Cdc34p Ubiquitin-Conjugating Enzyme Is a Component of the Tombusvirus Replicase Complex and Ubiquitinates p33 Replication Protein

ABSTRACT To identify host proteins interacting with Tomato bushy stunt virus (TBSV) replication proteins in a genome-wide scale, we have used a yeast (Saccharomyces cerevisiae) proteome microarray carrying 4,088 purified proteins. This approach led to the identification of 58 yeast proteins that interacted with p33 replication protein. The identified host proteins included protein chaperones, ubiquitin-associated proteins, translation factors, RNA-modifying enzymes, and other proteins with yet-unknown functions. We confirmed that 19 of the identified host proteins bound to p33 in vitro or in a split-ubiquitin-based two-hybrid assay. Further analysis of Cdc34p E2 ubiquitin-conjugating enzyme, which is one of the host proteins interacting with p33, revealed that Cdc34p is a novel component of the purified viral replicase. Downregulation of Cdc34p expression in yeast, which supports replication of a TBSV replicon RNA (repRNA), reduced repRNA accumulation and the activity of the tombusvirus replicase by up to fivefold. Overexpression of wild-type Cdc34p, but not that of an E2-defective mutant of Cdc34p, increased repRNA accumulation, suggesting a significant role for the ubiquitin-conjugating enzyme function of Cdc34p in TBSV replication. Also, Cdc34p was able to ubiquitinate p33 in vitro. In addition, we have shown that p33 becomes ubiquitinated in vivo. We propose that ubiquitination of p33 likely alters its function or affects the recruitment of host factors during TBSV replication.

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OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.004677 ◽

2018 ◽

Vol 293 (47) ◽

pp. 18285-18295 ◽

Cited By ~ 11

Author(s):

Nagesh Pasupala ◽

Marie E. Morrow ◽

Lauren T. Que ◽

Barbara A. Malynn ◽

Averil Ma ◽

...

Keyword(s):

Polyubiquitin Chains ◽

Protein Ubiquitination ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzymes ◽

E2 Enzymes ◽

Ubiquitin Conjugating Enzymes ◽

In Cells ◽

Conjugating Enzyme

OTUB1 is a deubiquitinating enzyme that cleaves Lys-48–linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2∼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1–E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1−/− knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.

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HNF4α pathway mapping identifies wild-type IDH1 as a targetable metabolic node in gastric cancer

Gut ◽

10.1136/gutjnl-2018-318025 ◽

2019 ◽

Vol 69 (2) ◽

pp. 231-242 ◽

Cited By ~ 2

Author(s):

Chang Xu ◽

Wen Fong Ooi ◽

Aditi Qamra ◽

Jing Tan ◽

Benjamin Yan-Jiang Chua ◽

...

Keyword(s):

Gastric Cancer ◽

Target Genes ◽

Wild Type ◽

Isocitrate Dehydrogenase 1 ◽

Oncogenic Pathways ◽

A Genome ◽

Direct Target Gene ◽

Wide Scale

ObjectiveGastric cancer (GC) is a leading cause of cancer mortality. Previous studies have shown that hepatocyte nuclear factor-4α (HNF4α) is specifically overexpressed in GC and functionally required for GC development. In this study, we investigated, on a genome-wide scale, target genes of HNF4α and oncogenic pathways driven by HNF4α and HNF4α target genes.DesignWe performed HNF4α chromatin immunoprecipitation followed by sequencing across multiple GC cell lines, integrating HNF4α occupancy data with (epi)genomic and transcriptome data of primary GCs to define HNF4α target genes of in vitro and in vivo relevance. To investigate mechanistic roles of HNF4α and HNF4α targets, we performed cancer metabolic measurements, drug treatments and functional assays including murine xenograft experiments.ResultsGene expression analysis across 19 tumour types revealed HNF4α to be specifically upregulated in GCs. Unbiased pathway analysis revealed organic acid metabolism as the top HNF4α-regulated pathway, orthogonally supported by metabolomic analysis. Isocitrate dehydrogenase 1 (IDH1) emerged as a convergent HNF4α direct target gene regulating GC metabolism. We show that wild-type IDH1 is essential for GC cell survival, and that certain GC cells can be targeted by IDH1 inhibitors.ConclusionsOur results highlight a role for HNF4α in sustaining GC oncogenic metabolism, through the regulation of IDH1. Drugs targeting wild-type IDH1 may thus have clinical utility in GCs exhibiting HNF4α overexpression, expanding the role of IDH1 in cancer beyond IDH1/2 mutated malignancies.

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Functional Interaction between Human Herpesvirus 6 Immediate-Early 2 Protein and Ubiquitin-Conjugating Enzyme 9 in the Absence of Sumoylation

Journal of Virology ◽

10.1128/jvi.00375-06 ◽

2006 ◽

Vol 80 (20) ◽

pp. 10218-10228 ◽

Cited By ~ 21

Author(s):

Andru Tomoiu ◽

Annie Gravel ◽

Robert M. Tanguay ◽

Louis Flamand

Keyword(s):

Human Herpesvirus ◽

Immediate Early ◽

Human Herpesvirus 6 ◽

Interacting Protein ◽

Ubiquitin Conjugating Enzyme ◽

Herpesvirus 6 ◽

Transcriptional Complex ◽

Conjugating Enzyