquitination-dependent Manner*

PubMed Central

Wang, Yetao; Tong, Xiaomei; Omoregie, Ehimwenma Sheena; Liu, Wenjun; Meng, Songdong; Ye, Xin

2012-01-01

The recognition between retinoic acid-inducible gene I-like receptors (RLRs) and viral RNA triggers an intracellular cascade of signaling to induce the expression of type I IFNs. Both positive and negative regulation of the RLR signaling pathway are important for the host antiviral immune response. Here, we demonstrate that the tetraspanin protein TSPAN6 inhibits RLR signaling by affecting the formation of the adaptor MAVS (mitochondrial antiviral signaling)-centered signalosome. We found that overexpression of TSPAN6 impaired RLR-mediated activation of IFN-stimulated response element, NF-κB, and IFN-β promoters, whereas knockdown of TSPAN6 enhanced the RLR-mediated signaling pathway. Interestingly, as the RLR pathway was activated, TSPAN6 underwent Lys-63-linked ubiquitination, which promoted its association with MAVS. The interaction of TSPAN6 and MAVS interfered with the recruitment of RLR downstream molecules TRAF3, MITA, and IRF3 to MAVS. Further study revealed that the first transmembrane domain of TSPAN6 is critical for its ubiquitination and association with MAVS as well as its inhibitory effect on RLR signaling. We concluded that TSPAN6 functions as a negative regulator of the RLR pathway by interacting with MAVS in a ubiquitination-dependent manner. PMID:22908223

Ubiquitin vinyl methyl ester binding orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive conformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boudreaux, David A.; Maiti, Tushar K.; Davies, Christopher W.

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a Parkinson disease-associated, putative cysteine protease found abundantly and selectively expressed in neurons. The crystal structure of apo UCHL1 showed that the active-site residues are not aligned in a canonical form, with the nucleophilic cysteine being 7.7 {angstrom} from the general base histidine, an arrangement consistent with an inactive form of the enzyme. Here we report the crystal structures of the wild type and two Parkinson disease-associated variants of the enzyme, S18Y and I93M, bound to a ubiquitin-based suicide substrate, ubiquitin vinyl methyl ester. These structures reveal that ubiquitin vinyl methyl ester binds primarilymore » at two sites on the enzyme, with its carboxy terminus at the active site and with its amino-terminal {beta}-hairpin at the distal site - a surface-exposed hydrophobic crevice 17 {angstrom} away from the active site. Binding at the distal site initiates a cascade of side-chain movements in the enzyme that starts at a highly conserved, surface-exposed phenylalanine and is relayed to the active site resulting in the reorientation and proximal placement of the general base within 4 {angstrom} of the catalytic cysteine, an arrangement found in productive cysteine proteases. Mutation of the distal-site, surface-exposed phenylalanine to alanine reduces ubiquitin binding and severely impairs the catalytic activity of the enzyme. These results suggest that the activity of UCHL1 may be regulated by its own substrate.« less

Ubiquitin Chains Modified by the Bacterial Ligase SdeA Are Protected from Deubiquitinase Hydrolysis.

PubMed

Puvar, Kedar; Zhou, Yiyang; Qiu, Jiazhang; Luo, Zhao-Qing; Wirth, Mary J; Das, Chittaranjan

2017-09-12

The SidE family of Legionella pneumophila effectors is a unique group of ubiquitin-modifying enzymes. Along with catalyzing NAD + -dependent ubiquitination of certain host proteins independent of the canonical E1/E2/E3 pathway, they have also been shown to produce phosphoribosylated free ubiquitin. This modified ubiquitin product is incompatible with conventional E1/E2/E3 ubiquitination processes, with the potential to lock down various cellular functions that are dependent on ubiquitin signaling. Here, we show that in addition to free ubiquitin, Lys63-, Lys48-, Lys11-, and Met1-linked diubiquitin chains are also modified by SdeA in a similar fashion. Both the proximal and distal ubiquitin moieties are targeted in the phosphoribosylation reaction. Furthermore, this renders the ubiquitin chains unable to be processed by a variety of deubiquitinating enzymes. These observations broaden the scope of SdeA's modulatory functions during Legionella infection.

Substrate phosphorylation and feedback regulation in JFK-promoted p53 destabilization.

PubMed

Sun, Luyang; Shi, Lei; Wang, Feng; Huangyang, Peiwei; Si, Wenzhe; Yang, Jie; Yao, Zhi; Shang, Yongfeng

2011-02-11

The p53 tumor suppressor plays a central role in integrating cellular responses to various stresses. Tight regulation of p53 is thus essential for the maintenance of genome integrity and normal cell proliferation. Previously, we reported that JFK, the only Kelch domain-containing F-box protein in human, promotes ubiquitination and degradation of p53 and that unlike the other E3 ligases for p53, all of which possess an intrinsic ubiquitin ligase activity, JFK destabilizes p53 through the assembly of a Skp1-Cul1-F-box complex. Here, we report that the substrate recognition by JFK requires phosphorylation of p53 in its central core region by CSN (COP9 signalosome)-associated kinase. Significantly, inhibition of CSN-associated kinase activity or knockdown of CSN5 impairs JFK-promoted p53 degradation, enhances p53-dependent transcription, and promotes cell growth suppression, G(1) arrest, and apoptosis. Moreover, we showed that JFK is transcriptionally regulated by p53 and forms an auto-regulatory negative feedback loop with p53. These data may shed new light on the functional connection between CSN, Skp1-Cul1-F-box ubiquitin ligase, and p53 and provide a molecular mechanism for the regulation of JFK-promoted p53 degradation.

Substrate Phosphorylation and Feedback Regulation in JFK-promoted p53 Destabilization*

PubMed Central

Sun, Luyang; Shi, Lei; Wang, Feng; Huangyang, Peiwei; Si, Wenzhe; Yang, Jie; Yao, Zhi; Shang, Yongfeng

2011-01-01

The p53 tumor suppressor plays a central role in integrating cellular responses to various stresses. Tight regulation of p53 is thus essential for the maintenance of genome integrity and normal cell proliferation. Previously, we reported that JFK, the only Kelch domain-containing F-box protein in human, promotes ubiquitination and degradation of p53 and that unlike the other E3 ligases for p53, all of which possess an intrinsic ubiquitin ligase activity, JFK destabilizes p53 through the assembly of a Skp1-Cul1-F-box complex. Here, we report that the substrate recognition by JFK requires phosphorylation of p53 in its central core region by CSN (COP9 signalosome)-associated kinase. Significantly, inhibition of CSN-associated kinase activity or knockdown of CSN5 impairs JFK-promoted p53 degradation, enhances p53-dependent transcription, and promotes cell growth suppression, G1 arrest, and apoptosis. Moreover, we showed that JFK is transcriptionally regulated by p53 and forms an auto-regulatory negative feedback loop with p53. These data may shed new light on the functional connection between CSN, Skp1-Cul1-F-box ubiquitin ligase, and p53 and provide a molecular mechanism for the regulation of JFK-promoted p53 degradation. PMID:21127074

Forskolin stimulation promotes urea transporter UT-A1 ubiquitination, endocytosis, and degradation in MDCK cells

PubMed Central

Su, Hua; Carter, Conner B.; Laur, Oskar; Sands, Jeff M.

2012-01-01

The adenylyl cyclase stimulator forskolin (FSK) stimulates UT-A1 phosphorylation, membrane trafficking, and urea transport activity. Here, we found that FSK stimulation induces UT-A1 ubiquitination in UT-A1 Madin-Darby canine kidney (MDCK) cells. This suggests that phosphorylation by FSK also triggers the protein degradation machinery for UT-A1. UT-A1-MDCK cells were treated with 100 μg/ml cycloheximide to inhibit protein synthesis, with or without 10 μM FSK. Total UT-A1 protein abundance was significantly reduced after FSK treatment, concomitantly ubiquitinated UT-A1 was increased. We then specifically investigated the effect of FSK on UT-A1 expressed on the cell plasma membrane. FSK treatment accelerated UT-A1 removal from the cell plasma membrane by increasing UT-A1 endocytosis as judged by biotinylation/MesNa treatment and confocal microscopy. We further found that inhibition of the clathrin-mediated endocytic pathway, but not the caveolin-mediated endocytic pathway, significantly blocks FSK-stimulated UT-A1 endocytosis. The PKA inhibitor H89 and the proteasome inhibitors MG132 and lactacystin reduced FSK-induced membrane UT-A1 reduction. Our study shows that FSK activates the UT-A1 urea transporter and the activation/phosphorylation subsequently triggers the downregulation of UT-A1, which represents an important mechanism for the cell to return to the basal conditions after vasopressin stimulation. PMID:22914781

The pineal gland: A model for adrenergic modulation of ubiquitin ligases.

PubMed

Vriend, Jerry; Liu, Wenjun; Reiter, Russel J

2017-01-01

A recent study of the pineal gland of the rat found that the expression of more than 3000 genes showed significant day/night variations (The Hartley dataset). The investigators of this report made available a supplemental table in which they tabulated the expression of many genes that they did not discuss, including those coding for components of the ubiquitin proteasome system. Herein we identify the genes of the ubiquitin proteasome system whose expression were significantly influenced by environmental lighting in the Hartley dataset, those that were stimulated by DBcAMP in pineal glands in culture, and those that were stimulated by norepinephrine. Using the Ubiquitin and Ubiquitin-like Conjugation Database (UUCA) we identified ubiquitin ligases and conjugases, and deubiquitinases in the Hartley dataset for the purpose of determining whether expression of genes of the ubiquitin proteasome pathway were significantly influenced by day/night variations and if these variations were regulated by autonomic innervation of the pineal gland from the superior cervical ganglia. In the Hartley experiments pineal glands groups of rats sacrificed during the day and groups sacrificed during the night were examined for gene expression. Additional groups of rats had their superior cervical ganglia removed surgically or surgically decentralized and the pineal glands likewise examined for gene expression. The genes with at least a 2-fold day/night significant difference in expression included genes for 5 ubiquitin conjugating enzymes, genes for 58 ubiquitin E3 ligases and genes for 6 deubiquitinases. A 35-fold day/night difference was noted in the expression of the gene Sik1, which codes for a protein containing both an ubiquitin binding domain (UBD) and an ubiquitin-associated (UBA) domain. Most of the significant differences in these genes were prevented by surgical removal, or disconnection, of the superior cervical ganglia, and most were responsive, in vitro, to treatment with

The pineal gland: A model for adrenergic modulation of ubiquitin ligases

PubMed Central

Liu, Wenjun; Reiter, Russel J.

2017-01-01

Introduction A recent study of the pineal gland of the rat found that the expression of more than 3000 genes showed significant day/night variations (The Hartley dataset). The investigators of this report made available a supplemental table in which they tabulated the expression of many genes that they did not discuss, including those coding for components of the ubiquitin proteasome system. Herein we identify the genes of the ubiquitin proteasome system whose expression were significantly influenced by environmental lighting in the Hartley dataset, those that were stimulated by DBcAMP in pineal glands in culture, and those that were stimulated by norepinephrine. Purpose Using the Ubiquitin and Ubiquitin-like Conjugation Database (UUCA) we identified ubiquitin ligases and conjugases, and deubiquitinases in the Hartley dataset for the purpose of determining whether expression of genes of the ubiquitin proteasome pathway were significantly influenced by day/night variations and if these variations were regulated by autonomic innervation of the pineal gland from the superior cervical ganglia. Methods In the Hartley experiments pineal glands groups of rats sacrificed during the day and groups sacrificed during the night were examined for gene expression. Additional groups of rats had their superior cervical ganglia removed surgically or surgically decentralized and the pineal glands likewise examined for gene expression. Results The genes with at least a 2-fold day/night significant difference in expression included genes for 5 ubiquitin conjugating enzymes, genes for 58 ubiquitin E3 ligases and genes for 6 deubiquitinases. A 35-fold day/night difference was noted in the expression of the gene Sik1, which codes for a protein containing both an ubiquitin binding domain (UBD) and an ubiquitin-associated (UBA) domain. Most of the significant differences in these genes were prevented by surgical removal, or disconnection, of the superior cervical ganglia, and most were

Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65

PubMed Central

Kazlauskaite, Agne; Kondapalli, Chandana; Gourlay, Robert; Campbell, David G.; Ritorto, Maria Stella; Hofmann, Kay; Alessi, Dario R.; Knebel, Axel; Trost, Matthias; Muqit, Miratul M. K.

2014-01-01

We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser65 within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser65 that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser65 in ubiquitin lies in a similar motif to Ser65 in the Ubl domain of Parkin. Remarkably, PINK1 directly phosphorylates Ser65 of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser65-phosphorylated ubiquitin (ubiquitinPhospho−Ser65) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser65 (ΔUbl-Parkin) is robustly activated by ubiquitinPhospho−Ser65, but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser65 (UblPhospho−Ser65) can also activate ΔUbl-Parkin similarly to ubiquitinPhospho−Ser65. Thirdly, we establish that ubiquitinPhospho−Ser65, but not non-phosphorylated ubiquitin or UblPhospho−Ser65, activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser65 and ubiquitin at Ser65, since only mutation of both proteins at Ser65 completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser65, but also by phosphorylating ubiquitin at Ser65

The ubiquitin ligase Siah2 regulates obesity-induced adipose tissue inflammation.

PubMed

Kilroy, Gail; Carter, Lauren E; Newman, Susan; Burk, David H; Manuel, Justin; Möller, Andreas; Bowtell, David D; Mynatt, Randall L; Ghosh, Sujoy; Floyd, Z Elizabeth

2015-11-01

Chronic, low-grade adipose tissue inflammation associated with adipocyte hypertrophy is an important link in the relationship between obesity and insulin resistance. Although ubiquitin ligases regulate inflammatory processes, the role of these enzymes in metabolically driven adipose tissue inflammation is relatively unexplored. Herein, the effect of the ubiquitin ligase Siah2 on obesity-related adipose tissue inflammation was examined. Wild-type and Siah2KO mice were fed a low- or high-fat diet for 16 weeks. Indirect calorimetry, body composition, and glucose and insulin tolerance were assayed along with glucose and insulin levels. Gene and protein expression, immunohistochemistry, adipocyte size distribution, and lipolysis were also analyzed. Enlarged adipocytes in obese Siah2KO mice were not associated with obesity-induced insulin resistance. Proinflammatory gene expression, stress kinase signaling, fibrosis, and crown-like structures were reduced in the Siah2KO adipose tissue, and Siah2KO adipocytes were more responsive to insulin-dependent inhibition of lipolysis. Loss of Siah2 increased expression of PPARγ target genes involved in lipid metabolism and decreased expression of proinflammatory adipokines regulated by PPARγ. Siah2 links adipocyte hypertrophy with adipocyte dysfunction and recruitment of proinflammatory immune cells to adipose tissue. Selective regulation of PPARγ activity is a Siah2-mediated mechanism contributing to obesity-induced adipose tissue inflammation. © 2015 The Obesity Society.

Galectin-3 Inhibits Galectin-8/Parkin-Mediated Ubiquitination of Group A Streptococcus.

PubMed

Cheng, Yi-Lin; Wu, Yan-Wei; Kuo, Chih-Feng; Lu, Shiou-Ling; Liu, Fu-Tong; Anderson, Robert; Lin, Chiou-Feng; Liu, Yi-Ling; Wang, Wan-Yu; Chen, Ying-Da; Zheng, Po-Xing; Wu, Jiunn-Jong; Lin, Yee-Shin

2017-07-25

Group A streptococcus (GAS) is an important human pathogen that causes a wide variety of cutaneous and systemic infections. Although originally thought to be an extracellular bacterium, numerous studies have demonstrated that GAS can trigger internalization into nonimmune cells to escape from immune surveillance or antibiotic-mediated killing. Epithelial cells possess a defense mechanism involving autophagy-mediated targeting and killing of GAS within lysosome-fused autophagosomes. In endothelial cells, in contrast, we previously showed that autophagy is not sufficient for GAS killing. In the present study, we showed higher galectin-3 (Gal-3) expression and lower Gal-8 expression in endothelial cells than in epithelial cells. The recruitment of Gal-3 to GAS is higher and the recruitment of Gal-8 to GAS is lower in endothelial cells than in epithelial cells. We further showed that Gal-3 promotes GAS replication and diminishes the recruitment of Gal-8 and ubiquitin, the latter of which is a critical protein for autophagy sequestration. After knockdown of Gal-3 in endothelial cells, the colocalization of Gal-8, parkin, and ubiquitin-decorated GAS is significantly increased, as is the interaction of Gal-8 and parkin, an E3 ligase. Furthermore, inhibition of Gal-8 in epithelial cells attenuates recruitment of parkin; both Gal-8 and parkin contribute to ubiquitin recruitment and GAS elimination. Animal studies confirmed that Gal-3-knockout mice develop less-severe skin damage and that GAS replication can be detected only in the air pouch and not in organs and endothelial cells. These results demonstrate that Gal-3 inhibits ubiquitin recruitment by blocking Gal-8 and parkin recruitment, resulting in GAS replication in endothelial cells. IMPORTANCE In epithelial cells, GAS can be efficiently killed within the lysosome-fused autophaosome compartment. However, we previously showed that, in spite of LC-3 recruitment, the autophagic machinery is not sufficient for GAS killing

Ubiquitin and Parkinson's disease through the looking glass of genetics.

PubMed

Walden, Helen; Muqit, Miratul M K

2017-04-13

Biochemical alterations found in the brains of Parkinson's disease (PD) patients indicate that cellular stress is a major driver of dopaminergic neuronal loss. Oxidative stress, mitochondrial dysfunction, and ER stress lead to impairment of the homeostatic regulation of protein quality control pathways with a consequent increase in protein misfolding and aggregation and failure of the protein degradation machinery. Ubiquitin signalling plays a central role in protein quality control; however, prior to genetic advances, the detailed mechanisms of how impairment in the ubiquitin system was linked to PD remained mysterious. The discovery of mutations in the α-synuclein gene, which encodes the main protein misfolded in PD aggregates, together with mutations in genes encoding ubiquitin regulatory molecules, including PTEN-induced kinase 1 (PINK1), Parkin, and FBX07, has provided an opportunity to dissect out the molecular basis of ubiquitin signalling disruption in PD, and this knowledge will be critical for developing novel therapeutic strategies in PD that target the ubiquitin system. © 2017 The Author(s).

Regulation of the Hippo signaling pathway by ubiquitin modification.

PubMed

Kim, Youngeun; Jho, Eek-Hoon

2018-03-01

The Hippo signaling pathway plays an essential role in adult tissue homeostasis and organ size control. Abnormal regulation of Hippo signaling can be a cause for multiple types of human cancers. Since the awareness of the importance of the Hippo signaling in a wide range of biological fields has been continually grown, it is also understood that a thorough and well-rounded comprehension of the precise dynamics could provide fundamental insights for therapeutic applications. Several components in the Hippo signaling pathway are known to be targeted for proteasomal degradation via ubiquitination by E3 ligases. β-TrCP is a well-known E3 ligase of YAP/TAZ, which leads to the reduction of YAP/TAZ levels. The Hippo signaling pathway can also be inhibited by the E3 ligases (such as ITCH) which target LATS1/2 for degradation. Regulation via ubiquitination involves not only complex network of E3 ligases but also deubiquitinating enzymes (DUBs), which remove ubiquitin from its targets. Interestingly, non-degradative ubiquitin modifications are also known to play important roles in the regulation of Hippo signaling. Although there has been much advanced progress in the investigation of ubiquitin modifications acting as regulators of the Hippo signaling pathway, research done to date still remains inadequate due to the sheer complexity and diversity of the subject. Herein, we review and discuss recent developments that implicate ubiquitin-mediated regulatory mechanisms at multiple steps of the Hippo signaling pathway. [BMB Reports 2018; 51(3): 143-150].

The effect of acetaminophen on ubiquitin homeostasis in Saccharomyces cerevisiae

PubMed Central

Huseinovic, Angelina; van Leeuwen, Jolanda S.; van Welsem, Tibor; Stulemeijer, Iris; van Leeuwen, Fred; Vermeulen, Nico P. E.; Kooter, Jan M.; Vos, J. Chris

2017-01-01

Acetaminophen (APAP), although considered a safe drug, is one of the major causes of acute liver failure by overdose, and therapeutic chronic use can cause serious health problems. Although the reactive APAP metabolite N-acetyl-p-benzoquinoneimine (NAPQI) is clearly linked to liver toxicity, toxicity of APAP is also found without drug metabolism of APAP to NAPQI. To get more insight into mechanisms of APAP toxicity, a genome-wide screen in Saccharomyces cerevisiae for APAP-resistant deletion strains was performed. In this screen we identified genes related to the DNA damage response. Next, we investigated the link between genotype and APAP-induced toxicity or resistance by performing a more detailed screen with a library containing mutants of 1522 genes related to nuclear processes, like DNA repair and chromatin remodelling. We identified 233 strains that had an altered growth rate relative to wild type, of which 107 showed increased resistance to APAP and 126 showed increased sensitivity. Gene Ontology analysis identified ubiquitin homeostasis, regulation of transcription of RNA polymerase II genes, and the mitochondria-to-nucleus signalling pathway to be associated with APAP resistance, while histone exchange and modification, and vesicular transport were connected to APAP sensitivity. Indeed, we observed a link between ubiquitin levels and APAP resistance, whereby ubiquitin deficiency conferred resistance to APAP toxicity while ubiquitin overexpression resulted in sensitivity. The toxicity profile of various chemicals, APAP, and its positional isomer AMAP on a series of deletion strains with ubiquitin deficiency showed a unique resistance pattern for APAP. Furthermore, exposure to APAP increased the level of free ubiquitin and influenced the ubiquitination of proteins. Together, these results uncover a role for ubiquitin homeostasis in APAP-induced toxicity. PMID:28291796

Quantitation and immunocytochemical localization of ubiquitin conjugates within rat red and white skeletal muscles

NASA Technical Reports Server (NTRS)

Riley, Danny A.; Bain, James L. W.; Haas, Arthur L.; Ellis, Stanley

1988-01-01

Solid-phase immunochemical methods were employed to probe the dynamics of ubiquitin pools within selected rat skeletal muscles. The total ubiquitin content of red muscles was greater than that of white muscles, even though the fractional conjugation was similar for both types of muscles. The specificity for conjugated ubiquitin in solid-phase applications, previously demonstrated for an affinity-purified antibody against SDS-denatured ubiquitin, was retained when used as a probe for ubiquitin-protein adducts in tissue sections. Immunohistochemical localization revealed that differences in ubiquitin pools derived from the relative content of red (oxidative) vs white (glycolytic) fibers, with the former exhibiting a higher content of ubiquitin conjugates. Subsequent immunogold labeling demonstrated statistically significant enhanced localization of ubiquitin conjugates to the Z-lines in both red and white muscle fiber types.

Ubiquitination of exposed glycoproteins by SCFFBXO27 directs damaged lysosomes for autophagy

PubMed Central

Yoshida, Yukiko; Yasuda, Sayaka; Fujita, Toshiharu; Hamasaki, Maho; Murakami, Arisa; Kawawaki, Junko; Iwai, Kazuhiro; Saeki, Yasushi; Yoshimori, Tamotsu; Matsuda, Noriyuki; Tanaka, Keiji

2017-01-01

Ubiquitination functions as a signal to recruit autophagic machinery to damaged organelles and induce their clearance. Here, we report the characterization of FBXO27, a glycoprotein-specific F-box protein that is part of the SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex, and demonstrate that SCFFBXO27 ubiquitinates glycoproteins in damaged lysosomes to regulate autophagic machinery recruitment. Unlike F-box proteins in other SCF complexes, FBXO27 is subject to N-myristoylation, which localizes it to membranes, allowing it to accumulate rapidly around damaged lysosomes. We also screened for proteins that are ubiquitinated upon lysosomal damage, and identified two SNARE proteins, VAMP3 and VAMP7, and five lysosomal proteins, LAMP1, LAMP2, GNS, PSAP, and TMEM192. Ubiquitination of all glycoproteins identified in this screen increased upon FBXO27 overexpression. We found that the lysosomal protein LAMP2, which is ubiquitinated preferentially on lysosomal damage, enhances autophagic machinery recruitment to damaged lysosomes. Thus, we propose that SCFFBXO27 ubiquitinates glycoproteins exposed upon lysosomal damage to induce lysophagy. PMID:28743755

Identification and expression of the protein ubiquitination system in Giardia intestinalis.

PubMed

Gallego, Eva; Alvarado, Magda; Wasserman, Moises

2007-06-01

Giardia intestinalis is a single-cell eukaryotic microorganism, regarded as one of the earliest divergent eukaryotes and thus an attractive model to study the evolution of regulatory systems. Giardia has two different forms throughout its life cycle, cyst and trophozoite, and changes from one to the other in response to environmental signals. The two differentiation processes involve a differential gene expression as well as a quick and specific protein turnover that may be mediated by the ubiquitin/proteasome system. The aim of this work was to search for unreported components of the ubiquitination system and to experimentally demonstrate their expression in the parasite and during the two differentiation processes. We found activity of protein ubiquitination in G. intestinalis trophozoites and analyzed the transcription of the ubiquitin gene, as well as that of the activating (E1), conjugating (E2), and ligase (E3) ubiquitin enzymes during encystation and excystation. A constant ubiquitin expression persisted during the parasite's differentiation processes, whereas variation in transcription was observed in the other genes under study.

The ubiquitin-proteasome pathway an emerging anticancer strategy for therapeutics: a patent analysis.

PubMed

Jain, Chakresh K; Arora, Shivam; Khanna, Aparna; Gupta, Money; Wadhwa, Gulshan; Sharma, Sanjeev K

2015-01-01

The degradation of intracellular proteins is targeted by ubiquitin via non-lysosomal proteolytic pathway in the cell system. These ubiquitin molecules have been found to be conserved from yeast to humans. Ubiquitin proteasome machinery utilises ATP and other mechanisms for degrading proteins of cytosol as well as nucleus. This process of ubiquitination is regulated by activating the E3 enzyme ligase, involved in phosphorylation. In humans, proteins which regulate the cell cycle are controlled by ubiquitin; therefore the ubiquitin-proteasome pathway can be targeted for novel anti-cancer strategies. Dysregulation of the components of the ubiquitin system has been linked to many diseases like cancer and inflammation. The primary triggering mechanism (apoptosis) of these diseases can also be induced when TNF-related apoptosis-inducing ligand (TRAIL) binds to its specific receptor DR4 and DR5. In this review, the emerging prospects and importance of ubiquitin proteasome pathway as an evolving anticancer strategy have been discussed. Current challenges in the field of drug discovery have also been discussed on the basis of recent patents on cancer diagnosis and therapeutics.

Conformational Space and Stability of ETD Charge Reduction Products of Ubiquitin

NASA Astrophysics Data System (ADS)

Lermyte, Frederik; Łącki, Mateusz Krzysztof; Valkenborg, Dirk; Gambin, Anna; Sobott, Frank

2017-01-01

Owing to its versatility, electron transfer dissociation (ETD) has become one of the most commonly utilized fragmentation techniques in both native and non-native top-down mass spectrometry. However, several competing reactions—primarily different forms of charge reduction—occur under ETD conditions, as evidenced by the distorted isotope patterns usually observed. In this work, we analyze these isotope patterns to compare the stability of nondissociative electron transfer (ETnoD) products, specifically noncovalent c/ z fragment complexes, across a range of ubiquitin conformational states. Using ion mobility, we find that more extended states are more prone to fragment release. We obtain evidence that for a given charge state, populations of ubiquitin ions formed either directly by electrospray ionization or through collapse of more extended states upon charge reduction, span a similar range of collision cross-sections. Products of gas-phase collapse are, however, less stabilized towards unfolding than the native conformation, indicating that the ions retain a memory of previous conformational states. Furthermore, this collapse of charge-reduced ions is promoted if the ions are `preheated' using collisional activation, with possible implications for the kinetics of gas-phase compaction.

Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress.

PubMed

Han, Ziying; Sagum, Cari A; Takizawa, Fumio; Ruthel, Gordon; Berry, Corbett T; Kong, Jing; Sunyer, J Oriol; Freedman, Bruce D; Bedford, Mark T; Sidhu, Sachdev S; Sudol, Marius; Harty, Ronald N

2017-10-15

virus (EBOV) is a high-priority, emerging human pathogen that can cause severe outbreaks of hemorrhagic fever with high mortality rates. As there are currently no approved vaccines or treatments for EBOV, a better understanding of the biology and functions of EBOV-host interactions that promote or inhibit viral budding is warranted. Here, we describe a physical and functional interaction between EBOV VP40 (eVP40) and WWP1, a host E3 ubiquitin ligase that ubiquitinates VP40 and regulates VLP egress. This viral PPXY-host WW domain-mediated interaction represents a potential new target for host-oriented inhibitors of EBOV egress. Copyright © 2017 American Society for Microbiology.

Ubiquitin-proteasome pathway and cellular responses to oxidative stress

USDA-ARS?s Scientific Manuscript database

The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Subs...

DNA-binding regulates site-specific ubiquitination of IRF-1.

PubMed

Landré, Vivien; Pion, Emmanuelle; Narayan, Vikram; Xirodimas, Dimitris P; Ball, Kathryn L

2013-02-01

Understanding the determinants for site-specific ubiquitination by E3 ligase components of the ubiquitin machinery is proving to be a challenge. In the present study we investigate the role of an E3 ligase docking site (Mf2 domain) in an intrinsically disordered domain of IRF-1 [IFN (interferon) regulatory factor-1], a short-lived IFNγ-regulated transcription factor, in ubiquitination of the protein. Ubiquitin modification of full-length IRF-1 by E3 ligases such as CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and MDM2 (murine double minute 2), which dock to the Mf2 domain, was specific for lysine residues found predominantly in loop structures that extend from the DNA-binding domain, whereas no modification was detected in the more conformationally flexible C-terminal half of the protein. The E3 docking site was not available when IRF-1 was in its DNA-bound conformation and cognate DNA-binding sequences strongly suppressed ubiquitination, highlighting a strict relationship between ligase binding and site-specific modification at residues in the DNA-binding domain. Hyperubiquitination of a non-DNA-binding mutant supports a mechanism where an active DNA-bound pool of IRF-1 is protected from polyubiquitination and degradation.

FANCL ubiquitinates β-catenin and enhances its nuclear function

PubMed Central

Rotelli, Michael D.; Petersen, Curtis L.; Kaech, Stefanie; Nelson, Whitney D.; Yates, Jane E.; Hanlon Newell, Amy E.; Olson, Susan B.; Druker, Brian J.; Bagby, Grover C.

2012-01-01

Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of β-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates β-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, β-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate β-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34+ stem and progenitor cells results in fewer β-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/β-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss. PMID:22653977

FANCL ubiquitinates β-catenin and enhances its nuclear function.

PubMed

Dao, Kim-Hien T; Rotelli, Michael D; Petersen, Curtis L; Kaech, Stefanie; Nelson, Whitney D; Yates, Jane E; Hanlon Newell, Amy E; Olson, Susan B; Druker, Brian J; Bagby, Grover C

2012-07-12

Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of β-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates β-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, β-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate β-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34(+) stem and progenitor cells results in fewer β-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/β-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss.

Altered social behavior and neuronal development in mice lacking the Uba6-Use1 ubiquitin transfer system

PubMed Central

Lee, Peter C. W.; Dodart, Jean-Cosme; Aron, Liviu; Finley, Lydia W.; Bronson, Roderick T.; Haigis, Marcia C.; Yankner, Bruce A.; Harper, J. Wade

2013-01-01

The Uba6 (E1)-Use1 (E2) ubiquitin transfer cascade is a poorly understood alternative arm of the ubiquitin proteasome system (UPS) required for mouse embryonic development, independent of the canonical Uba1-E2-E3 pathway. Loss of neuronal Uba6 during embryonic development results in altered patterning of neurons in the hippocampus and the amygdala, decreased dendritic spine density, and numerous behavioral disorders. The levels of the E3 ubiquitin ligase Ube3a (E6-AP) and Shank3, both linked with dendritic spine function, are elevated in the amygdala of Uba6-deficient mice, while levels of the Ube3a substrate Arc are reduced. Uba6 and Use1 promote proteasomal turnover of Ube3a in mouse embryo fibroblasts (MEFs) and catalyze Ube3a ubiquitylation in vitro. These activities occur in parallel with an independent pathway involving Uba1-UbcH7, but in a spatially distinct manner in MEFs. These data reveal an unanticipated role for Uba6 in neuronal development, spine architecture, mouse behavior, and turnover of Ube3a. PMID:23499007

A20 restricts ubiquitination of pro-interleukin-1β protein complexes and suppresses NLRP3 inflammasome activity

PubMed Central

Duong, Bao H.; Onizawa, Michio; Oses-Prieto, Juan A.; Advincula, Rommel; Burlingame, Alma; Malynn, Barbara A.; Ma, Averil

2015-01-01

SUMMARY Inappropriate inflammasome activation contributes to multiple human diseases, but the mechanisms by which inflammasomes are suppressed are poorly understood. The NFκB inhibitor A20 is a ubiquitin-modifying enzyme that may prevent human inflammatory diseases and lymphomas. Here, we report that A20-deficient macrophages, unlike normal cells, exhibit spontaneous NLRP3 inflammasome activity to LPS alone. The kinase RIPK3, but not the adaptor MyD88, is required for this response. In normal cells, A20 constitutively associates with caspase-1 and pro-IL-1β, and NLRP3 activation further promotes A20 recruitment to the inflammasome. Pro-IL-1β also co-immunoprecipitates with RIPK1, RIPK3, caspase-1 and caspase-8 in a complex that is modified with K63-linked and unanchored polyubiquitin. In A20-deficient macrophages, this pro-IL-1β-associated ubiquitination is markedly increased in a RIPK3-dependent manner. Mass spectrometric and mutational analyses reveal that K133 of pro-IL-1β is a physiological ubiquitination site that supports processing. Our study reveals a novel mechanism by which A20 prevents inflammatory diseases. PMID:25607459

Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains.

PubMed

Harper, Stephen; Gratton, Hayley E; Cornaciu, Irina; Oberer, Monika; Scott, David J; Emsley, Jonas; Dreveny, Ingrid

2014-05-13

The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGFβ signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal "domain present in USPs" (DUSP) and "ubiquitin-like" (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15. A conserved VEVY motif is a signature feature at the two-domain interface that shapes a potential protein interaction site. Small angle X-ray scattering and gel filtration experiments are consistent with the USP11DU domains and full-length USP11 being monomeric. Unexpectedly, we reveal, through kinetic assays of a series of deletion mutants, that the catalytic activity of USP11 is not regulated through intramolecular autoinhibition or activation by the N-terminal DU or UBL domains. Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys(63)-, Lys(6)-, Lys(33)-, and Lys(11)-linked chains over Lys(27)-, Lys(29)-, and Lys(48)-linked and linear chains consistent with USP11's function in DNA repair pathways that is mediated by the protease domain. Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity. This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation.

Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation*♦

PubMed Central

Yamano, Koji; Queliconi, Bruno B.; Koyano, Fumika; Saeki, Yasushi; Hirokawa, Takatsugu; Tanaka, Keiji; Matsuda, Noriyuki

2015-01-01

Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser65 by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity. PMID:26260794

Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit.

PubMed

Chojnacki, Michal; Mansour, Wissam; Hameed, Dharjath S; Singh, Rajesh K; El Oualid, Farid; Rosenzweig, Rina; Nakasone, Mark A; Yu, Zanlin; Glaser, Fabian; Kay, Lewis E; Fushman, David; Ovaa, Huib; Glickman, Michael H

2017-04-20

Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The best documented Ub signaling pathway is the one that delivers polyUb proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (Ub PT ), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb conjugates. Our work demonstrates the value of Ub PT , and we expect that its future uses will help define and investigate the ubiquitin interactome. Copyright © 2017 Elsevier Ltd. All rights reserved.

Activation of the Yeast UBI4 Polyubiquitin Gene by Zap1 Transcription Factor via an Intragenic Promoter Is Critical for Zinc-deficient Growth*

PubMed Central

MacDiarmid, Colin W.; Taggart, Janet; Jeong, Jeeyon; Kerdsomboon, Kittikhun; Eide, David J.

2016-01-01

Stability of many proteins requires zinc. Zinc deficiency disrupts their folding, and the ubiquitin-proteasome system may help manage this stress. In Saccharomyces cerevisiae, UBI4 encodes five tandem ubiquitin monomers and is essential for growth in zinc-deficient conditions. Although UBI4 is only one of four ubiquitin-encoding genes in the genome, a dramatic decrease in ubiquitin was observed in zinc-deficient ubi4Δ cells. The three other ubiquitin genes were strongly repressed under these conditions, contributing to the decline in ubiquitin. In a screen for ubi4Δ suppressors, a hypomorphic allele of the RPT2 proteasome regulatory subunit gene (rpt2E301K) suppressed the ubi4Δ growth defect. The rpt2E301K mutation also increased ubiquitin accumulation in zinc-deficient cells, and by using a ubiquitin-independent proteasome substrate we found that proteasome activity was reduced. These results suggested that increased ubiquitin supply in suppressed ubi4Δ cells was a consequence of more efficient ubiquitin release and recycling during proteasome degradation. Degradation of a ubiquitin-dependent substrate was restored by the rpt2E301K mutation, indicating that ubiquitination is rate-limiting in this process. The UBI4 gene was induced ∼5-fold in low zinc and is regulated by the zinc-responsive Zap1 transcription factor. Surprisingly, Zap1 controls UBI4 by inducing transcription from an intragenic promoter, and the resulting truncated mRNA encodes only two of the five ubiquitin repeats. Expression of a short transcript alone complemented the ubi4Δ mutation, indicating that it is efficiently translated. Loss of Zap1-dependent UBI4 expression caused a growth defect in zinc-deficient conditions. Thus, the intragenic UBI4 promoter is critical to preventing ubiquitin deficiency in zinc-deficient cells. PMID:27432887

Solution structure of lysine-free (K0) ubiquitin

PubMed Central

Huang, Tao; Li, Jess; Byrd, R Andrew

2014-01-01

Lysine-free ubiquitin (K0-Ub) is commonly used to study the ubiquitin-signaling pathway, where it is assumed to have the same structure and function as wild-type ubiquitin (wt-Ub). However, the K0-Ub 15N heteronuclear single quantum correlation NMR spectrum differs significantly from wt-Ub and the melting temperature is depressed by 19°C, raising the question of the structural integrity and equivalence to wt-Ub. The three-dimensional structure of K0-Ub was determined by solution NMR, using chemical shift and residual dipolar coupling data. K0-Ub adopts the same backbone structure as wt-Ub, and all significant chemical shifts can be related to interactions impacted by the K to R mutations. PMID:24591328

Autographa californica Nucleopolyhedrovirus AC141 (Exon0), a Potential E3 Ubiquitin Ligase, Interacts with Viral Ubiquitin and AC66 To Facilitate Nucleocapsid Egress.

PubMed

Biswas, Siddhartha; Willis, Leslie G; Fang, Minggang; Nie, Yingchao; Theilmann, David A

2018-02-01

During the infection cycle of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), two forms of virions are produced, budded virus (BV) and occlusion-derived virus (ODV). Nucleocapsids that form BV have to egress from the nucleus, whereas nucleocapsids that form ODV remain inside the nucleus. The molecular mechanism that determines whether nucleocapsids remain inside or egress from the nucleus is unknown. AC141 (a predicted E3 ubiquitin ligase) and viral ubiquitin (vUbi) have both been shown to be required for efficient BV production. In this study, it was hypothesized that vUbi interacts with AC141, and in addition, that this interaction was required for BV production. Deletion of both ac141 and vubi restricted viral infection to a single cell, and BV production was completely eliminated. AC141 was ubiquitinated by either vUbi or cellular Ubi, and this interaction was required for optimal BV production. Nucleocapsids in BV, but not ODV, were shown to be specifically ubiquitinated by vUbi, including a 100-kDa protein, as well as high-molecular-weight conjugates. The viral ubiquitinated 100-kDa BV-specific nucleocapsid protein was identified as AC66, which is known to be required for BV production and was shown by coimmunoprecipitation and mass spectrometry to interact with AC141. Confocal microscopy also showed that AC141, AC66, and vUbi interact at the nuclear periphery. These results suggest that ubiquitination of nucleocapsid proteins by vUbi functions as a signal to determine if a nucleocapsid will egress from the nucleus and form BV or remain in the nucleus to form ODV. IMPORTANCE Baculoviruses produce two types of virions called occlusion-derived virus (ODV) and budded virus (BV). ODVs are required for oral infection, whereas BV enables the systemic spread of virus to all host tissues, which is critical for killing insects. One of the important steps for BV production is the export of nucleocapsids out of the nucleus. This study investigated the

USP19-Mediated Deubiquitination Facilitates the Stabilization of HRD1 Ubiquitin Ligase.

PubMed

Harada, Kumi; Kato, Masako; Nakamura, Nobuhiro

2016-11-02

In the endoplasmic reticulum (ER), misfolded and unfolded proteins are eliminated by a process called ER-associated protein degradation (ERAD) in order to maintain cell homeostasis. In the ERAD pathway, several ER-localized E3 ubiquitin ligases target ERAD substrate proteins for ubiquitination and subsequent proteasomal degradation. However, little is known about how the functions of the ERAD ubiquitin ligases are regulated. Recently, USP19, an ER-anchored deubiquitinating enzyme (DUB), has been suggested to be involved in the regulation of ERAD. In this study, HRD1, an ERAD ubiquitin ligase, is shown to be a novel substrate for USP19. We demonstrate that USP19 rescues HRD1 from proteasomal degradation by deubiquitination of K48-linked ubiquitin chains. In addition, the altered expression of USP19 affects the steady-state levels of HRD1. These results suggest that USP19 regulates the stability of HRD1 and provide insight into the regulatory mechanism of the ERAD ubiquitin ligases.

Natural products inhibiting the ubiquitin-proteasome proteolytic pathway, a target for drug development.

PubMed

Tsukamoto, Sachiko; Yokosawa, Hideyoshi

2006-01-01

The ubiquitin-proteasome proteolytic pathway plays a major role in selective protein degradation and regulates various cellular events including cell cycle progression, transcription, DNA repair, signal transduction, and immune response. Ubiquitin, a highly conserved small protein in eukaryotes, attaches to a target protein prior to degradation. The polyubiquitin chain tagged to the target protein is recognized by the 26S proteasome, a high-molecular-mass protease subunit complex, and the protein portion is degraded by the 26S proteasome. The potential of specific proteasome inhibitors, which act as anti-cancer agents, is now under intensive investigation, and bortezomib (PS-341), a proteasome inhibitor, has been recently approved by FDA for multiple myeloma treatment. Since ubiquitination of proteins requires the sequential action of three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3), and polyubiquitination is a prerequisite for proteasome-mediated protein degradation, inhibitors of E1, E2, and E3 are reasonably thought to be drug candidates for treatment of diseases related to ubiquitination. Recently, various compounds inhibiting the ubiquitin-proteasome pathway have been isolated from natural resources. We also succeeded in isolating inhibitors against the proteasome and E1 enzyme from marine natural resources. In this review, we summarize the structures and biological activities of natural products that inhibit the ubiquitin-proteasome proteolytic pathway.

Structural basis for ubiquitin-mediated antiviral signal activation by RIG-I.

PubMed

Peisley, Alys; Wu, Bin; Xu, Hui; Chen, Zhijian J; Hur, Sun

2014-05-01

Ubiquitin (Ub) has important roles in a wide range of intracellular signalling pathways. In the conventional view, ubiquitin alters the signalling activity of the target protein through covalent modification, but accumulating evidence points to the emerging role of non-covalent interaction between ubiquitin and the target. In the innate immune signalling pathway of a viral RNA sensor, RIG-I, both covalent and non-covalent interactions with K63-linked ubiquitin chains (K63-Ubn) were shown to occur in its signalling domain, a tandem caspase activation and recruitment domain (hereafter referred to as 2CARD). Non-covalent binding of K63-Ubn to 2CARD induces its tetramer formation, a requirement for downstream signal activation. Here we report the crystal structure of the tetramer of human RIG-I 2CARD bound by three chains of K63-Ub2. 2CARD assembles into a helical tetramer resembling a 'lock-washer', in which the tetrameric surface serves as a signalling platform for recruitment and activation of the downstream signalling molecule, MAVS. Ubiquitin chains are bound along the outer rim of the helical trajectory, bridging adjacent subunits of 2CARD and stabilizing the 2CARD tetramer. The combination of structural and functional analyses reveals that binding avidity dictates the K63-linkage and chain-length specificity of 2CARD, and that covalent ubiquitin conjugation of 2CARD further stabilizes the Ub-2CARD interaction and thus the 2CARD tetramer. Our work provides unique insights into the novel types of ubiquitin-mediated signal-activation mechanism, and previously unexpected synergism between the covalent and non-covalent ubiquitin interaction modes.

CacyBP/SIP promotes the proliferation of colon cancer cells

PubMed Central

Chen, Xiong; Wang, Jun; Lu, Yuanyuan; Zhang, Faming; Liu, Zhengxiong; Lei, Ting; Fan, Daiming

2017-01-01

CacyBP/SIP is a component of the ubiquitin pathway and is overexpressed in several transformed tumor tissues, including colon cancer, which is one of the most common cancers worldwide. It is unknown whether CacyBP/SIP promotes the proliferation of colon cancer cells. This study examined the expression level, subcellular localization, and binding activity of CacyBP/SIP in human colon cancer cells in the presence and absence of the hormone gastrin. We found that CacyBP/SIP was expressed in a high percentage of colon cancer cells, but not in normal colonic surface epithelium. CacyBP/SIP promoted the cell proliferation of colon cancer cells under both basal and gastrin stimulated conditions as shown by knockdown studies. Gastrin stimulation triggered the translocation of CacyBP/SIP to the nucleus, and enhanced interaction between CacyBP/SIP and SKP1, a key component of ubiquitination pathway which further mediated the proteasome-dependent degradation of p27kip1 protein. The gastrin induced reduction in p27kip1 was prevented when cells were treated with the proteasome inhibitor MG132. These results suggest that CacyBP/SIP may be promoting growth of colon cancer cells by enhancing ubiquitin-mediated degradation of p27kip1. PMID:28196083

Characterization and identification of ubiquitin conjugation sites with E3 ligase recognition specificities.

PubMed

Nguyen, Van-Nui; Huang, Kai-Yao; Huang, Chien-Hsun; Chang, Tzu-Hao; Bretaña, Neil; Lai, K; Weng, Julia; Lee, Tzong-Yi

2015-01-01

In eukaryotes, ubiquitin-conjugation is an important mechanism underlying proteasome-mediated degradation of proteins, and as such, plays an essential role in the regulation of many cellular processes. In the ubiquitin-proteasome pathway, E3 ligases play important roles by recognizing a specific protein substrate and catalyzing the attachment of ubiquitin to a lysine (K) residue. As more and more experimental data on ubiquitin conjugation sites become available, it becomes possible to develop prediction models that can be scaled to big data. However, no development that focuses on the investigation of ubiquitinated substrate specificities has existed. Herein, we present an approach that exploits an iteratively statistical method to identify ubiquitin conjugation sites with substrate site specificities. In this investigation, totally 6259 experimentally validated ubiquitinated proteins were obtained from dbPTM. After having filtered out homologous fragments with 40% sequence identity, the training data set contained 2658 ubiquitination sites (positive data) and 5532 non-ubiquitinated sites (negative data). Due to the difficulty in characterizing the substrate site specificities of E3 ligases by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. The profile hidden Markov model (profile HMM) was adopted to construct the predictive models learned from the identified substrate motifs. A five-fold cross validation was then used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 73.07%, 65.46%, and 67.93%, respectively. Additionally, an independent testing set, completely blind to the training data of the predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (76.13%) and outperform other ubiquitination site prediction tool. A case study demonstrated the effectiveness of the characterized substrate motifs for

Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

PubMed Central

Xu, Daichao; Shan, Bing; Lee, Byung-Hoon; Zhu, Kezhou; Zhang, Tao; Sun, Huawang; Liu, Min; Shi, Linyu; Liang, Wei; Qian, Lihui; Xiao, Juan; Wang, Lili; Pan, Lifeng; Finley, Daniel; Yuan, Junying

2015-01-01

Regulation of ubiquitin-proteasome system (UPS), which controls the turnover of short-lived proteins in eukaryotic cells, is critical in maintaining cellular proteostasis. Here we show that USP14, a major deubiquitinating enzyme that regulates the UPS, is a substrate of Akt, a serine/threonine-specific protein kinase critical in mediating intracellular signaling transducer for growth factors. We report that Akt-mediated phosphorylation of USP14 at Ser432, which normally blocks its catalytic site in the inactive conformation, activates its deubiquitinating activity in vitro and in cells. We also demonstrate that phosphorylation of USP14 is critical for Akt to regulate proteasome activity and consequently global protein degradation. Since Akt can be activated by a wide range of growth factors and is under negative control by phosphoinosotide phosphatase PTEN, we suggest that regulation of UPS by Akt-mediated phosphorylation of USP14 may provide a common mechanism for growth factors to control global proteostasis and for promoting tumorigenesis in PTEN-negative cancer cells. DOI: http://dx.doi.org/10.7554/eLife.10510.001 PMID:26523394

Newly translated proteins are substrates for ubiquitin, ISG15, and FAT10.

PubMed

Spinnenhirn, Valentina; Bitzer, Annegret; Aichem, Annette; Groettrup, Marcus

2017-01-01

The ubiquitin-like modifier, FAT10, is involved in proteasomal degradation and antigen processing. As ubiquitin and the ubiquitin-like modifier, ISG15, cotranslationally modify proteins, we investigated whether FAT10 could also be conjugated to newly synthesized proteins. Indeed, we found that nascent proteins are modified with FAT10, but not with the same preference for newly synthesized proteins as observed for ISG15. Our data show that puromycin-labeled polypeptides are strongly modified by ISG15 and less intensely by ubiquitin and FAT10. Nevertheless, conjugates of all three modifiers copurify with ribosomes. Taken together, we show that unlike ISG15, ubiquitin and FAT10 are conjugated to a similar degree to newly translated and pre-existing proteins. © 2016 Federation of European Biochemical Societies.

Distinct Ubiquitin Binding Modes Exhibited by SH3 Domains: Molecular Determinants and Functional Implications

PubMed Central

Ortega Roldan, Jose L.; Casares, Salvador; Ringkjøbing Jensen, Malene; Cárdenes, Nayra; Bravo, Jerónimo; Blackledge, Martin; Azuaga, Ana I.; van Nuland, Nico A. J.

2013-01-01

SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination. PMID:24039852

Subunit-Specific Labeling of Ubiquitin Chains by Using Sortase: Insights into the Selectivity of Deubiquitinases.

PubMed

Crowe, Sean O; Pham, Grace H; Ziegler, Jacob C; Deol, Kirandeep K; Guenette, Robert G; Ge, Ying; Strieter, Eric R

2016-08-17

Information embedded in different ubiquitin chains is transduced by proteins with ubiquitin-binding domains (UBDs) and erased by a set of hydrolytic enzymes referred to as deubiquitinases (DUBs). Understanding the selectivity of UBDs and DUBs is necessary for decoding the functions of different ubiquitin chains. Critical to these efforts is the access to chemically defined ubiquitin chains bearing site-specific fluorescent labels. One approach toward constructing such molecules involves peptide ligation by sortase (SrtA), a bacterial transpeptidase responsible for covalently attaching cell surface proteins to the cell wall. Here, we demonstrate the utility of SrtA in modifying individual subunits of ubiquitin chains. Using ubiquitin derivatives in which an N-terminal glycine is unveiled after protease-mediated digestion, we synthesized ubiquitin dimers, trimers, and tetramers with different isopeptide linkages. SrtA was then used in combination with fluorescent depsipeptide substrates to effect the modification of each subunit in a chain. By constructing branched ubiquitin chains with individual subunits tagged with a fluorophore, we provide evidence that the ubiquitin-specific protease USP15 prefers ubiquitin trimers but has little preference for a particular isopeptide linkage. Our results emphasize the importance of subunit-specific labeling of ubiquitin chains when studying how DUBs process these chains. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional assessment of ubiquitin-depended processes under microgravity conditions

NASA Astrophysics Data System (ADS)

Zhabereva, Anastasia; Shenkman, Boris S.; Gainullin, Murat; Gurev, Eugeny; Kondratieva, Ekaterina; Kopylov, Arthur

Ubiquitylation, a widespread and important posttranslational modification of eukaryotic proteins, controls a multitude of critical cellular processes, both in normal and pathological conditions. The present work aims to study involvement of ubiquitin-dependent regulation in adaptive response to the external stimuli. Experiments were carried out on C57BL/6 mice. The microgravity state under conditions of real spaceflight on the biosatellite “BION-M1” was used as a model of stress impact. Additionally, number of control series including the vivarium control and experiments in Ground-based analog were also studied. The aggregate of endogenously ubiquitylated proteins was selected as specific feature of ubiquitin-dependent processes. Dynamic changes of modification pattern were characterized in liver tissue by combination of some methods, particularly by specific isolation of explicit protein pool, followed by immunodetection and/or mass spectrometry-based identification. The main approach includes specific extraction of proteins, modified by multiubiquitin chains of different length and topology. For this purpose two techniques were applied: 1) immunoprecipitation with antibodies against ubiquitin and/or multiubiquitin chains; 2) pull-down using synthetic protein construct termed Tandem Ubiquitin Binding Entities (TUBE, LifeSensors). TUBE represents fusion protein, composed of well characterized ubiquitin-binding domains, and thereby allows specific high-affinity binding and extraction of ubiquitylated proteins. Resulting protein fractions were analyzed by immunoblotting with antibodies against different types of multiubiquitin chains. Using this method we mapped endogenously modified proteins involved in two different types of ubiquitin-dependent processes, namely catabolic and non-catabolic ubiquitylation, in liver tissues, obtained from both control as well as experimental groups of animals, mentioned above. Then, isolated fractions of ubiquitylated proteins

Structural and functional characterization of a ubiquitin variant engineered for tight and specific binding to an alpha-helical ubiquitin interacting motif.

PubMed

Manczyk, Noah; Yates, Bradley P; Veggiani, Gianluca; Ernst, Andreas; Sicheri, Frank; Sidhu, Sachdev S

2017-05-01

Ubiquitin interacting motifs (UIMs) are short α-helices found in a number of eukaryotic proteins. UIMs interact weakly but specifically with ubiquitin conjugated to other proteins, and in so doing, mediate specific cellular signals. Here we used phage display to generate ubiquitin variants (UbVs) targeting the N-terminal UIM of the yeast Vps27 protein. Selections yielded UbV.v27.1, which recognized the cognate UIM with high specificity relative to other yeast UIMs and bound with an affinity more than two orders of magnitude higher than that of ubiquitin. Structural and mutational studies of the UbV.v27.1-UIM complex revealed the molecular details for the enhanced affinity and specificity of UbV.v27.1, and underscored the importance of changes at the binding interface as well as at positions that do not contact the UIM. Our study highlights the power of the phage display approach for selecting UbVs with unprecedented affinity and high selectivity for particular α-helical UIM domains within proteomes, and it establishes a general approach for the development of inhibitors targeting interactions of this type. © 2017 The Protein Society.

Regulation of synaptic structure by ubiquitin C-terminal hydrolase L1.

PubMed

Cartier, Anna E; Djakovic, Stevan N; Salehi, Afshin; Wilson, Scott M; Masliah, Eliezer; Patrick, Gentry N

2009-06-17

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We found that UCH-L1 activity is rapidly upregulated by NMDA receptor activation, which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of presynaptic and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1-inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling, most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner.

Quantitative regulation of histone variant H2A.Z during cell cycle by ubiquitin proteasome system and SUMO-targeted ubiquitin ligases.

PubMed

Takahashi, Daisuke; Orihara, Yuki; Kitagawa, Saho; Kusakabe, Masayuki; Shintani, Takahiro; Oma, Yukako; Harata, Masahiko

2017-08-01

Quantitative control of histones and histone variants during cell cycle is relevant to their epigenetic functions. We found that the level of yeast histone variant H2A.Z in the G2/M-phase is actively kept low by the ubiquitin proteasome system and SUMO-targeted ubiquitin ligases. Overexpression of H2A.Z induced defects in mitotic progression, suggesting functional importance of this quantitative control.

Chemical approaches to targeted protein degradation through modulation of the ubiquitin-proteasome pathway.

PubMed

Collins, Ian; Wang, Hannah; Caldwell, John J; Chopra, Raj

2017-03-15

Manipulation of the ubiquitin-proteasome system to achieve targeted degradation of proteins within cells using chemical tools and drugs has the potential to transform pharmacological and therapeutic approaches in cancer and other diseases. An increased understanding of the molecular mechanism of thalidomide and its analogues following their clinical use has unlocked small-molecule modulation of the substrate specificity of the E3 ligase cereblon (CRBN), which in turn has resulted in the advancement of new immunomodulatory drugs (IMiDs) into the clinic. The degradation of multiple context-specific proteins by these pleiotropic small molecules provides a means to uncover new cell biology and to generate future drug molecules against currently undruggable targets. In parallel, the development of larger bifunctional molecules that bring together highly specific protein targets in complexes with CRBN, von Hippel-Lindau, or other E3 ligases to promote ubiquitin-dependent degradation has progressed to generate selective chemical compounds with potent effects in cells and in vivo models, providing valuable tools for biological target validation and with future potential for therapeutic use. In this review, we survey recent breakthroughs achieved in these two complementary methods and the discovery of new modes of direct and indirect engagement of target proteins with the proteasome. We discuss the experimental characterisation that validates the use of molecules that promote protein degradation as chemical tools, the preclinical and clinical examples disclosed to date, and the future prospects for this exciting area of chemical biology. © 2017 The Author(s).

The ubiquitin conjugating enzyme UbcH7, controls cell migration

USDA-ARS?s Scientific Manuscript database

Post translational modification by ubiquitination can target proteins for degradation, allow the interaction of proteins to form complexes or direct relocalization of proteins to different subcellular compartments. As such, ubiquitin controls a variety of essential cellular processes. Previously we ...

Phosphorylation of ubiquitin at Ser65 affects its polymerization, targets, and proteome-wide turnover

PubMed Central

Swaney, Danielle L; Rodríguez-Mias, Ricard A; Villén, Judit

2015-01-01

Ubiquitylation is an essential post-translational modification that regulates numerous cellular processes, most notably protein degradation. Ubiquitin can itself be phosphorylated at nearly every serine, threonine, and tyrosine residue. However, the effect of this modification on ubiquitin function is largely unknown. Here, we characterized the effects of phosphorylation of yeast ubiquitin at serine 65 in vivo and in vitro. We find this post-translational modification to be regulated under oxidative stress, occurring concomitantly with the restructuring of the ubiquitin landscape into a highly polymeric state. Phosphomimetic mutation of S65 recapitulates the oxidative stress phenotype, causing a dramatic accumulation of ubiquitylated proteins and a proteome-wide reduction of protein turnover rates. Importantly, this mutation impacts ubiquitin chain disassembly, chain linkage distribution, ubiquitin interactions, and substrate targeting. These results demonstrate that phosphorylation is an additional mode of ubiquitin regulation with broad implications in cellular physiology. PMID:26142280

Recognition of Poly-Ubiquitins by the Proteasome through Protein Refolding Guided by Electrostatic and Hydrophobic Interactions.

PubMed

Zhang, Yi; Vuković, Lela; Rudack, Till; Han, Wei; Schulten, Klaus

2016-08-25

Specificity of protein degradation by cellular proteasomes comes from tetra-ubiquitin recognition. We carry out molecular dynamics simulations to characterize how the ubiquitin receptor Rpn10 recognizes in the 26S proteasome K48-linked tetra-ubiquitin. In the binding pose, ubiquitin and Rpn10 interact primarily through hydrophobic patches. However, K48-linked tetra-ubiquitin mostly assumes a closed form in solution prior to binding, in which its hydrophobic patches are not exposed to solvent. Likewise, the hydrophobic ubiquitin interacting motifs (UIMs) of Rpn10 are mostly protected prior to binding. As a result, ubiquitin recognition in the proteasome requires refolding of both K48-linked tetra-ubiquitin and Rpn10. Simulations suggest that conserved complementary electrostatic patterns of Rpn10 and ubiquitins guide protein association (stage 1 in the recognition process), which induces refolding (stage 2), and then facilitates formation of hydrophobic contacts (stage 3). The simulations also explain why Rpn10 has a higher affinity for K48-linked tetra-ubiquitin than for mono-ubiquitin and K48-linked di- and tri-ubiquitins. Simulation results expand on the current view that the flexible arm of Rpn10 acts as an extended fragment of α-helices and flexible coils in the recognition process.

Qualitative ubiquitome unveils the potential significances of protein lysine ubiquitination in hyphal growth of Aspergillus nidulans.

PubMed

Chu, Xin-Ling; Feng, Ming-Guang; Ying, Sheng-Hua

2016-02-01

Protein ubiquitination is an evolutionarily conserved post-translational modification process in eukaryotes, and it plays an important role in many biological processes. Aspergillus nidulans, a model filamentous fungus, contributes to our understanding of cellular physiology, metabolism and genetics, but its ubiquitination is not completely revealed. In this study, the ubiquitination sites in the proteome of A. nidulans were identified using a highly sensitive mass spectrometry combined with immuno-affinity enrichment of the ubiquitinated peptides. The 4816 ubiquitination sites were identified in 1913 ubiquitinated proteins, accounting for 18.1% of total proteins in A. nidulans. Bioinformatic analysis suggested that the ubiquitinated proteins associated with a number of biological functions and displayed various sub-cellular localisations. Meanwhile, seven motifs were revealed from the ubiquitinated peptides, and significantly over-presented in the different pathways. Comparison of the enriched functional catalogues indicated that the ubiquitination functions divergently during growth of A. nidulans and Saccharomyces cerevisiae. Additionally, the proteins in A. nidulans-specific sub-category (cell growth/morphogenesis) were subjected to the protein interaction analysis which demonstrated that ubiquitination is involved in the comprehensive protein interactions. This study presents a first proteomic view of ubiquitination in the filamentous fungus, and provides an initial framework for exploring the physiological roles of ubiquitination in A. nidulans.

Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation

PubMed Central

Wei, Xiaofan; Wang, Xiang; Zhan, Jun; Chen, Yuhan; Fang, Weigang; Zhang, Lingqiang

2017-01-01

Integrin activation is an indispensable step for various integrin-mediated biological functions. Kindlin-2 is known to coactivate integrins with Talin; however, molecules that restrict integrin activation are elusive. Here, we demonstrate that the E3 ubiquitin ligase Smurf1 controls the amount of Kindlin-2 protein in cells and hinders integrin activation. Smurf1 interacts with and promotes Kindlin-2 ubiquitination and degradation. Smurf1 selectively mediates degradation of Kindlin-2 but not Talin, leading to inhibition of αIIbβ3 integrin activation in Chinese hamster ovary cells and β1 integrin activation in fibroblasts. Enhanced activation of β1 integrin was found in Smurf1-knockout mouse embryonic fibroblasts, which correlates with an increase in Kindlin-2 protein levels. Similarly, a reciprocal relationship between Smurf1 and Kindlin-2 protein levels is found in tissues from colon cancer patients, suggesting that Smurf1 mediates Kindlin-2 degradation in vivo. Collectively, we demonstrate that Smurf1 acts as a brake for integrin activation by controlling Kindlin-2 protein levels, a new mechanism that permits precise modulation of integrin-mediated cellular functions. PMID:28408404

Rines E3 ubiquitin ligase regulates MAO-A levels and emotional responses.

PubMed

Kabayama, Miyuki; Sakoori, Kazuto; Yamada, Kazuyuki; Ornthanalai, Veravej G; Ota, Maya; Morimura, Naoko; Katayama, Kei-ichi; Murphy, Niall P; Aruga, Jun

2013-08-07

Monoamine oxidase A (MAO-A), the catabolic enzyme of norepinephrine and serotonin, plays a critical role in emotional and social behavior. However, the control and impact of endogenous MAO-A levels in the brain remains unknown. Here we show that the RING finger-type E3 ubiquitin ligase Rines/RNF180 regulates brain MAO-A subset, monoamine levels, and emotional behavior. Rines interacted with MAO-A and promoted its ubiquitination and degradation. Rines knock-out mice displayed impaired stress responses, enhanced anxiety, and affiliative behavior. Norepinephrine and serotonin levels were altered in the locus ceruleus, prefrontal cortex, and amygdala in either stressed or resting conditions, and MAO-A enzymatic activity was enhanced in the locus ceruleus in Rines knock-out mice. Treatment of Rines knock-out mice with MAO inhibitors showed genotype-specific effects on some of the abnormal affective behaviors. These results indicated that the control of emotional behavior by Rines is partly due to the regulation of MAO-A levels. These findings verify that Rines is a critical regulator of the monoaminergic system and emotional behavior and identify a promising candidate drug target for treating diseases associated with emotion.

The Kinase Activity of Calcineurin B-like Interacting Protein Kinase 26 (CIPK26) Influences Its Own Stability and that of the ABA-regulated Ubiquitin Ligase, Keep on Going (KEG)

PubMed Central

Lyzenga, Wendy J.; Sullivan, Victoria; Liu, Hongxia; Stone, Sophia L.

2017-01-01

The Really Interesting New Gene (RING)-type E3 ligase, Keep on Going (KEG) plays a critical role in Arabidopsis growth after germination and the connections between KEG and hormone signaling pathways are expanding. With regards to abscisic acid (ABA) signaling, KEG targets ABA-responsive transcription factors abscisic acid insensitive 5, ABF1 and ABF3 for ubiquitination and subsequent degradation through the 26S proteasome. Regulation of E3 ligases through self-ubiquitination is common to RING-type E3 ligases and ABA promotes KEG self-ubiquitination and degradation. ABA-mediated degradation of KEG is phosphorylation-dependent; however, upstream signaling proteins that may regulate KEG stability have not been characterized. In this report, we show that CBL-Interacting Protein Kinase (CIPK) 26 can phosphorylate KEG in vitro. Using both in vitro and in planta degradation assays we provide evidence which suggests that the kinase activity of CIPK26 promotes the degradation of KEG. Furthermore, we found that the kinase activity of CIPK26 also influences its own stability; a constitutively active version is more stable than a wild type or a kinase dead version. Our results suggest a reciprocal regulation model wherein an activated and stable CIPK26 phosphorylates KEG to promote degradation of the E3. PMID:28443108

USP5/Leon deubiquitinase confines postsynaptic growth by maintaining ubiquitin homeostasis through Ubiquilin.

PubMed

Wang, Chien-Hsiang; Huang, Yi-Chun; Chen, Pei-Yi; Cheng, Ying-Ju; Kao, Hsiu-Hua; Pi, Haiwei; Chien, Cheng-Ting

2017-05-10

Synapse formation and growth are tightly controlled processes. How synaptic growth is terminated after reaching proper size remains unclear. Here, we show that Leon, the Drosophila USP5 deubiquitinase, controls postsynaptic growth. In leon mutants, postsynaptic specializations of neuromuscular junctions are dramatically expanded, including the subsynaptic reticulum, the postsynaptic density, and the glutamate receptor cluster. Expansion of these postsynaptic features is caused by a disruption of ubiquitin homeostasis with accumulation of free ubiquitin chains and ubiquitinated substrates in the leon mutant. Accumulation of Ubiquilin (Ubqn), the ubiquitin receptor whose human homolog ubiquilin 2 is associated with familial amyotrophic lateral sclerosis, also contributes to defects in postsynaptic growth and ubiquitin homeostasis. Importantly, accumulations of postsynaptic proteins cause different aspects of postsynaptic overgrowth in leon mutants. Thus, the deubiquitinase Leon maintains ubiquitin homeostasis and proper Ubqn levels, preventing postsynaptic proteins from accumulation to confine postsynaptic growth.

Activity‐Based Probes for HECT E3 Ubiquitin Ligases

PubMed Central

Byrne, Robert; Mund, Thomas

2017-01-01

Abstract Activity‐based probes (ABPs) have been used to dissect the biochemical/structural properties and cellular functions of deubiquitinases. However, their utility in studying cysteine‐based E3 ubiquitin ligases has been limited. In this study, we evaluate the use of ubiquitin‐ABPs (Ub‐VME and Ub‐PA) and a novel set of E2–Ub‐ABPs on a panel of HECT E3 ubiquitin ligases. Our in vitro data show that ubiquitin‐ABPs can label HECT domains. We also provide the first evidence that, in addition to the RBR E3 ubiquitin ligase Parkin, E2–Ub‐ABPs can also label the catalytic HECT domains of NEDD4, UBE3C, and HECTD1. Importantly, the endogenous proteasomal E3 ligase UBE3C was also successfully labelled by Ub‐PA and His‐UBE2D2–Ub‐ABP in lysate of cells grown under basal conditions. Our findings provide novel insights into the use of ABPs for the study of HECT E3 ubiquitin ligases. PMID:28425671

Structure of the Ubiquitin Hydrolase UCH-L3 Complexed with a Suicide Substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Misaghi, S.; Galardy, P.J.; Meester, W.J.

Ubiquitin C-terminal hydrolases (UCHs) comprise a family of small ubiquitin-specific proteases of uncertain function. Although no cellular substrates have been identified for UCHs, their highly tissue-specific expression patterns and the association of UCH-L1 mutations with human disease strongly suggest a critical role. The structure of the yeast UCH Yuh1-ubiquitin aldehyde complex identified an active site crossover loop predicted to limit the size of suitable substrates. We report the 1.45 {angstrom} resolution crystal structure of human UCH-L3 in complex with the inhibitor ubiquitin vinylmethylester, an inhibitor that forms a covalent adduct with the active site cysteine of ubiquitin-specific proteases. This structuremore » confirms the predicted mechanism of the inhibitor and allows the direct comparison of a UCH family enzyme in the free and ligand-bound state. We also show the efficient hydrolysis by human UCH-L3 of a 13-residue peptide in isopeptide linkage with ubiquitin, consistent with considerable flexibility in UCH substrate size. We propose a model for the catalytic cycle of UCH family members which accounts for the hydrolysis of larger ubiquitin conjugates.« less

Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

PubMed

Suzuki, Takashi; Ezure, Toru; Ando, Eiji; Nishimura, Osamu; Utsumi, Toshihiko; Tsunasawa, Susumu

2010-01-01

Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

The SCF ubiquitin ligase Slimb controls Nerfin-1 turnover in Drosophila.

PubMed

Lin, Xiaohui; Wang, Feng; Li, Yuanpei; Zhai, Chaojun; Wang, Guiping; Zhang, Xiaoting; Gao, Yang; Yi, Tao; Sun, Dan; Wu, Shian

2018-01-01

The C2H2 type zinc-finger transcription factor Nerfin-1 expresses dominantly in Drosophila nervous system and plays an important role in early axon guidance decisions and preventing neurons dedifferentiation. Recently, increasing reports indicated that INSM1 (homologue to nerfin-1 in mammals) is a useful marker for prognosis of neuroendocrine tumors. The dynamic expression of Nerfin-1 is regulated post-transcriptionally by multiple microRNAs; however, its post-translational regulation is still unclear. Here we showed that the protein turnover of Nerfin-1 is regulated by Slimb, the substrate adaptor of SCF Slimb ubiquitin ligase complex. Mechanistically, Slimb associates with Nerfin-1 and promotes it ubiquitination and degradation in Drosophila S2R + cells. Furthermore, we determined that the C-terminal half of Nerfin-1 (Nerfin-1 CT ) is required for its binding to Slimb. Genetic epistasis assays showed that Slimb misexpression antagonizes, while knock-down enhances the activity of Nerfin-1 CT in Drosophila eyes. Our data revealed a new link to understand the underlying mechanism for Nerfin-1 turnover in post-translational level, and provided useful insights in animal development and disease treatment by manipulating the activity of Slimb and Nerfin-1. Copyright © 2017 Elsevier Inc. All rights reserved.

Posttranslational Modification of HOIP Blocks Toll-Like Receptor 4-Mediated Linear-Ubiquitin-Chain Formation

PubMed Central

Bowman, James; Rodgers, Mary A.; Shi, Mude; Amatya, Rina; Hostager, Bruce; Iwai, Kazuhiro; Gao, Shou-Jiang

2015-01-01

ABSTRACT Linear ubiquitination is an atypical posttranslational modification catalyzed by the linear-ubiquitin-chain assembly complex (LUBAC), containing HOIP, HOIL-1L, and Sharpin. LUBAC facilitates NF-κB activation and inflammation upon receptor stimulation by ligating linear ubiquitin chains to critical signaling molecules. Indeed, linear-ubiquitination-dependent signaling is essential to prevent pyogenic bacterial infections that can lead to death. While linear ubiquitination is essential for intracellular receptor signaling upon microbial infection, this response must be measured and stopped to avoid tissue damage and autoimmunity. While LUBAC is activated upon bacterial stimulation, the mechanisms regulating LUBAC activity in response to bacterial stimuli have remained elusive. We demonstrate that LUBAC activity itself is downregulated through ubiquitination, specifically, ubiquitination of the catalytic subunit HOIP at the carboxyl-terminal lysine 1056. Ubiquitination of Lys1056 dynamically altered HOIP conformation, resulting in the suppression of its catalytic activity. Consequently, HOIP Lys1056-to-Arg mutation led not only to persistent LUBAC activity but also to prolonged NF-κB activation induced by bacterial lipopolysaccharide-mediated Toll-like receptor 4 (TLR4) stimulation, whereas it showed no effect on NF-κB activation induced by CD40 stimulation. This study describes a novel posttranslational regulation of LUBAC-mediated linear ubiquitination that is critical for specifically directing TLR4-mediated NF-κB activation. PMID:26578682

E3 ubiquitin ligases: key regulators of hormone signaling in plants.

PubMed

Kelley, Dior

2018-03-07

Ubiquitin-mediated control of protein stability is central to most aspects of plant hormone signaling. Attachment of ubiquitin to target proteins occurs via an enzymatic cascade with the final step being catalyzed by a family of enzymes known as E3 ubiquitin ligases, which have been classified based on their protein domains and structures. While E3 ubiquitin ligases are conserved among eukaryotes, in plants they are well-known to fulfill unique roles as central regulators of phytohormone signaling, including hormone perception and regulation of hormone biosynthesis. This review will highlight up-to-date findings that have refined well-known E3 ligase-substrate interactions and defined novel E3 ligase substrates that mediate numerous hormone signaling pathways. Additionally, examples of how particular E3 ligases may mediate hormone crosstalk will be discussed as an emerging theme. Looking forward, promising experimental approaches and methods that will provide deeper mechanistic insight into the roles of E3 ubiquitin ligases in plants will be considered. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Complete regression of human malignant mesothelioma xenografts following local injection of midkine promoter-driven oncolytic adenovirus

PubMed Central

Kubo, Shuji; Kawasaki, Yoshiko; Yamaoka, Norie; Tagawa, Masatoshi; Kasahara, Noriyuki; Terada, Nobuyuki; Okamura, Haruki

2010-01-01

Background Malignant mesothelioma is a highly aggressive tumor with poor prognosis. Conventional therapies for mesothelioma are generally non-curative, and new treatment paradigms are urgently needed. We hypothesized that the tumor-specific midkine (Mdk) promoter could confer transcriptional targeting to oncolytic adenoviruses for effective treatment of malignant mesothelioma. Methods We analyzed Mdk expression by quantitative RT-PCR in six human mesothelioma cell lines, and tested Mdk promoter activity by luciferase reporter assay. Based on these data, we constructed a replication-selective oncolytic adenovirus, designated AdMdk-E1-iresTK, which contains an Mdk promoter-driven adenoviral E1 gene and HSV-thymidine kinase (TK) suicide gene, and CMV promoter-driven green fluorescent protein (GFP) marker gene. Selectivity of viral replication and cytolysis were characterized in normal vs. mesothelioma cells in vitro, and intratumoral spread and antitumor efficacy were investigated in vivo. Results Mdk promoter activity was restricted in normal cells, but highly activated in mesothelioma cell lines. AdMdk-E1-iresTK was seen to efficiently replicate, produce viral progeny, and spread in multiple mesothelioma cell lines. Lytic spread of AdMdk-E1-iresTK mediated efficient killing of these mesothelioma cells, and its in vitro cytocidal effect was significantly enhanced by treatment with the prodrug, ganciclovir. Intratumoral injection of AdMdk-E1-iresTK caused complete regression of MESO4 and MSTO human mesothelioma xenografts in athymic mice. In vivo fluorescence imaging demonstrated intratumoral spread of AdMdk-E1-iresTK-derived signals, which vanished after tumor eradication. Conclusions These data indicate that transcriptional targeting of viral replication by the Mdk promoter represents a promising general strategy for oncolytic virotherapy of cancers with upregulated Mdk expression, including malignant mesothelioma. PMID:20635326

Ube2V2 Is a Rosetta Stone Bridging Redox and Ubiquitin Codes, Coordinating DNA Damage Responses.

PubMed

Zhao, Yi; Long, Marcus J C; Wang, Yiran; Zhang, Sheng; Aye, Yimon

2018-02-28

Posttranslational modifications (PTMs) are the lingua franca of cellular communication. Most PTMs are enzyme-orchestrated. However, the reemergence of electrophilic drugs has ushered mining of unconventional/non-enzyme-catalyzed electrophile-signaling pathways. Despite the latest impetus toward harnessing kinetically and functionally privileged cysteines for electrophilic drug design, identifying these sensors remains challenging. Herein, we designed "G-REX"-a technique that allows controlled release of reactive electrophiles in vivo. Mitigating toxicity/off-target effects associated with uncontrolled bolus exposure, G-REX tagged first-responding innate cysteines that bind electrophiles under true k cat / K m conditions. G-REX identified two allosteric ubiquitin-conjugating proteins-Ube2V1/Ube2V2-sharing a novel privileged-sensor-cysteine. This non-enzyme-catalyzed-PTM triggered responses specific to each protein. Thus, G-REX is an unbiased method to identify novel functional cysteines. Contrasting conventional active-site/off-active-site cysteine-modifications that regulate target activity, modification of Ube2V2 allosterically hyperactivated its enzymatically active binding-partner Ube2N, promoting K63-linked client ubiquitination and stimulating H2AX-dependent DNA damage response. This work establishes Ube2V2 as a Rosetta-stone bridging redox and ubiquitin codes to guard genome integrity.

Ubiquitin-Dependent Regulation of the Mammalian Hippo Pathway: Therapeutic Implications for Cancer

PubMed Central

Nguyen, Thanh Hung

2018-01-01

The Hippo pathway serves as a key barrier for oncogenic transformation. It acts by limiting the activity of the proto-oncogenes YAP and TAZ. Reduced Hippo signaling and elevated YAP/TAZ activities are frequently observed in various types of tumors. Emerging evidence suggests that the ubiquitin system plays an important role in regulating Hippo pathway activity. Deregulation of ubiquitin ligases and of deubiquitinating enzymes has been implicated in increased YAP/TAZ activity in cancer. In this article, we review recent insights into the ubiquitin-mediated regulation of the mammalian Hippo pathway, its deregulation in cancer, and possibilities for targeting the Hippo pathway through the ubiquitin system. PMID:29673168

Ubiquitin-Dependent Regulation of the Mammalian Hippo Pathway: Therapeutic Implications for Cancer.

PubMed

Nguyen, Thanh Hung; Kugler, Jan-Michael

2018-04-17

The Hippo pathway serves as a key barrier for oncogenic transformation. It acts by limiting the activity of the proto-oncogenes YAP and TAZ. Reduced Hippo signaling and elevated YAP/TAZ activities are frequently observed in various types of tumors. Emerging evidence suggests that the ubiquitin system plays an important role in regulating Hippo pathway activity. Deregulation of ubiquitin ligases and of deubiquitinating enzymes has been implicated in increased YAP/TAZ activity in cancer. In this article, we review recent insights into the ubiquitin-mediated regulation of the mammalian Hippo pathway, its deregulation in cancer, and possibilities for targeting the Hippo pathway through the ubiquitin system.

Constructing and decoding unconventional ubiquitin chains.

PubMed

Behrends, Christian; Harper, J Wade

2011-05-01

One of the most notable discoveries in the ubiquitin system during the past decade is the extensive use of diverse chain linkages to control signaling networks. Although the utility of Lys48- and Lys63-linked chains in protein turnover and molecular assembly, respectively, are well known, we are only beginning to understand how unconventional chain linkages are formed on target proteins and how such linkages are decoded by specific binding proteins. In this review, we summarize recent efforts to elucidate the machinery and mechanisms controlling assembly of Lys11-linked and linear (or Met1-linked) ubiquitin chains, and describe current models for how these chain types function in immune signaling and cell-cycle control.

UbSRD: The Ubiquitin Structural Relational Database.

PubMed

Harrison, Joseph S; Jacobs, Tim M; Houlihan, Kevin; Van Doorslaer, Koenraad; Kuhlman, Brian

2016-02-22

The structurally defined ubiquitin-like homology fold (UBL) can engage in several unique protein-protein interactions and many of these complexes have been characterized with high-resolution techniques. Using Rosetta's structural classification tools, we have created the Ubiquitin Structural Relational Database (UbSRD), an SQL database of features for all 509 UBL-containing structures in the PDB, allowing users to browse these structures by protein-protein interaction and providing a platform for quantitative analysis of structural features. We used UbSRD to define the recognition features of ubiquitin (UBQ) and SUMO observed in the PDB and the orientation of the UBQ tail while interacting with certain types of proteins. While some of the interaction surfaces on UBQ and SUMO overlap, each molecule has distinct features that aid in molecular discrimination. Additionally, we find that the UBQ tail is malleable and can adopt a variety of conformations upon binding. UbSRD is accessible as an online resource at rosettadesign.med.unc.edu/ubsrd. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ubiquitin-specific protease 14 regulates cell proliferation and apoptosis in oral squamous cell carcinoma.

PubMed

Chen, Xiangyun; Wu, Jingjing; Chen, Yitian; Ye, Dongxia; Lei, Hu; Xu, Hanzhang; Yang, Li; Wu, Yingli; Gu, Wenli

2016-10-01

Ubiquitin-specific protease 14, a deubiquitinating enzyme, has been implicated in the tumorigenesis and progression of several cancers, but its role in oral squamous cell carcinoma remains to be elucidated. The aim of this study was to explore the expression pattern and roles of Ubiquitin-specific protease 14 in the occurrence and development of oral squamous cell carcinoma. Interestingly, Ubiquitin-specific protease 14 was overexpressed in oral cancer tissues and cell lines at both mRNA and protein levels. b-AP15, a specific inhibitor of Ubiquitin-specific protease 14, significantly inhibited the growth of cancer cells and increased cell apoptosis in a dose-dependent manner. Moreover, knockdown of Ubiquitin-specific protease 14 by shRNA significantly inhibited the proliferation and migration of cancer cells in vitro. Finally, using a xenograft mouse model of oral squamous cell carcinoma, knockdown of Ubiquitin-specific protease 14 markedly inhibited tumor growth and triggered the cancer cell apoptosis in vivo, supporting previous results. In conclusion, for the first time we have demonstrated the expression pattern of Ubiquitin-specific protease 14 in oral squamous cell carcinoma and verified a relationship with tumor growth and metastasis. These results may highlight new therapeutic strategies for tumor treatment, application of Ubiquitin-specific protease 14 selective inhibitor, such as b-AP15, or knockdown by shRNA. Collectively, Ubiquitin-specific protease 14 could be a potential therapeutic target for oral squamous cell carcinoma patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation

PubMed Central

Lechtenberg, Bernhard C.; Rajput, Akhil; Sanishvili, Ruslan; Dobaczewska, Małgorzata K.; Ware, Carl F.; Mace, Peter D.; Riedl, Stefan J.

2015-01-01

Ubiquitination is a central process affecting all facets of cellular signaling and function1. A critical step in ubiquitination is the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate or a growing ubiquitin chain, which is mediated by E3 ubiquitin ligases. RING-type E3 ligases typically facilitate the transfer of ubiquitin from the E2 directly to the substrate2,3. The RBR family of RING-type E3 ligases, however, breaks this paradigm by forming a covalent intermediate with ubiquitin similarly to HECT-type E3 ligases4–6. The RBR family includes Parkin4 and HOIP, the central catalytic factor of the linear ubiquitin chain assembly complex (LUBAC)7. While structural insights into the RBR E3 ligases Parkin and HHARI in their overall autoinhibited forms are available8–13, no structures exist of intact fully active RBR E3 ligases or any of their complexes. Thus, the RBR mechanism of action has remained largely enigmatic. Here we present the first structure of the fully active HOIP-RBR in its transfer complex with an E2~ubiquitin conjugate, which elucidates the intricate nature of RBR E3 ligases. The active HOIP-RBR adopts a conformation markedly different from that of autoinhibited RBRs. HOIP-RBR binds the E2~ubiquitin conjugate in an elongated fashion, with the E2 and E3 catalytic centers ideally aligned for ubiquitin transfer, which structurally both requires and enables a HECT-like mechanism. In addition, surprisingly, three distinct helix–IBR-fold motifs inherent to RBRs form ubiquitin-binding regions that engage the activated ubiquitin of the E2~Ub conjugate as well as an additional regulatory ubiquitin molecule. The features uncovered reveal critical states of the HOIP-RBR E3 ligase cycle, and comparison with Parkin and HHARI suggests a general mechanism for RBR E3 ligases. PMID:26789245

Expression of a polyubiquitin promoter isolated from Gladiolus.

PubMed

Joung, Young Hee; Kamo, Kathryn

2006-10-01

A polyubiquitin promoter (GUBQ1) including its 5'UTR and intron was isolated from the floral monocot Gladiolus because high levels of expression could not be obtained using publicly available promoters isolated from either cereals or dicots. Sequencing of the promoter revealed highly conserved 5' and 3' intron splicing sites for the 1.234 kb intron. The coding sequence of the first two ubiquitin genes showed the highest homology (87 and 86%, respectively) to the ubiquitin genes of Nicotiana tabacum and Oryza sativa RUBQ2. Transient expression following gene gun bombardment showed that relative levels of GUS activity with the GUBQ1 promoter were comparable to the CaMV 35S promoter in gladiolus, tobacco, rose, rice, and the floral monocot freesia. The highest levels of GUS expression with GUBQ1 were attained with Gladiolus. The full-length GUBQ1 promoter including 5'UTR and intron were necessary for maximum GUS expression in Gladiolus. The relative GUS activity for the promoter only was 9%, and the activity for the promoter with 5'UTR and 399 bp of the full-length 1.234 kb intron was 41%. Arabidopsis plants transformed with uidA under GUBQ1 showed moderate GUS expression throughout young leaves and in the vasculature of older leaves. The highest levels of transient GUS expression in Gladiolus have been achieved using the GUBQ1 promoter. This promoter should be useful for genetic engineering of disease resistance in Gladiolus, rose, and freesia, where high levels of gene expression are important.

Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1

PubMed Central

Burton, Janet L; Xiong, Yong; Solomon, Mark J

2011-01-01

The anaphase promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators by the 26S proteasome. Cdc20 and Cdh1 are WD40-containing APC co-activators that bind destruction boxes (DB) and KEN boxes within substrates to recruit them to the APC for ubiquitination. Acm1 is an APCCdh1 inhibitor that utilizes a DB and a KEN box to bind Cdh1 and prevent substrate binding, although Acm1 itself is not a substrate. We investigated what differentiates an APC substrate from an inhibitor. We identified the Acm1 A-motif that interacts with Cdh1 and together with the DB and KEN box is required for APCCdh1 inhibition. A genetic screen identified Cdh1 WD40 domain residues important for Acm1 A-motif interaction and inhibition that appears to reside near Cdh1 residues important for DB recognition. Specific lysine insertion mutations within Acm1 promoted its ubiquitination by APCCdh1 whereas lysine removal from the APC substrate Hsl1 converted it into a potent APCCdh1 inhibitor. These findings suggest that tight Cdh1 binding combined with the inaccessibility of ubiquitinatable lysines contributes to pseudosubstrate inhibition of APCCdh1. PMID:21460798

Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII.

PubMed

Inturi, Raviteja; Mun, Kwangchol; Singethan, Katrin; Schreiner, Sabrina; Punga, Tanel

2018-02-01

Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections. IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and

A Versatile Strategy for the Semisynthetic Production of Ser65 Phosphorylated Ubiquitin and Its Biochemical and Structural Characterisation

PubMed Central

Han, Cong; Pao, Kuan-Chuan; Kazlauskaite, Agne; Muqit, Miratul M K; Virdee, Satpal

2015-01-01

Ubiquitin phosphorylation is emerging as an important regulatory layer in the ubiquitin system. This is exemplified by the phosphorylation of ubiquitin on Ser65 by the Parkinson's disease-associated kinase PINK1, which mediates the activation of the E3 ligase Parkin. Additional phosphorylation sites on ubiquitin might also have important cellular roles. Here we report a versatile strategy for preparing phosphorylated ubiquitin. We biochemically and structurally characterise semisynthetic phospho-Ser65-ubiquitin. Unexpectedly, we observed disulfide bond formation between ubiquitin molecules, and hence a novel crystal form. The method outlined provides a direct approach to study the combinatorial effects of phosphorylation on ubiquitin function. Our analysis also suggests that disulfide engineering of ubiquitin could be a useful strategy for obtaining alternative crystal forms of ubiquitin species thereby facilitating structural validation. PMID:26010437

USP8 regulates mitophagy by removing K6-linked ubiquitin conjugates from parkin

PubMed Central

Durcan, Thomas M; Tang, Matthew Y; Pérusse, Joëlle R; Dashti, Eman A; Aguileta, Miguel A; McLelland, Gian-Luca; Gros, Priti; Shaler, Thomas A; Faubert, Denis; Coulombe, Benoit; Fon, Edward A

2014-01-01

Mutations in the Park2 gene, encoding the E3 ubiquitin-ligase parkin, are responsible for a familial form of Parkinson's disease (PD). Parkin-mediated ubiquitination is critical for the efficient elimination of depolarized dysfunctional mitochondria by autophagy (mitophagy). As damaged mitochondria are a major source of toxic reactive oxygen species within the cell, this pathway is believed to be highly relevant to the pathogenesis of PD. Little is known about how parkin-mediated ubiquitination is regulated during mitophagy or about the nature of the ubiquitin conjugates involved. We report here that USP8/UBPY, a deubiquitinating enzyme not previously implicated in mitochondrial quality control, is critical for parkin-mediated mitophagy. USP8 preferentially removes non-canonical K6-linked ubiquitin chains from parkin, a process required for the efficient recruitment of parkin to depolarized mitochondria and for their subsequent elimination by mitophagy. This work uncovers a novel role for USP8-mediated deubiquitination of K6-linked ubiquitin conjugates from parkin in mitochondrial quality control. PMID:25216678

RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheren, Jamie E.; Kassenbrock, C. Kenneth, E-mail: ken.kassenbrock@ucdenver.edu; Department of Biology, Colorado State University, Fort Collins, CO 80523-1878

2013-11-01

Highlights: •RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. •RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. •We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. •Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. •Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequencemore » (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.« less

Prohibitin-mediated mitochondrial ubiquitination during spermiogenesis in Chinese mitten crab Eriocheir sinensis

PubMed Central

Hou, Cong-Cong; Wei, Chao-Guang; Lu, Cheng-Peng; Gao, Xin-Ming; Yang, Wan-Xi; Zhu, Jun-Quan

2017-01-01

The sperm of Eriocheir sinensis has a cup-shaped nucleus that contains several mitochondria embedded at the opening of the cup. The acrosome vesicle also contains derivants of mitochondria. The mitochondria distribution pattern involves a decrease in the number and changes in the structure and transportation of these organelles. The decreased number of sperm mitochondria is achieved through autophagy or the ubiquitination pathway. Prohibitin (PHB), the mitochondria inner membrane protein, is an evolutionarily highly conserved protein, is closely associated with spermatogenesis and sperm quality control and is also a potential substrate of ubiquitination. However, whether PHB protein mediates the ubiquitination pathway of sperm mitochondria in crustacean animals remains poorly understood. In the present study, we revealed that PHB, a substrate of ubiquitin, participates in the ubiquitination and degradation of mitochondria during spermiogenesis in E. sinensis. To confirm this finding, we used shRNA interference to reduce PHB expression and an overexpression technique to increase PHB expression in vitro. The interference experiment showed that the reduced PHB expression directly affected the polyubiquitination level and mitochondria status, whereas PHB overexpression markedly increased the polyubiquitination level. In vitro experiments also showed that PHB and its ubiquitination decide the fate of mitochondria. PMID:29228727

BAG2 Interferes with CHIP-Mediated Ubiquitination of HSP72.

PubMed

Schönbühler, Bianca; Schmitt, Verena; Huesmann, Heike; Kern, Andreas; Gamerdinger, Martin; Behl, Christian

2016-12-30

The maintenance of cellular proteostasis is dependent on molecular chaperones and protein degradation pathways. Chaperones facilitate protein folding, maturation, and degradation, and the particular fate of a misfolded protein is determined by the interaction of chaperones with co-chaperones. The co-factor CHIP (C-terminus of HSP70-inteacting protein, STUB1) ubiquitinates chaperone substrates and directs proteins to the cellular degradation systems. The activity of CHIP is regulated by two co-chaperones, BAG2 and HSPBP1, which are potent inhibitors of the E3 ubiquitin ligase activity. Here, we examined the functional correlation of HSP72, CHIP, and BAG2, employing human primary fibroblasts. We showed that HSP72 is a substrate of CHIP and that BAG2 efficiently prevented the ubiquitination of HSP72 in young cells as well as aged cells. Aging is associated with a decline in proteostasis and we observed increased protein levels of CHIP as well as BAG2 in senescent cells. Interestingly, the ubiquitination of HSP72 was strongly reduced during aging, which revealed that BAG2 functionally counteracted the increased levels of CHIP. Interestingly, HSPBP1 protein levels were down-regulated during aging. The data presented here demonstrates that the co-chaperone BAG2 influences HSP72 protein levels and is an important modulator of the ubiquitination activity of CHIP in young as well as aged cells.

Alanine scan of core positions in ubiquitin reveals links between dynamics, stability, and function

PubMed Central

Lee, Shirley Y.; Pullen, Lester; Virgil, Daniel J.; Castañeda, Carlos A.; Abeykoon, Dulith; Bolon, Daniel N. A.; Fushman, David

2014-01-01

Mutations at solvent inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast. The majority (13 of 15) of alanine substitutions supported yeast growth as the sole ubiquitin. The two null mutants (I30A and L43A) were both less stable to temperature-induced unfolding in vitro than wild-type, but were well folded at physiological temperatures. Heteronuclear NMR studies indicated that the L43A mutation reduces temperature stability while retaining a ground-state structure similar to wild-type. This structure enables L43A to bind to common ubiquitin receptors in vitro. Many of the core alanine ubiquitin mutants, including one of the null variants (I30A), exhibited an increased accumulation of high molecular weight species, suggesting that these mutants caused a defect in the processing of ubiquitin-substrate conjugates. In contrast, L43A exhibited a unique accumulation pattern with reduced levels of high molecular weight species and undetectable levels of free ubiquitin. When conjugation to other proteins was blocked, L43A ubiquitin accumulated as free ubiquitin in yeast. Based on these findings we speculate that ubiquitin's stability to unfolding may be required for efficient recycling during proteasome-mediated substrate degradation. PMID:24361330

Ubiquitinated Sirtuin 1 (SIRT1) Function Is Modulated during DNA Damage-induced Cell Death and Survival*

PubMed Central

Peng, Lirong; Yuan, Zhigang; Li, Yixuan; Ling, Hongbo; Izumi, Victoria; Fang, Bin; Fukasawa, Kenji; Koomen, John; Chen, Jiandong; Seto, Edward

2015-01-01

Downstream signaling of physiological and pathological cell responses depends on post-translational modification such as ubiquitination. The mechanisms regulating downstream DNA damage response (DDR) signaling are not completely elucidated. Sirtuin 1 (SIRT1), the founding member of Class III histone deacetylases, regulates multiple steps in DDR and is closely associated with many physiological and pathological processes. However, the role of post-translational modification or ubiquitination of SIRT1 during DDR is unclear. We show that SIRT1 is dynamically and distinctly ubiquitinated in response to DNA damage. SIRT1 was ubiquitinated by the MDM2 E3 ligase in vitro and in vivo. SIRT1 ubiquitination under normal conditions had no effect on its enzymatic activity or rate of degradation; hypo-ubiquitination, however, reduced SIRT1 nuclear localization. Ubiquitination of SIRT1 affected its function in cell death and survival in response to DNA damage. Our results suggest that ubiquitination is required for SIRT1 function during DDR. PMID:25670865

Effects of covalent modification by 4-hydroxy-2-nonenal on the noncovalent oligomerization of ubiquitin.

PubMed

Grasso, Giuseppe; Axelsen, Paul H

2017-01-01

When lipid membranes containing ω-6 polyunsaturated fatty acyl chains are subjected to oxidative stress, one of the reaction products is 4-hydroxy-2-nonenal (HNE)-a chemically reactive short chain alkenal that can covalently modify proteins. The ubiquitin proteasome system is involved in the clearing of proteins modified by oxidation products such as HNE, but the chemical structure, stability and function of ubiquitin may be impaired by HNE modification. To evaluate this possibility, the susceptibility of ubiquitin to modification by HNE has been characterized over a range of concentrations where ubiquitin forms non-covalent oligomers. Results indicate that HNE modifies ubiquitin at only two of the many possible sites, and that HNE modification at these two sites alters the ubiquitin oligomerization equilibrium. These results suggest that any role ubiquitin may have in clearing proteins damaged by oxidative stress may itself be impaired by oxidative lipid degradation products. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Pichia anomala DBVPG 3003 Secretes a Ubiquitin-Like Protein That Has Antimicrobial Activity▿

PubMed Central

De Ingeniis, Jessica; Raffaelli, Nadia; Ciani, Maurizio; Mannazzu, Ilaria

2009-01-01

The yeast strain Pichia anomala DBVPG 3003 secretes a killer toxin (Pikt) that has antifungal activity against Brettanomyces/Dekkera sp. yeasts. Pikt interacts with β-1,6-glucan, consistent with binding to the cell wall of sensitive targets. In contrast to that of toxin K1, secreted by Saccharomyces cerevisiae, Pikt killer activity is not mediated by an increase in membrane permeability. Purification of the toxin yielded a homogeneous protein of about 8 kDa, which showed a marked similarity to ubiquitin in terms of molecular mass and N-terminal sequences. Pikt is also specifically recognized by anti-bovine ubiquitin antibodies and, similar to ubiquitin-like peptides, is not absorbed by DEAE-cellulose. However, Pikt differs from ubiquitin in its sensitivity to proteolytic enzymes. Therefore, Pikt appears to be a novel ubiquitin-like peptide that has killer activity. PMID:19114528

Expression of TRAF6 and ubiquitin mRNA in skeletal muscle of gastric cancer patients

PubMed Central

2012-01-01

Objective To investigate the prognostic significance of tumor necrosis factor receptor (TNFR),-associated factor 6 (TRAF6),-and ubiquitin in gastric cancer patients. Methods Biopsies of the rectus abdominis muscle were obtained intra operatively from 102 gastric cancer patients and 29 subjects undergoing surgery for benign abdominal diseases, and muscle TRAF6 and ubiquitin mRNA expression and proteasome proteolytic activities were assessed. Results TRAF6 was significantly upregulated in muscle of gastric cancer compared with the control muscles. TRAF6 was upregulated in 67.65% (69/102) muscle of gastric cancer. Over expression of TRAF6 in muscles of gastric cancer were associated with TNM stage, level of serum albumin and percent of weight loss. Ubiquitin was significantly upregulated in muscle of gastric cancer compared with the control muscles. Ubiquitin was upregulated in 58.82% (60/102) muscles of gastric cancer. Over expression of ubiquitin in muscles of gastric cancer were associated with TNM (Tumor-Node-Metastasis) stage and weight loss. There was significant relation between TRAF6 and ubiquitin expression. Conclusions We found a positive correlation between TRAF6 and ubiquitin expression, suggesting that TRAF6 may up regulates ubiquitin activity in cancer cachexia. While more investigations are required to understand its mechanisms of TRAF6 and ubiquitin in skeletal muscle. Correct the catabolic-anabolic imbalance is essential for the effective treatment of cancer cachexia. PMID:23013936

Target Specificity of the E3 Ligase LUBAC for Ubiquitin and NEMO Relies on Different Minimal Requirements*

PubMed Central

Smit, Judith J.; van Dijk, Willem J.; El Atmioui, Dris; Merkx, Remco; Ovaa, Huib; Sixma, Titia K.

2013-01-01

The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway. NEMO ubiquitination requires a dual target specificity of LUBAC, priming on a lysine on NEMO and chain elongation on the N terminus of the priming ubiquitin. Here we explore the minimal requirements for these specificities. Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin. Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP. Consequently, target specificity toward NEMO is determined by multiple LUBAC components, whereas linear ubiquitin chain elongation is realized by a specific interplay between HOIP and ubiquitin. PMID:24030825

The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo.

PubMed

Su, Jianguo; Zhu, Zuoyan; Wang, Yaping; Xiong, Feng; Zou, Jun

2008-01-01

The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. In the present study, we used a cytomegalovirus (CMV) promoter-driven DNA template approach to induce short hairpin RNA (shRNA) triggered RNAi to block exogenous Enhanced Green Fluorescent Protein (EGFP) and endogenous No Tail (NTL) gene expressions. We constructed three plasmids, pCMV-EGFP-CMV-shGFP-SV40, pCMV-EGFP-CMV-shNTL-SV40, and pCMV-EGFP-CMV-shScrambled-SV40, each containing a CMV promoter driving an EGFP reporter cDNA and DNA coding for one shRNA under the control of another CMV promoter. The three shRNA-generating plasmids and pCMV-EGFP control plasmid were introduced into zebrafish embryos by microinjection. Samples were collected at 48 h after injection. Results were evaluated by phenotype observation and real-time fluorescent quantitative reverse-transcription polymerase chain reaction (Q-PCR). The shGFP-generating plasmid significantly inhibited the EGFP expression viewed under fluorescent microscope and reduced by 70.05 +/- 1.26% of exogenous EGFP gene mRNA levels compared with controls by Q-PCR. The shRNA targeting endogenous NTL gene resulted in obvious NTL phenotype of 30 +/- 4% and decreased the level of their corresponding mRNAs up to 54.52 +/- 2.05% compared with nontargeting control shRNA. These data proved the feasibility of the CMV promoter-driven shRNA expression technique to be used to inhibit exogenous and endogenous gene expressions in zebrafish in vivo.

Identification of ubiquitin/ubiquitin-like protein modification from tandem mass spectra with various PTMs

PubMed Central

2011-01-01

Background Various solutions have been introduced for the identification of post-translational modification (PTM) from tandem mass spectrometry (MS/MS) in proteomics field but the identification of peptide modifiers, such as Ubiquitin (Ub) and ubiquitin-like proteins (Ubls), is still a challenge. The fragmentation of peptide modifier produce complex shifted ion mass patterns in combination with other PTMs, which makes it difficult to identify and locate the PTMs on a protein sequence. Currently, most PTM identification methods do not consider the complex fragmentation of peptide modifier or deals it separately from the other PTMs. Results We developed an advanced PTM identification method that inspects possible ion patterns of the most known peptide modifiers as well as other known biological and chemical PTMs to make more comprehensive and accurate conclusion. The proposed method searches all detectable mass differences of measured peaks from their theoretical values and the mass differences within mass tolerance range are grouped as mass shift classes. The most possible locations of multiple PTMs including peptide modifiers can be determined by evaluating all possible scenarios generated by the combination of the qualified mass shift classes.The proposed method showed excellent performance in the test with simulated spectra having various PTMs including peptide modifiers and in the comparison with recently developed methods such as QuickMod and SUMmOn. In the analysis of HUPO Brain Proteome Project (BPP) datasets, the proposed method could find the ubiquitin modification sites that were not identified by other conventional methods. Conclusions This work presents a novel method for identifying bothpeptide modifiers that generate complex fragmentation patternsand PTMs that are not fragmented during fragmentation processfrom tandem mass spectra. PMID:22373085

TRIM65 negatively regulates p53 through ubiquitination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yang; Ma, Chengyuan; Zhou, Tong

2016-04-22

Tripartite-motif protein family member 65 (TRIM65) is an important protein involved in white matter lesion. However, the role of TRIM65 in human cancer remains less understood. Through the Cancer Genome Atlas (TCGA) gene alteration database, we found that TRIM65 is upregulated in a significant portion of non-small cell lung carcinoma (NSCLC) patients. Our cell growth assay revealed that TRIM65 overexpression promotes cell proliferation, while knockdown of TRIM65 displays opposite effect. Mechanistically, TRIM65 binds to p53, one of the most critical tumor suppressors, and serves as an E3 ligase toward p53. Consequently, TRIM65 inactivates p53 through facilitating p53 poly-ubiquitination and proteasome-mediatedmore » degradation. Notably, chemotherapeutic reagent cisplatin induction of p53 is markedly attenuated in response to ectopic expression of TRIM65. Cell growth inhibition by TRIM65 knockdown is more significant in p53 positive H460 than p53 negative H1299 cells, and knockdown of p53 in H460 cells also shows compromised cell growth inhibition by TRIM65 knockdown, indicating that p53 is required, at least in part, for TRIM65 function. Our findings demonstrate TRIM65 as a potential oncogenic protein, highly likely through p53 inactivation, and provide insight into development of novel approaches targeting TRIM65 for NSCLC treatment, and also overcoming chemotherapy resistance. - Highlights: • TRIM65 expression is elevated in NSCLC. • TRIM65 inactivates p53 through mediating p53 ubiquitination and degradation. • TRIM65 attenuates the response of NSCLC cells to cisplatin.« less

Autophagy promotes synapse development in Drosophila.

PubMed

Shen, Wei; Ganetzky, Barry

2009-10-05

Autophagy, a lysosome-dependent degradation mechanism, mediates many biological processes, including cellular stress responses and neuroprotection. In this study, we demonstrate that autophagy positively regulates development of the Drosophila melanogaster larval neuromuscular junction (NMJ). Autophagy induces an NMJ overgrowth phenotype closely resembling that of highwire (hiw), an E3 ubiquitin ligase mutant. Moreover, like hiw, autophagy-induced NMJ overgrowth is suppressed by wallenda (wnd) and by a dominant-negative c-Jun NH(2)-terminal kinase (bsk(DN)). We show that autophagy promotes NMJ growth by reducing Hiw levels. Thus, autophagy and the ubiquitin-proteasome system converge in regulating synaptic development. Because autophagy is triggered in response to many environmental cues, our findings suggest that it is perfectly positioned to link environmental conditions with synaptic growth and plasticity.

Adaptor Protein Complex-2 (AP-2) and Epsin-1 Mediate Protease-activated Receptor-1 Internalization via Phosphorylation- and Ubiquitination-dependent Sorting Signals*

PubMed Central

Chen, Buxin; Dores, Michael R.; Grimsey, Neil; Canto, Isabel; Barker, Breann L.; Trejo, JoAnn

2011-01-01

Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs. PMID:21965661

Identification and characterization of a novel ISG15-ubiquitin mixed chain and its role in regulating protein homeostasis

PubMed Central

Fan, Jun-Bao; Arimoto, Kei-lchiro; Motamedchaboki, Khatereh; Yan, Ming; Wolf, Dieter A.; Zhang, Dong-Er

2015-01-01

As a ubiquitin-like modifier, ISG15 is conjugated to many cellular proteins in a process termed protein ISGylation. However, the crosstalk between protein ISGylation and the ubiquitin proteasome system is not fully understood. Here, we report that cellular ubiquitin is a substrate of ISG15 and Lys 29 on ubiquitin is the major ISG15 acceptor site. Using a model substrate, we demonstrate that ISG15 can modify ubiquitin, which is immobilized on its substrate, to form ISG15-ubiquitin mixed chains. Furthermore, our results indicate that ISG15-ubiquitin mixed chains do not serve as degradation signals for a ubiquitin fusion degradation substrate. Accordingly, an ISG15-ubiquitin fusion protein, which mimics an ISG15-ubiquitin mixed chain, negatively regulates cellular turnover of ubiquitylated proteins. In addition, ISG15-ubiquitin mixed chains, which are detectable on endogenously ubiquitylated proteins, dampen cellular turnover of these proteins. Thus, our studies unveil an unanticipated interplay between two protein modification systems and highlight its role in coordinating protein homeostasis. PMID:26226047

Inhibition of the ubiquitin-proteasome system by natural products for cancer therapy.

PubMed

Tsukamoto, Sachiko; Yokosawa, Hideyoshi

2010-08-01

The ubiquitin-proteasome system plays a critical role in selective protein degradation and regulates almost all cellular events such as cell cycle progression, signal transduction, cell death, immune responses, metabolism, protein quality control, development, and neuronal function. The recent approval of bortezomib, a synthetic proteasome inhibitor, for the treatment of relapsed multiple myeloma has opened the way to the discovery of drugs targeting the proteasome and ubiquitinating and deubiquitinating enzymes as well as the delivery system. To date, various synthetic and natural products have been reported to inhibit the components of the ubiquitin-proteasome system. Here, we review natural products targeting the ubiquitin-proteasome system as well as synthetic compounds with potent inhibitory effects. Georg Thieme Verlag KG Stuttgart-New York.

Inhibitors of ubiquitin E3 ligase as potential new antimalarial drug leads.

PubMed

Jain, Jagrati; Jain, Surendra K; Walker, Larry A; Tekwani, Babu L

2017-06-02

Protein ubiquitylation is an important post-translational regulation, which has been shown to be necessary for life cycle progression and survival of Plasmodium falciparum. Ubiquitin is a highly conserved 76 amino acid polypeptide, which attaches covalently to target proteins through combined action of three classes of enzymes namely, the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin-protein ligase (E3). Ubiquitin E1 and E2 are highly conserved within eukaryotes. However, the P. falciparum E3 ligase is substantially variable and divergent compared to the homologs from other eukaryotes, which make the E3 ligase a parasite-specific target. A set of selected E3 ubiquitin ligase inhibitors was tested in vitro against a chloroquine-sensitive P. falciparum D6 strain (PfD6) and a chloroquine-resistant P. falciparum W2 strain (PfW2). The inhibitors were also tested against Vero and transformed THP1 cells for cytotoxicity. The lead antimalarial E3 ubiquitin ligase inhibitors were further evaluated for the stage-specific antimalarial action and effects on cellular development of P. falciparum in vitro. Statistics analysis was done by two-way ANOVA followed by Tukey and Sidak multiple comparison test using GraphPad Prism 6. E3 ligase inhibitors namely, JNJ 26854165, HLI 373 and Nutlin 3 showed prominent antimalarial activity against PfD6 and PfW2. These inhibitors were considerably less cytotoxic to mammalian Vero cells. JNJ 26854165, HLI 373 and Nutlin 3 blocked the development of P. falciparum parasite at the trophozoite and schizont stages, resulting in accumulation of distorted trophozoites and immature schizonts. Interruption of trophozoites and schizont maturation by the antimalarial E3 ligase inhibitors suggest the role of ubiquitin/proteasome functions in the intraerythrocytic development of malaria parasite. The ubiquitin/proteasome functions may be critical for schizont maturation. Further investigations on the lead E3 ligase

Hidden targets of ubiquitin proteasome system: To prevent diabetic nephropathy.

PubMed

Goru, Santosh Kumar; Kadakol, Almesh; Gaikwad, Anil Bhanudas

2017-06-01

Diabetic nephropathy (DN) is the major cause of end stage renal failure. Although, several therapeutic targets have emerged to prevent the progression of DN, the number of people with DN still continues to rise worldwide, suggesting an urgent need of novel targets to prevent DN completely. Currently, the role of ubiquitin proteasome system (UPS) has been highlighted in the pathogenesis and progression of various diseases like obesity, insulin resistance, atherosclerosis, cancers, neurodegerative disorders and including secondary complications of diabetes. UPS mainly involves in protein homeostatis through ubiquitination (post translational modification) and proteasomal degradation of various proteins. Ubiquitination, not only involves in proteasomal degradation, but also directs the substrate proteins to participate in multitude of cell signalling pathways. However, very little is known about ubiquitination and UPS in the progression of DN. This review mainly focuses on UPS and its components including E2 conjugating enzymes, E3 ligases and deubiquitinases (DUBs) in the development of DN and thus may help us to find novel therapeutic targets with in UPS to prevent DN completely in future. Copyright © 2017 Elsevier Ltd. All rights reserved.

Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes.

PubMed

Ramadan, Abdelaziz; Nemoto, Keiichirou; Seki, Motoaki; Shinozaki, Kazuo; Takeda, Hiroyuki; Takahashi, Hirotaka; Sawasaki, Tatsuya

2015-11-10

Protein ubiquitination is a ubiquitous mechanism in eukaryotes. In Arabidopsis, ubiquitin modification is mainly mediated by two ubiquitin activating enzymes (E1s), 37 ubiquitin conjugating enzymes (E2s), and more than 1300 predicted ubiquitin ligase enzymes (E3s), of which ~470 are RING-type E3s. A large proportion of the RING E3's gene products have yet to be characterised in vitro, likely because of the laborious work involved in large-scale cDNA cloning and protein expression, purification, and characterisation. In addition, several E2s, which might be necessary for the activity of certain E3 ligases, cannot be expressed by Escherichia coli or cultured insect cells and, therefore, remain uncharacterised. Using the RIKEN Arabidopsis full-length cDNA library (RAFL) with the 'split-primer' PCR method and a wheat germ cell-free system, we established protein libraries of Arabidopsis E2 and RING E3 enzymes. We expressed 35 Arabidopsis E2s including six enzymes that have not been previously expressed, and 204 RING proteins, most of which had not been functionally characterised. Thioester assays using dithiothreitol (DTT) showed DTT-sensitive ubiquitin thioester formation for all E2s expressed. In expression assays of RING proteins, 31 proteins showed high molecular smears, which are probably the result of their functional activity. The activities of another 27 RING proteins were evaluated with AtUBC10 and/or a group of different E2s. All the 27 RING E3s tested showed ubiquitin ligase activity, including 17 RING E3s. Their activities are reported for the first time. The wheat germ cell-free system used in our study, which is a eukaryotic expression system and more closely resembles the endogenous expression of plant proteins, is very suitable for expressing Arabidopsis E2s and RING E3s in their functional form. In addition, the protein libraries described here can be used for further understanding E2-E3 specificities and as platforms for protein-protein interaction

Ubiquitin ligase CHIP functions as an oncogene and activates the AKT signaling pathway in prostate cancer.

PubMed

Cheng, Li; Zang, Jin; Dai, Han-Jue; Li, Feng; Guo, Feng

2018-07-01

Carboxyl terminus of Hsc-70-interacting protein (CHIP) is an E3 ubiquitin ligase that induces the ubiquitination and degradation of numerous tumor-associated proteins and serves as a suppressor or promoter in tumor progression. To date, the molecular mechanism of CHIP in prostate cancer remains unknown. Therefore, the present study investigated the biological function of CHIP in prostate cancer cells and obtained evidence that CHIP expression is upregulated in prostate cancer tissues. The CHIP vector was introduced into DU145 cancer cells and the cell biological behaviour was examined through a series of experiments, including cell growth, cell apoptosis and migration and invasion assays. The results indicated that the overexpression of CHIP in DU145 prostatic cancer cells promoted cell proliferation through activation of the protein kinase B (AKT) signaling pathway, which subsequently increased cyclin D1 protein levels and decreased p21 and p27 protein levels. The overexpression of CHIP significantly increased the migration and invasion of the DU145 cells, which is possible due to activation of the AKT signaling pathway and upregulation of vimentin. The expression level of CHIP was observed to be increased in human prostate cancer tissues compared with the adjacent normal tissue. Furthermore, the CHIP expression level exhibited a positively association with the Gleason score of the patents. These findings indicate that CHIP functions as an oncogene in prostate cancer.

The Arabidopsis COP9 SIGNALOSOME INTERACTING F-BOX KELCH 1 protein forms an SCF ubiquitin ligase and regulates hypocotyl elongation.

PubMed

Franciosini, Anna; Lombardi, Benedetta; Iafrate, Silvia; Pecce, Valeria; Mele, Giovanni; Lupacchini, Leonardo; Rinaldi, Gianmarco; Kondou, Youichi; Gusmaroli, Giuliana; Aki, Shiori; Tsuge, Tomohiko; Deng, Xing-Wang; Matsui, Minami; Vittorioso, Paola; Costantino, Paolo; Serino, Giovanna

2013-09-01

The regulation of protein turnover by the ubiquitin proteasome system (UPS) is a major posttranslational mechanism in eukaryotes. One of the key components of the UPS, the COP9 signalosome (CSN), regulates 'cullin-ring' E3 ubiquitin ligases. In plants, CSN participates in diverse cellular and developmental processes, ranging from light signaling to cell cycle control. In this work, we isolated a new plant-specific CSN-interacting F-box protein, which we denominated CFK1 (COP9 INTERACTING F-BOX KELCH 1). We show that, in Arabidopsis thaliana, CFK1 is a component of a functional ubiquitin ligase complex. We also show that CFK1 stability is regulated by CSN and by proteasome-dependent proteolysis, and that light induces accumulation of the CFK1 transcript in the hypocotyl. Analysis of CFK1 knockdown, mutant, and overexpressing seedlings indicates that CFK1 promotes hypocotyl elongation by increasing cell size. Reduction of CSN levels enhances the short hypocotyl phenotype of CFK1-depleted seedlings, while complete loss of CSN activity suppresses the long-hypocotyl phenotype of CFK1-overexpressing seedlings. We propose that CFK1 (and its regulation by CSN) is a novel component of the cellular mechanisms controlling hypocotyl elongation.

Lys48 ubiquitination during the intraerythrocytic cycle of the rodent malaria parasite, Plasmodium chabaudi.

PubMed

González-López, Lorena; Carballar-Lejarazú, Rebeca; Arrevillaga Boni, Gerardo; Cortés-Martínez, Leticia; Cázares-Raga, Febe Elena; Trujillo-Ocampo, Abel; Rodríguez, Mario H; James, Anthony A; Hernández-Hernández, Fidel de la Cruz

2017-01-01

Ubiquitination tags proteins for different functions within the cell. One of the most abundant and studied ubiquitin modification is the Lys48 polyubiquitin chain that modifies proteins for their destruction by proteasome. In Plasmodium is proposed that post-translational regulation is fundamental for parasite development during its complex life-cycle; thus, the objective of this work was to analyze the ubiquitination during Plasmodium chabaudi intraerythrocytic stages. Ubiquitinated proteins were detected during intraerythrocytic stages of Plasmodium chabaudi by immunofluorescent microscopy, bidimensional electrophoresis (2-DE) combined with immunoblotting and mass spectrometry. All the studied stages presented protein ubiquitination and Lys48 polyubiquitination with more abundance during the schizont stage. Three ubiquitinated proteins were identified for rings, five for trophozoites and twenty for schizonts. Only proteins detected with a specific anti- Lys48 polyubiquitin antibody were selected for Mass Spectrometry analysis and two of these identified proteins were selected in order to detect the specific amino acid residues where ubiquitin is placed. Ubiquitinated proteins during the ring and trophozoite stages were related with the invasion process and in schizont proteins were related with nucleic acid metabolism, glycolysis and protein biosynthesis. Most of the ubiquitin detection was during the schizont stage and the Lys48 polyubiquitination during this stage was related to proteins that are expected to be abundant during the trophozoite stage. The evidence that these Lys48 polyubiquitinated proteins are tagged for destruction by the proteasome complex suggests that this type of post-translational modification is important in the regulation of protein abundance during the life-cycle and may also contribute to the parasite cell-cycle progression.

Hydrophobic Collapse of Ubiquitin Generates Rapid Protein-Water Motions.

PubMed

Wirtz, Hanna; Schäfer, Sarah; Hoberg, Claudius; Reid, Korey M; Leitner, David M; Havenith, Martina

2018-06-04

We report time-resolved measurements of the coupled protein-water modes of solvated ubiquitin during protein folding. Kinetic terahertz absorption (KITA) spectroscopy serves as a label-free technique for monitoring large scale conformational changes and folding of proteins subsequent to a sudden T-jump. We report here KITA measurements at an unprecedented time resolution of 500 ns, a resolution 2 orders of magnitude better than those of any previous KITA measurements, which reveal the coupled ubiquitin-solvent dynamics even in the initial phase of hydrophobic collapse. Complementary equilibrium experiments and molecular simulations of ubiquitin solutions are performed to clarify non-equilibrium contributions and reveal the molecular picture upon a change in structure, respectively. On the basis of our results, we propose that in the case of ubiquitin a rapid (<500 ns) initial phase of the hydrophobic collapse from the elongated protein to a molten globule structure precedes secondary structure formation. We find that these very first steps, including large-amplitude changes within the unfolded manifold, are accompanied by a rapid (<500 ns) pronounced change of the coupled protein-solvent response. The KITA response upon secondary structure formation exhibits an opposite sign, which indicates a distinct effect on the solvent-exposed surface.

Emerging Role of Ubiquitination in Antiviral RIG-I Signaling

PubMed Central

Maelfait, Jonathan

2012-01-01

Summary: Detection of viruses by the innate immune system involves the action of specialized pattern recognition receptors. Intracellular RIG-I receptors sense the presence of viral nucleic acids in infected cells and trigger signaling pathways that lead to the production of proinflammatory and antiviral proteins. Over the past few years, posttranslational modification of RIG-I and downstream signaling proteins by different types of ubiquitination has been found to be a key event in the regulation of RIG-I-induced NF-κB and interferon regulatory factor 3 (IRF3) activation. Multiple ubiquitin ligases, deubiquitinases, and ubiquitin binding scaffold proteins contribute to both positive and negative regulation of the RIG-I-induced antiviral immune response. A better understanding of the function and activity of these proteins might eventually lead to the development of novel therapeutic approaches for management of viral diseases. PMID:22390971

Autoregulation of Parkin activity through its ubiquitin-like domain

PubMed Central

Chaugule, Viduth K; Burchell, Lynn; Barber, Kathryn R; Sidhu, Ateesh; Leslie, Simon J; Shaw, Gary S; Walden, Helen

2011-01-01

Parkin is an E3-ubiquitin ligase belonging to the RBR (RING–InBetweenRING–RING family), and is involved in the neurodegenerative disorder Parkinson's disease. Autosomal recessive juvenile Parkinsonism, which is one of the most common familial forms of the disease, is directly linked to mutations in the parkin gene. However, the molecular mechanisms of Parkin dysfunction in the disease state remain to be established. We now demonstrate that the ubiquitin-like domain of Parkin functions to inhibit its autoubiquitination. Moreover pathogenic Parkin mutations disrupt this autoinhibition, resulting in a constitutively active molecule. In addition, we show that the mechanism of autoregulation involves ubiquitin binding by a C-terminal region of Parkin. Our observations provide important molecular insights into the underlying basis of Parkinson's disease, and in the regulation of RBR E3-ligase activity. PMID:21694720

Structural basis of ubiquitin modification by the Legionella effector SdeA.

PubMed

Dong, Yanan; Mu, Yajuan; Xie, Yongchao; Zhang, Yupeng; Han, Youyou; Zhou, Yu; Wang, Wenhe; Liu, Zihe; Wu, Mei; Wang, Hao; Pan, Man; Xu, Ning; Xu, Cong-Qiao; Yang, Maojun; Fan, Shilong; Deng, Haiteng; Tan, Tianwei; Liu, Xiaoyun; Liu, Lei; Li, Jun; Wang, Jiawei; Fang, Xianyang; Feng, Yue

2018-05-01

Protein ubiquitination is a multifaceted post-translational modification that controls almost every process in eukaryotic cells. Recently, the Legionella effector SdeA was reported to mediate a unique phosphoribosyl-linked ubiquitination through successive modifications of the Arg42 of ubiquitin (Ub) by its mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains. However, the mechanisms of SdeA-mediated Ub modification and phosphoribosyl-linked ubiquitination remain unknown. Here we report the structures of SdeA in its ligand-free, Ub-bound and Ub-NADH-bound states. The structures reveal that the mART and PDE domains of SdeA form a catalytic domain over its C-terminal region. Upon Ub binding, the canonical ADP-ribosyltransferase toxin turn-turn (ARTT) and phosphate-nicotinamide (PN) loops in the mART domain of SdeA undergo marked conformational changes. The Ub Arg72 might act as a 'probe' that interacts with the mART domain first, and then movements may occur in the side chains of Arg72 and Arg42 during the ADP-ribosylation of Ub. Our study reveals the mechanism of SdeA-mediated Ub modification and provides a framework for further investigations into the phosphoribosyl-linked ubiquitination process.

Histone deacetylase and Cullin3-REN(KCTD11) ubiquitin ligase interplay regulates Hedgehog signalling through Gli acetylation.

PubMed

Canettieri, Gianluca; Di Marcotullio, Lucia; Greco, Azzura; Coni, Sonia; Antonucci, Laura; Infante, Paola; Pietrosanti, Laura; De Smaele, Enrico; Ferretti, Elisabetta; Miele, Evelina; Pelloni, Marianna; De Simone, Giuseppina; Pedone, Emilia Maria; Gallinari, Paola; Giorgi, Alessandra; Steinkühler, Christian; Vitagliano, Luigi; Pedone, Carlo; Schinin, M Eugenià; Screpanti, Isabella; Gulino, Alberto

2010-02-01

Hedgehog signalling is crucial for development and is deregulated in several tumours, including medulloblastoma. Regulation of the transcriptional activity of Gli (glioma-associated oncogene) proteins, effectors of the Hedgehog pathway, is poorly understood. We show here that Gli1 and Gli2 are acetylated proteins and that their HDAC-mediated deacetylation promotes transcriptional activation and sustains a positive autoregulatory loop through Hedgehog-induced upregulation of HDAC1. This mechanism is turned off by HDAC1 degradation through an E3 ubiquitin ligase complex formed by Cullin3 and REN, a Gli antagonist lost in human medulloblastoma. Whereas high HDAC1 and low REN expression in neural progenitors and medulloblastomas correlates with active Hedgehog signalling, loss of HDAC activity suppresses Hedgehog-dependent growth of neural progenitors and tumour cells. Consistent with this, abrogation of Gli1 acetylation enhances cellular proliferation and transformation. These data identify an integrated HDAC- and ubiquitin-mediated circuitry, where acetylation of Gli proteins functions as an unexpected key transcriptional checkpoint of Hedgehog signalling.

Activation of duck RIG-I by TRIM25 is independent of anchored ubiquitin.

PubMed

Miranzo-Navarro, Domingo; Magor, Katharine E

2014-01-01

Retinoic acid inducible gene I (RIG-I) is a viral RNA sensor crucial in defense against several viruses including measles, influenza A and hepatitis C. RIG-I activates type-I interferon signalling through the adaptor for mitochondrial antiviral signaling (MAVS). The E3 ubiquitin ligase, tripartite motif containing protein 25 (TRIM25), activates human RIG-I through generation of anchored K63-linked polyubiquitin chains attached to lysine 172, or alternatively, through the generation of unanchored K63-linked polyubiquitin chains that interact non-covalently with RIG-I CARD domains. Previously, we identified RIG-I of ducks, of interest because ducks are the host and natural reservoir of influenza viruses, and showed it initiates innate immune signaling leading to production of interferon-beta (IFN-β). We noted that K172 is not conserved in RIG-I of ducks and other avian species, or mouse. Because K172 is important for both mechanisms of activation of human RIG-I, we investigated whether duck RIG-I was activated by TRIM25, and if other residues were the sites for attachment of ubiquitin. Here we show duck RIG-I CARD domains are ubiquitinated for activation, and ubiquitination depends on interaction with TRIM25, as a splice variant that cannot interact with TRIM25 is not ubiquitinated, and cannot be activated. We expressed GST-fusion proteins of duck CARD domains and characterized TRIM25 modifications of CARD domains by mass spectrometry. We identified two sites that are ubiquitinated in duck CARD domains, K167 and K193, and detected K63 linked polyubiquitin chains. Site directed mutagenesis of each site alone, does not alter the ubiquitination profile of the duck CARD domains. However, mutation of both sites resulted in loss of all attached ubiquitin and polyubiquitin chains. Remarkably, the double mutant duck RIG-I CARD still interacts with TRIM25, and can still be activated. Our results demonstrate that anchored ubiquitin chains are not necessary for TRIM25

Activation of Duck RIG-I by TRIM25 Is Independent of Anchored Ubiquitin

PubMed Central

Miranzo-Navarro, Domingo; Magor, Katharine E.

2014-01-01

Retinoic acid inducible gene I (RIG-I) is a viral RNA sensor crucial in defense against several viruses including measles, influenza A and hepatitis C. RIG-I activates type-I interferon signalling through the adaptor for mitochondrial antiviral signaling (MAVS). The E3 ubiquitin ligase, tripartite motif containing protein 25 (TRIM25), activates human RIG-I through generation of anchored K63-linked polyubiquitin chains attached to lysine 172, or alternatively, through the generation of unanchored K63-linked polyubiquitin chains that interact non-covalently with RIG-I CARD domains. Previously, we identified RIG-I of ducks, of interest because ducks are the host and natural reservoir of influenza viruses, and showed it initiates innate immune signaling leading to production of interferon-beta (IFN-β). We noted that K172 is not conserved in RIG-I of ducks and other avian species, or mouse. Because K172 is important for both mechanisms of activation of human RIG-I, we investigated whether duck RIG-I was activated by TRIM25, and if other residues were the sites for attachment of ubiquitin. Here we show duck RIG-I CARD domains are ubiquitinated for activation, and ubiquitination depends on interaction with TRIM25, as a splice variant that cannot interact with TRIM25 is not ubiquitinated, and cannot be activated. We expressed GST-fusion proteins of duck CARD domains and characterized TRIM25 modifications of CARD domains by mass spectrometry. We identified two sites that are ubiquitinated in duck CARD domains, K167 and K193, and detected K63 linked polyubiquitin chains. Site directed mutagenesis of each site alone, does not alter the ubiquitination profile of the duck CARD domains. However, mutation of both sites resulted in loss of all attached ubiquitin and polyubiquitin chains. Remarkably, the double mutant duck RIG-I CARD still interacts with TRIM25, and can still be activated. Our results demonstrate that anchored ubiquitin chains are not necessary for TRIM25

Structural insight into SUMO chain recognition and manipulation by the ubiquitin ligase RNF4

PubMed Central

Xu, Yingqi; Plechanovová, Anna; Simpson, Peter; Marchant, Jan; Leidecker, Orsolya; Kraatz, Sebastian; Hay, Ronald T.; Matthews, Steve J.

2014-01-01

The small ubiquitin-like modifier (SUMO) can form polymeric chains that are important signals in cellular processes such as meiosis, genome maintenance and stress response. The SUMO-targeted ubiquitin ligase RNF4 engages with SUMO chains on linked substrates and catalyses their ubiquitination, which targets substrates for proteasomal degradation. Here we use a segmental labelling approach combined with solution nuclear magnetic resonance (NMR) spectroscopy and biochemical characterization to reveal how RNF4 manipulates the conformation of the SUMO chain, thereby facilitating optimal delivery of the distal SUMO domain for ubiquitin transfer. PMID:24969970

SUMOylation Regulates the Homologous to E6-AP Carboxyl Terminus (HECT) Ubiquitin Ligase Rsp5p*

PubMed Central

Novoselova, Tatiana Vladislavovna; Rose, Ruth-Sarah; Marks, Helen Margaret; Sullivan, James Andrew

2013-01-01

The post-translational modifiers ubiquitin and small ubiquitin-related modifier (SUMO) regulate numerous critical signaling pathways and are key to controlling the cellular fate of proteins in eukaryotes. The attachment of ubiquitin and SUMO involves distinct, but related, machinery. However, it is now apparent that many substrates can be modified by both ubiquitin and SUMO and that some regulatory interaction takes place between the respective attachment machinery. Here, we demonstrate that the Saccharomyces cerevisiae ubiquitin ligase Rsp5p, a member of the highly conserved Nedd4 family of ubiquitin ligases, is SUMOylated in vivo. We further show that Rsp5p SUMOylation is mediated by the SUMO ligases Siz1p and Siz2p, members of the conserved family of PIAS SUMO ligases that are, in turn, substrates for Rsp5p-mediated ubiquitylation. Our experiments show that SUMOylated Rsp5p has reduced ubiquitin ligase activity, and similarly, ubiquitylated Siz1p demonstrates reduced SUMO ligase activity leading to respective changes in both ubiquitin-mediated sorting of the manganese transporter Smf1p and polySUMO chain formation. This reciprocal regulation of these highly conserved ligases represents an exciting and previously unidentified system of cross talk between the ubiquitin and SUMO systems. PMID:23443663

An essential role of ubiquitination in Cbl-mediated negative regulation of the Src-family kinase Fyn

PubMed Central

Rao, Navin; Ghosh, Amiya K.; Douillard, Patrice; Andoniou, Christopher E.; Zhou, Pengcheng; Band, Hamid

2009-01-01

SUMMARY The Cbl family of ubiquitin ligases function as negative regulators of activated receptor tyrosine kinases by facilitating their ubiquitination and subsequent lysosomal targeting. Here, we have investigated the role of Cbl ubiquitin ligase activity in the negative regulation of a non-receptor tyrosine kinase, the Src-family kinase Fyn. Using primary embryonic fibroblasts from Cbl+/+ and Cbl−/− mice, we demonstrate that endogenous Cbl mediates the ubiquitination of Fyn and dictates the rate of Fyn turnover. By analyzing CHO-TS20 cells with a temperature-sensitive ubiquitin activating enzyme, we demonstrate that intact cellular ubiquitin machinery is required for Cbl-induced degradation of Fyn. Analyses of Cbl mutants, with mutations in or near the RING finger domain, in 293T cells revealed that the ubiquitin ligase activity of Cbl is essential for Cbl-induced degradation of Fyn by the proteasome pathway. Finally, use of a SRE-luciferase reporter demonstrated that Cbl-dependent negative regulation of Fyn function requires the region of Cbl that mediates the ubiquitin ligase activity. Given the conservation of structure between various Src-family kinases and the ability of Cbl to interact with multiple members of this family, Cbl-dependent ubiquitination could serve a general role to negatively regulate activated Src-family kinases. PMID:19966925

The Superimposed Deubiquitination Effect of OTULIN and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Nsp11 Promotes Multiplication of PRRSV.

PubMed

Su, Yanxin; Shi, Peidian; Zhang, Lilin; Lu, Dong; Zhao, Chengxue; Li, Ruiqiao; Zhang, Lei; Huang, Jinhai

2018-05-01

Linear ubiquitination plays an important role in the regulation of the immune response by regulating nuclear factor κB (NF-κB). The linear ubiquitination-specific deubiquitinase ovarian tumor domain deubiquitinase with linear linkage specificity (OTULIN) can control the immune signaling transduction pathway by restricting the Met1-linked ubiquitination process. In our study, the porcine OTLLIN gene was cloned and deubiquitin functions were detected in a porcine reproductive and respiratory syndrome virus (PRRSV)-infected-cell model. PRRSV infection promotes the expression of the OTULIN gene; in turn, overexpression of OTULIN contributes to PRRSV proliferation. There is negative regulation of innate immunity with OTULIN during viral infection. The cooperative effects of swine OTULIN and PRRSV Nsp11 potentiate the ability to reduce levels of cellular protein ubiquitin associated with innate immunity. Importantly, PRRSV Nsp11 recruits OTULIN through a nonenzymatic combination to enhance its ability to remove linear ubiquitination targeting NEMO, resulting in a superimposed effect that inhibits the production of type I interferons (IFNs). Our report presents a new model of virus utilization of the ubiquitin-protease system in vivo from the perspective of the viral proteins that interact with cell deubiquitination enzymes, providing new ideas for prevention and control of PRRSV. IMPORTANCE Deubiquitination effects of swine OTULIN were identified. The interaction between porcine OTULIN and PRRSV Nsp11 is dependent on the OTU domain. PRRSV Nsp11 recruits OTULIN through a nonenzymatic combination to promote removal of linear ubiquitination targeting NEMO, resulting in a superimposed effect that inhibits the production of type I IFNs. Copyright © 2018 American Society for Microbiology.

Ubiquitin recognition by FAAP20 expands the complex interface beyond the canonical UBZ domain

PubMed Central

Wojtaszek, Jessica L.; Wang, Su; Kim, Hyungjin; Wu, Qinglin; D'Andrea, Alan D.; Zhou, Pei

2014-01-01

FAAP20 is an integral component of the Fanconi anemia core complex that mediates the repair of DNA interstrand crosslinks. The ubiquitin-binding capacity of the FAAP20 UBZ is required for recruitment of the Fanconi anemia complex to interstrand DNA crosslink sites and for interaction with the translesion synthesis machinery. Although the UBZ–ubiquitin interaction is thought to be exclusively encapsulated within the ββα module of UBZ, we show that the FAAP20–ubiquitin interaction extends beyond such a canonical zinc-finger motif. Instead, ubiquitin binding by FAAP20 is accompanied by transforming a disordered tail C-terminal to the UBZ of FAAP20 into a rigid, extended β-loop that latches onto the complex interface of the FAAP20 UBZ and ubiquitin, with the invariant C-terminal tryptophan emanating toward I44Ub for enhanced binding specificity and affinity. Substitution of the C-terminal tryptophan with alanine in FAAP20 not only abolishes FAAP20–ubiquitin binding in vitro, but also causes profound cellular hypersensitivity to DNA interstrand crosslink lesions in vivo, highlighting the indispensable role of the C-terminal tail of FAAP20, beyond the compact zinc finger module, toward ubiquitin recognition and Fanconi anemia complex-mediated DNA interstrand crosslink repair. PMID:25414354

Derlin-1 promotes ubiquitylation and degradation of the epithelial Na+ channel, ENaC.

PubMed

You, Hui; Ge, Yamei; Zhang, Jian; Cao, Yizhi; Xing, Jing; Su, Dongming; Huang, Yujie; Li, Min; Qu, Shen; Sun, Fei; Liang, Xiubin

2017-03-15

Ubiquitylation of the epithelial Na + channel (ENaC) plays a critical role in cellular functions, including transmembrane transport of Na + , Na + and water balance, and blood pressure stabilization. Published studies have suggested that ENaC subunits are targets of ER-related degradation (ERAD) in yeast systems. However, the molecular mechanism underlying proteasome-mediated degradation of ENaC subunits remains to be established. Derlin-1, an E3 ligase mediator, links recognized target proteins to ubiquitin-mediated proteasomal degradation in the cytosol. In the present study, we found that derlin-1 suppressed the expression of ENaC at the protein level and that the subunit α-ENaC (also known as SCNN1A) physically interacted with derlin-1 at the membrane-anchored domains or the loop regions, and that derlin-1 initiated α-ENaC retrotranslocation. In addition, HUWE1, an endoplasmic reticulum (ER)-resident E3 ubiquitin ligase, was recruited and promoted K11-linked polyubiquitylation of α-ENaC and, hence, formation of an α-ENaC ubiquitin-mediated degradation complex. These findings suggest that derlin-1 promotes ENaC ubiquitylation and enhances ENaC ubiquitin- mediated proteasome degradation. The derlin-1 pathway therefore may represent a significant early checkpoint in the recognition and degradation of ENaC in mammalian cells. © 2017. Published by The Company of Biologists Ltd.

Regulation of Synaptic Structure by the Ubiquitin C-terminal Hydrolase UCH-L1

PubMed Central

Cartier, Anna E.; Djakovic, Stevan N.; Salehi, Afshin; Wilson, Scott M.; Masliah, Eliezer; Patrick, Gentry N.

2009-01-01

UCH-L1 is a de-ubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We have found that UCH-L1 activity is rapidly up-regulated by NMDA receptor activation which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of pre and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1 inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner. PMID:19535597

The autoimmunity-associated gene PTPN22 potentiates toll-like receptor-driven, type 1 interferon-dependent immunity.

PubMed

Wang, Yaya; Shaked, Iftach; Stanford, Stephanie M; Zhou, Wenbo; Curtsinger, Julie M; Mikulski, Zbigniew; Shaheen, Zachary R; Cheng, Genhong; Sawatzke, Kristy; Campbell, Amanda M; Auger, Jennifer L; Bilgic, Hatice; Shoyama, Fernanda M; Schmeling, David O; Balfour, Henry H; Hasegawa, Kiminori; Chan, Andrew C; Corbett, John A; Binstadt, Bryce A; Mescher, Matthew F; Ley, Klaus; Bottini, Nunzio; Peterson, Erik J

2013-07-25

Immune cells sense microbial products through Toll-like receptors (TLR), which trigger host defense responses including type 1 interferons (IFNs) secretion. A coding polymorphism in the protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene is a susceptibility allele for human autoimmune and infectious disease. We report that Ptpn22 selectively regulated type 1 IFN production after TLR engagement in myeloid cells. Ptpn22 promoted host antiviral responses and was critical for TLR agonist-induced, type 1 IFN-dependent suppression of inflammation in colitis and arthritis. PTPN22 directly associated with TNF receptor-associated factor 3 (TRAF3) and promotes TRAF3 lysine 63-linked ubiquitination. The disease-associated PTPN22W variant failed to promote TRAF3 ubiquitination, type 1 IFN upregulation, and type 1 IFN-dependent suppression of arthritis. The findings establish a candidate innate immune mechanism of action for a human autoimmunity "risk" gene in the regulation of host defense and inflammation. Copyright © 2013 Elsevier Inc. All rights reserved.

Keap1/Cullin3 Modulates p62/SQSTM1 Activity via UBA domain Ubiquitination

PubMed Central

Lee, YouJin; Chou, Tsui-Fen; Pittman, Sara K.; Keith, Amy L.; Razani, Babak; Weihl, Conrad C.

2017-01-01

Summary p62/SQSTM1 (p62) is a scaffolding protein that facilitates the formation and degradation of ubiquitinated aggregates via its self-interaction and ubiquitin binding domains. The regulation of this process is unclear but may relate to the post-translational modification of p62. In the present study, we find that Keap1/Cullin3 ubiquitinates p62 at lysine 420 within its UBA domain. Substitution of lysine 420 with an arginine diminishes p62 sequestration and degradation activity similar to that seen when the UBA domain is deleted. Overexpression of Keap1/Cullin3 in p62-WT expressing cells increases ubiquitinated inclusion formation, p62’s association with LC3 and rescues proteotoxicity. This effect is not seen in cells expressing a mutant p62 that fails to interact with Keap1. Interestingly, p62 disease mutants have diminished or absent UBA domain ubiquitination. These data suggest that the ubiquitination of p62’s UBA domain at lysine 420 may regulate p62’s function and be disrupted in p62 associated disease. PMID:28380357

Ubiquitin-like domains can target to the proteasome but proteolysis requires a disordered region.

PubMed

Yu, Houqing; Kago, Grace; Yellman, Christopher M; Matouschek, Andreas

2016-07-15

Ubiquitin and some of its homologues target proteins to the proteasome for degradation. Other ubiquitin-like domains are involved in cellular processes unrelated to the proteasome, and proteins containing these domains remain stable in the cell. We find that the 10 yeast ubiquitin-like domains tested bind to the proteasome, and that all 11 identified domains can target proteins for degradation. Their apparent proteasome affinities are not directly related to their stabilities or functions. That is, ubiquitin-like domains in proteins not part of the ubiquitin proteasome system may bind the proteasome more tightly than domains in proteins that are bona fide components. We propose that proteins with ubiquitin-like domains have properties other than proteasome binding that confer stability. We show that one of these properties is the absence of accessible disordered regions that allow the proteasome to initiate degradation. In support of this model, we find that Mdy2 is degraded in yeast when a disordered region in the protein becomes exposed and that the attachment of a disordered region to Ubp6 leads to its degradation. © 2016 The Authors.

The catalytic region and PEST domain of PTPN18 distinctly regulate the HER2 phosphorylation and ubiquitination barcodes.

PubMed

Wang, Hong-Mei; Xu, Yun-Fei; Ning, Shang-Lei; Yang, Du-Xiao; Li, Yi; Du, Yu-Jie; Yang, Fan; Zhang, Ya; Liang, Nan; Yao, Wei; Zhang, Ling-Li; Gu, Li-Chuan; Gao, Cheng-Jiang; Pang, Qi; Chen, Yu-Xin; Xiao, Kun-Hong; Ma, Rong; Yu, Xiao; Sun, Jin-Peng

2014-09-01

The tyrosine phosphorylation barcode encoded in C-terminus of HER2 and its ubiquitination regulate diverse HER2 functions. PTPN18 was reported as a HER2 phosphatase; however, the exact mechanism by which it defines HER2 signaling is not fully understood. Here, we demonstrate that PTPN18 regulates HER2-mediated cellular functions through defining both its phosphorylation and ubiquitination barcodes. Enzymologic characterization and three crystal structures of PTPN18 in complex with HER2 phospho-peptides revealed the molecular basis for the recognition between PTPN18 and specific HER2 phosphorylation sites, which assumes two distinct conformations. Unique structural properties of PTPN18 contribute to the regulation of sub-cellular phosphorylation networks downstream of HER2, which are required for inhibition of HER2-mediated cell growth and migration. Whereas the catalytic domain of PTPN18 blocks lysosomal routing and delays the degradation of HER2 by dephosphorylation of HER2 on pY(1112), the PEST domain of PTPN18 promotes K48-linked HER2 ubiquitination and its rapid destruction via the proteasome pathway and an HER2 negative feedback loop. In agreement with the negative regulatory role of PTPN18 in HER2 signaling, the HER2/PTPN18 ratio was correlated with breast cancer stage. Taken together, our study presents a structural basis for selective HER2 dephosphorylation, a previously uncharacterized mechanism for HER2 degradation and a novel function for the PTPN18 PEST domain. The new regulatory role of the PEST domain in the ubiquitination pathway will broaden our understanding of the functions of other important PEST domain-containing phosphatases, such as LYP and PTPN12.

E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue.

PubMed

Lim, Jung Hwa; Shin, Hee Won; Chung, Kyung-Sook; Kim, Nam-Soon; Kim, Ju Hee; Jung, Hong-Ryul; Im, Dong-Soo; Jung, Cho-Rok

Here, we show that E2-EPF ubiquitin carrier protein (UCP) elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL) and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196). A UCP mutant in which Cys118 was changed to alanine (UCPC118A) did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation.

E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

PubMed Central

Lim, Jung Hwa; Shin, Hee Won; Chung, Kyung-Sook; Kim, Nam-Soon; Kim, Ju Hee; Jung, Hong-Ryul; Im, Dong-Soo; Jung, Cho-Rok

2016-01-01

Here, we show that E2-EPF ubiquitin carrier protein (UCP) elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL) and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196). A UCP mutant in which Cys118 was changed to alanine (UCPC118A) did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation. PMID:27685940

Discovery of a Keap1-dependent peptide PROTAC to knockdown Tau by ubiquitination-proteasome degradation pathway.

PubMed

Lu, Mengchen; Liu, Tian; Jiao, Qiong; Ji, Jianai; Tao, Mengmin; Liu, Yijun; You, Qidong; Jiang, Zhengyu

2018-02-25

Induced protein degradation by PROTACs has emerged as a promising strategy to target nonenzymatic proteins inside the cell. The aim of this study was to identify Keap1, a substrate adaptor protein for ubiquitin E3 ligase involved in oxidative stress regulation, as a novel candidate for PROTACs that can be applied in the degradation of the nonenzymatic protein Tau. A peptide PROTAC by recruiting Keap1-Cul3 ubiquitin E3 ligase was developed and applied in the degradation of intracellular Tau. Peptide 1 showed strong in vitro binding with Keap1 and Tau. With proper cell permeability, peptide 1 was found to colocalize with cellular Keap1 and resulted in the coimmunoprecipitation of Tau and Keap1. The results of flow cytometry and western blotting assays showed that peptide 1 can downregulate the intracellular Tau level in both time- and concentration-dependent manner. The application of Keap1 siRNA silencing and the proteasome inhibitor MG132 confirmed that peptide 1 could promote the Keap1-dependent poly-ubiquitination and proteasome-dependent degradation of Tau. The results suggested that using PROTACs to recruit Keap1 to induce the degradation of Tau may show promising character in the treatment of neurodegenerative disease. Besides, our research demonstrated that Keap1 should be a promising E3 ligase adaptor to be used in the design of novel PROTACs. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Enzyme-substrate relationships in the ubiquitin system: approaches for identifying substrates of ubiquitin ligases.

PubMed

O'Connor, Hazel F; Huibregtse, Jon M

2017-09-01

Protein ubiquitylation is an important post-translational modification, regulating aspects of virtually every biochemical pathway in eukaryotic cells. Hundreds of enzymes participate in the conjugation and deconjugation of ubiquitin, as well as the recognition, signaling functions, and degradation of ubiquitylated proteins. Regulation of ubiquitylation is most commonly at the level of recognition of substrates by E3 ubiquitin ligases. Characterization of the network of E3-substrate relationships is a major goal and challenge in the field, as this expected to yield fundamental biological insights and opportunities for drug development. There has been remarkable success in identifying substrates for some E3 ligases, in many instances using the standard protein-protein interaction techniques (e.g., two-hybrid screens and co-immunoprecipitations paired with mass spectrometry). However, some E3s have remained refractory to characterization, while others have simply not yet been studied due to the sheer number and diversity of E3s. This review will discuss the range of tools and techniques that can be used for substrate profiling of E3 ligases.

Dual RING E3 Architectures Regulate Multiubiquitination and Ubiquitin Chain Elongation by APC/C.

PubMed

Brown, Nicholas G; VanderLinden, Ryan; Watson, Edmond R; Weissmann, Florian; Ordureau, Alban; Wu, Kuen-Phon; Zhang, Wei; Yu, Shanshan; Mercredi, Peter Y; Harrison, Joseph S; Davidson, Iain F; Qiao, Renping; Lu, Ying; Dube, Prakash; Brunner, Michael R; Grace, Christy R R; Miller, Darcie J; Haselbach, David; Jarvis, Marc A; Yamaguchi, Masaya; Yanishevski, David; Petzold, Georg; Sidhu, Sachdev S; Kuhlman, Brian; Kirschner, Marc W; Harper, J Wade; Peters, Jan-Michael; Stark, Holger; Schulman, Brenda A

2016-06-02

Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3-E2-substrate-UB architectures control different forms of polyubiquitination. Copyright © 2016 Elsevier Inc. All rights reserved.

The role of spartin and its novel ubiquitin binding region in DALIS occurrence

PubMed Central

Karlsson, Amelia B.; Washington, Jacqueline; Dimitrova, Valentina; Hooper, Christopher; Shekhtman, Alexander; Bakowska, Joanna C.

2014-01-01

Troyer syndrome is an autosomal recessive hereditary spastic paraplegia (HSP) caused by frameshift mutations in the SPG20 gene that results in a lack of expression of the truncated protein. Spartin is a multifunctional protein, yet only two conserved domains—a microtubule-interacting and trafficking domain and a plant-related senescence domain involved in cytokinesis and mitochondrial physiology, respectively—have been defined. We have shown that overexpressed spartin binds to the Ile44 hydrophobic pocket of ubiquitin, suggesting spartin might contain a ubiquitin-binding domain. In the present study, we demonstrate that spartin contributes to the formation of dendritic aggresome-like induced structures (DALIS) through a unique ubiquitin-binding region (UBR). Using short hairpin RNA, we knocked down spartin in RAW264.7 cells and found that DALIS frequency decreased; conversely, overexpression of spartin increased the percentage of cells containing DALIS. Using nuclear magnetic resonance spectroscopy, we characterized spartin's UBR and defined the UBR's amino acids that are key for ubiquitin binding. We also found that spartin, via the UBR, binds Lys-63–linked ubiquitin chains but does not bind Lys-48–linked ubiquitin chains. Finally, we demonstrate that spartin's role in DALIS formation depends on key residues within its UBR. PMID:24523286

Rates of ubiquitin conjugation increase when muscles atrophy, largely through activation of the N-end rule pathway

NASA Technical Reports Server (NTRS)

Solomon, V.; Baracos, V.; Sarraf, P.; Goldberg, A. L.

1998-01-01

The rapid loss of muscle mass that accompanies many disease states, such as cancer or sepsis, is primarily a result of increased protein breakdown in muscle, and several observations have suggested an activation of the ubiquitin-proteasome system. Accordingly, in extracts of atrophying muscles from tumor-bearing or septic rats, rates of 125I-ubiquitin conjugation to endogenous proteins were found to be higher than in control extracts. On the other hand, in extracts of muscles from hypothyroid rats, where overall proteolysis is reduced below normal, the conjugation of 125I-ubiquitin to soluble proteins decreased by 50%, and treatment with triiodothyronine (T3) restored ubiquitination to control levels. Surprisingly, the N-end rule pathway, which selectively degrades proteins with basic or large hydrophobic N-terminal residues, was found to be responsible for most of these changes in ubiquitin conjugation. Competitive inhibitors of this pathway that specifically block the ubiquitin ligase, E3alpha, suppressed most of the increased ubiquitin conjugation in the muscle extracts from tumor-bearing and septic rats. These inhibitors also suppressed ubiquitination in normal extracts toward levels in hypothyroid extracts, which showed little E3alpha-dependent ubiquitination. Thus, the inhibitors eliminated most of the differences in ubiquitination under these different pathological conditions. Moreover, 125I-lysozyme, a model N-end rule substrate, was ubiquitinated more rapidly in extracts from tumor-bearing and septic rats, and more slowly in those from hypothyroid rats, than in controls. Thus, the rate of ubiquitin conjugation increases in atrophying muscles, and these hormone- and cytokine-dependent responses are in large part due to activation of the N-end rule pathway.

Parkin-catalyzed Ubiquitin-Ester Transfer Is Triggered by PINK1-dependent Phosphorylation*

PubMed Central

Iguchi, Masahiro; Kujuro, Yuki; Okatsu, Kei; Koyano, Fumika; Kosako, Hidetaka; Kimura, Mayumi; Suzuki, Norihiro; Uchiyama, Shinichiro; Tanaka, Keiji; Matsuda, Noriyuki

2013-01-01

PINK1 and PARKIN are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation. PMID:23754282

RNF8- and Ube2S-Dependent Ubiquitin Lysine 11-Linkage Modification in Response to DNA Damage.

PubMed

Paul, Atanu; Wang, Bin

2017-05-18

Ubiquitin modification of proteins plays pivotal roles in the cellular response to DNA damage. Given the complexity of ubiquitin conjugation due to the formation of poly-conjugates of different linkages, functional roles of linkage-specific ubiquitin modification at DNA damage sites are largely unclear. We identify that Lys11-linkage ubiquitin modification occurs at DNA damage sites in an ATM-dependent manner, and ubiquitin-modifying enzymes, including Ube2S E2-conjugating enzyme and RNF8 E3 ligase, are responsible for the assembly of Lys11-linkage conjugates on damaged chromatin, including histone H2A/H2AX. We show that RNF8- and Ube2S-dependent Lys11-linkage ubiquitin conjugation plays an important role in regulating DNA damage-induced transcriptional silencing, distinct from the role of Lys63-linkage ubiquitin in the recruitment of DNA damage repair proteins 53BP1 and BRCA1. Thus, our study highlights the importance of linkage-specific ubiquitination at DNA damage sites, and it reveals that Lys11-linkage ubiquitin modification plays a crucial role in the DNA damage response. Copyright © 2017 Elsevier Inc. All rights reserved.

Bioinformatic analyses implicate the collaborating meiotic crossover/chiasma proteins Zip2, Zip3, and Spo22/Zip4 in ubiquitin labeling

PubMed Central

Perry, Jason; Kleckner, Nancy; Börner, G. Valentin

2005-01-01

Zip2 and Zip3 are meiosis-specific proteins that, in collaboration with several partners, act at the sites of crossover-designated, axis-associated recombinational interactions to mediate crossover/chiasma formation. Here, Spo22 (also called Zip4) is identified as a probable functional collaborator of Zip2/3. The molecular roles of Zip2, Zip3, and Spo22/Zip4 are unknown. All three proteins are part of a small evolutionary cohort comprising similar homologs in four related yeasts. Zip3 is shown to contain a RING finger whose structural features most closely match those of known ubiquitin E3s. Further, Zip3 exhibits major domainal homologies to Rad18, a known DNA-binding ubiquitin E3. Also described is an approach to the identification and mapping of repeated protein sequence motifs, Alignment Based Repeat Annotation (ABRA), that we have developed. When ABRA is applied to Zip2 and Spo22/Zip4, they emerge as a 14-blade WD40-like repeat protein and a 22-unit tetratricopeptide repeat protein, respectively. WD40 repeats of Cdc20, Cdh1, and Cdc16 and tetratricopeptide repeats of Cdc16, Cdc23, and Cdc27, all components of the anaphase-promoting complex, are also analyzed. These and other findings suggest that Zip2, Zip3, and Zip4 act together to mediate a process that involves Zip3-mediated ubiquitin labeling, potentially as a unique type of ubiquitin-conjugating complex. PMID:16314568

Skeletal muscle and liver contain a soluble ATP + ubiquitin-dependent proteolytic system.

PubMed Central

Fagan, J M; Waxman, L; Goldberg, A L

1987-01-01

Although protein breakdown in most cells seems to require metabolic energy, it has only been possible to establish a soluble ATP-dependent proteolytic system in extracts of reticulocytes and erythroleukemia cells. We have now succeeded in demonstrating in soluble extracts and more purified preparations from rabbit skeletal muscle a 12-fold stimulation by ATP of breakdown of endogenous proteins and a 6-fold stimulation of 125I-lysozyme degradation. However, it has still not been possible to demonstrate such large effects of ATP in similar preparations from liver. Nevertheless, after fractionation by DEAE-chromatography and gel filtration, we found that extracts from liver as well as muscle contain both the enzymes which conjugate ubiquitin to 125I-lysozyme and an enzyme which specifically degrades the ubiquitin-protein conjugates. When this proteolytic activity was recombined with the conjugating enzymes, ATP + ubiquitin-dependent degradation of many proteins was observed. This proteinase is unusually large, approx. 1500 kDa, requires ATP hydrolysis for activity and resembles the ubiquitin-protein-conjugate degrading activity isolated from reticulocytes. Thus the ATP + ubiquitin-dependent pathway is likely to be present in all mammalian cells, although certain tissues may contain inhibitory factors. Images Fig. 2. PMID:2820375

Regulation of Protein Degradation by O-GlcNAcylation: Crosstalk with Ubiquitination*

PubMed Central

Ruan, Hai-Bin; Nie, Yongzhan; Yang, Xiaoyong

2013-01-01

The post-translational modification of intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc) regulates essential cellular processes such as signal transduction, transcription, translation, and protein degradation. Misfolded, damaged, and unwanted proteins are tagged with a chain of ubiquitin moieties for degradation by the proteasome, which is critical for cellular homeostasis. In this review, we summarize the current knowledge of the interplay between O-GlcNAcylation and ubiquitination in the control of protein degradation. Understanding the mechanisms of action of O-GlcNAcylation in the ubiquitin-proteosome system shall facilitate the development of therapeutics for human diseases such as cancer, metabolic syndrome, and neurodegenerative diseases. PMID:23824911

Inhibition of FOXO1/3 promotes vascular calcification.

PubMed

Deng, Liang; Huang, Lu; Sun, Yong; Heath, Jack M; Wu, Hui; Chen, Yabing

2015-01-01

Vascular calcification is a characteristic feature of atherosclerosis, diabetes mellitus, and end-stage renal disease. We have demonstrated that activation of protein kinase B (AKT) upregulates runt-related transcription factor 2 (Runx2), a key osteogenic transcription factor that is crucial for calcification of vascular smooth muscle cells (VSMC). Using mice with SMC-specific deletion of phosphatase and tensin homolog (PTEN), a major negative regulator of AKT, the present studies uncovered a novel molecular mechanism underlying PTEN/AKT/FOXO (forkhead box O)-mediated Runx2 upregulation and VSMC calcification. SMC-specific PTEN deletion mice were generated by crossing PTEN floxed mice with SM22α-Cre transgenic mice. The PTEN deletion resulted in sustained activation of AKT that upregulated Runx2 and promoted VSMC calcification in vitro and arterial calcification ex vivo. Runx2 knockdown did not affect proliferation but blocked calcification of the PTEN-deficient VSMC, suggesting that PTEN deletion promotes Runx2-depedent VSMC calcification that is independent of proliferation. At the molecular level, PTEN deficiency increased the amount of Runx2 post-transcriptionally by inhibiting Runx2 ubiquitination. AKT activation increased phosphorylation of FOXO1/3 that led to nuclear exclusion of FOXO1/3. FOXO1/3 knockdown in VSMC phenocopied the PTEN deficiency, demonstrating a novel function of FOXO1/3, as a downstream signaling of PTEN/AKT, in regulating Runx2 ubiquitination and VSMC calcification. Using heterozygous SMC-specific PTEN-deficient mice and atherogenic ApoE(-/-) mice, we further demonstrated AKT activation, FOXO phosphorylation, and Runx2 ubiquitination in vascular calcification in vivo. Our studies have determined a new causative effect of SMC-specific PTEN deficiency on vascular calcification and demonstrated that FOXO1/3 plays a crucial role in PTEN/AKT-modulated Runx2 ubiquitination and VSMC calcification. © 2014 American Heart Association, Inc.

Ubiquitin control of S phase: a new role for the ubiquitin conjugating enzyme, UbcH7

USDA-ARS?s Scientific Manuscript database

Events within and transitions between the phases of the eukaryotic cell cycle are tightly controlled by transcriptional and post-translational processes. Prominent among them is a profound role for the ubiquitin proteasome proteolytic pathway. The timely degradation of proteins balances the increase...

Ubiquitin phosphorylated at Ser57 hyper-activates parkin.

PubMed

George, Susanna; Wang, Sabrina M; Bi, Yumin; Treidlinger, Margot; Barber, Kathryn R; Shaw, Gary S; O'Donoghue, Patrick

2017-11-01

Malfunction of the ubiquitin (Ub) E3 ligase, parkin, leads to defects in mitophagy and protein quality control linked to Parkinson's disease. Parkin activity is stimulated by phosphorylation of Ub at Ser65 (pUb S65 ). Since the upstream kinase is only known for Ser65 (PINK1), the biochemical function of other phosphorylation sites on Ub remain largely unknown. We used fluorescently labelled and site-specifically phosphorylated Ub substrates to quantitatively relate the position and stoichiometry of Ub phosphorylation to parkin activation. Fluorescence measurements show that pUb S65 -stimulated parkin is 5-fold more active than auto-inhibited and un-stimulated parkin, which catalyzes a basal level of auto-ubiquitination. We consistently observed a low but detectable level of parkin activity with pUb S12 . Strikingly, pUb S57 hyper-activates parkin, and our data demonstrate that parkin is able to selectively synthesize poly-pUb S57 chains, even when 90% of the Ub in the reaction is un-phosphorylated. We further found that parkin ubiquitinates its physiological substrate Miro-1 with chains solely composed of pUb S65 and more efficiently with pUb S57 chains. Parkin hyper-activation by pUb S57 demonstrates the first PINK1-independent route to active parkin, revealing the roles of multiple ubiquitin phosphorylation sites in governing parkin stimulation and catalytic activity. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulation of amino acid transporter trafficking by mTORC1 in primary human trophoblast cells is mediated by the ubiquitin ligase Nedd4-2.

PubMed

Rosario, Fredrick J; Dimasuay, Kris Genelyn; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

2016-04-01

Changes in placental amino acid transfer directly contribute to altered fetal growth, which increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Placental amino acid transfer is critically dependent on the expression of specific transporters in the plasma membrane of the trophoblast, the transporting epithelium of the human placenta. However, the molecular mechanisms regulating this process are largely unknown. Nedd4-2 is an ubiquitin ligase that catalyses the ubiquitination of proteins, resulting in proteasomal degradation. We hypothesized that inhibition of mechanistic target of rapamycin complex 1 (mTORC1) decreases amino acid uptake in primary human trophoblast (PHT) cells by activation of Nedd4-2, which increases transporter ubiquitination resulting in decreased transporter expression in the plasma membrane. mTORC 1 inhibition increased the expression of Nedd4-2, promoted ubiquitination and decreased the plasma membrane expression of SNAT2 (an isoform of the System A amino acid transporter) and LAT1 (a System L amino acid transporter isoform), resulting in decreased cellular amino acid uptake. Nedd4-2 silencing markedly increased the trafficking of SNAT2 and LAT1 to the plasma membrane, which stimulated cellular amino acid uptake. mTORC1 inhibition by silencing of raptor failed to decrease amino acid transport following Nedd4-2 silencing. In conclusion, we have identified a novel link between mTORC1 signalling and ubiquitination, a common posttranslational modification. Because placental mTORC1 is inhibited in fetal growth restriction and activated in fetal overgrowth, we propose that regulation of placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth. © 2016 Authors; published by Portland Press Limited.

Human T Cell Leukemia Virus Type 2 Tax-Mediated NF-κB Activation Involves a Mechanism Independent of Tax Conjugation to Ubiquitin and SUMO

PubMed Central

Journo, Chloé; Bonnet, Amandine; Favre-Bonvin, Arnaud; Turpin, Jocelyn; Vinera, Jennifer; Côté, Emilie; Chevalier, Sébastien Alain; Kfoury, Youmna; Bazarbachi, Ali

2013-01-01

Permanent activation of the NF-κB pathway by the human T cell leukemia virus type 1 (HTLV-1) Tax (Tax1) viral transactivator is a key event in the process of HTLV-1-induced T lymphocyte immortalization and leukemogenesis. Although encoding a Tax transactivator (Tax2) that activates the canonical NF-κB pathway, HTLV-2 does not cause leukemia. These distinct pathological outcomes might be related, at least in part, to distinct NF-κB activation mechanisms. Tax1 has been shown to be both ubiquitinated and SUMOylated, and these two modifications were originally proposed to be required for Tax1-mediated NF-κB activation. Tax1 ubiquitination allows recruitment of the IKK-γ/NEMO regulatory subunit of the IKK complex together with Tax1 into centrosome/Golgi-associated cytoplasmic structures, followed by activation of the IKK complex and RelA/p65 nuclear translocation. Herein, we compared the ubiquitination, SUMOylation, and acetylation patterns of Tax2 and Tax1. We show that, in contrast to Tax1, Tax2 conjugation to endogenous ubiquitin and SUMO is barely detectable while both proteins are acetylated. Importantly, Tax2 is neither polyubiquitinated on lysine residues nor ubiquitinated on its N-terminal residue. Consistent with these observations, Tax2 conjugation to ubiquitin and Tax2-mediated NF-κB activation is not affected by overexpression of the E2 conjugating enzyme Ubc13. We further demonstrate that a nonubiquitinable, non-SUMOylable, and nonacetylable Tax2 mutant retains a significant ability to activate transcription from a NF-κB-dependent promoter after partial activation of the IKK complex and induction of RelA/p65 nuclear translocation. Finally, we also show that Tax2 does not interact with TRAF6, a protein that was shown to positively regulate Tax1-mediated activation of the NF-κB pathway. PMID:23135727

UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis.

PubMed

Xie, Youming; Varshavsky, Alexander

2002-12-01

The ubiquitin system recognizes degradation signals of protein substrates through E3-E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4(DeltaN), which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.

Structural and Functional Investigations of the N-Terminal Ubiquitin Binding Region of Usp25.

PubMed

Yang, Yuanyuan; Shi, Li; Ding, Yiluan; Shi, Yanhong; Hu, Hong-Yu; Wen, Yi; Zhang, Naixia

2017-05-23

Ubiquitin-specific protease 25 (Usp25) is a deubiquitinase that is involved in multiple biological processes. The N-terminal ubiquitin-binding region (UBR) of Usp25 contains one ubiquitin-associated domain, one small ubiquitin-like modifier (SUMO)-interacting motif and two ubiquitin-interacting motifs. Previous studies suggest that the covalent sumoylation in the UBR of Usp25 impairs its enzymatic activity. Here, we raise the hypothesis that non-covalent binding of SUMO, a prerequisite for efficient sumoylation, will impair Usp25's catalytic activity as well. To test our hypothesis and elucidate the underlying molecular mechanism, we investigated the structure and function of the Usp25 N-terminal UBR. The solution structure of Usp25 1-146 is obtained, and the key residues responsible for recognition of ubiquitin and SUMO2 are identified. Our data suggest inhibition of Usp25's catalytic activity upon the non-covalent binding of SUMO2 to the Usp25 SUMO-interacting motif. We also find that SUMO2 can competitively block the interaction between the Usp25 UBR and its ubiquitin substrates. Based on our findings, we have proposed a working model to depict the regulatory role of the Usp25 UBR in the functional display of the enzyme. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Proteasome-associated deubiquitinase ubiquitin-specific protease 14 regulates prostate cancer proliferation by deubiquitinating and stabilizing androgen receptor.

PubMed

Liao, Yuning; Liu, Ningning; Hua, Xianliang; Cai, Jianyu; Xia, Xiaohong; Wang, Xuejun; Huang, Hongbiao; Liu, Jinbao

2017-02-02

Androgen receptor (AR) is frequently over-expressed and plays a critical role in the growth and progression of human prostate cancer. The therapy attempting to target AR signalling was established in decades ago but the treatment of prostate cancer is far from being satisfactory. The assignable cause is that our understanding of the mechanism of AR regulation and re-activation remains incomplete. Increasing evidence suggests that deubiquitinases are involved in the regulation of cancer development and progression but the specific underlying mechanism often is not elucidated. In the current study, we have identified ubiquitin-specific protease 14 (USP14) as a novel regulator of AR, inhibiting the degradation of AR via deubiquitinating this oncoprotein in the androgen-responsive prostate cancer cells. We found that (i) USP14 could bind to AR, and additionally, both genetic and pharmacological inhibition of USP14 accelerated the ubiquitination and degradation of AR; (ii) downregulation or inhibition of USP14 suppressed cell proliferation and colony formation of LNcap cells and, conversely, overexpression of USP14 promoted the proliferation; and (iii) reduction or inhibition of USP14 induced G0/G1 phase arrest in LNcap prostate cancer cells. Hence, we conclude that USP14 promotes prostate cancer progression likely through stabilization of AR, suggesting that USP14 could be a promising therapeutic target for prostate cancer.

In Vivo Regulation of NGF-Mediated Functions by Nedd4-2 Ubiquitination of TrkA

PubMed Central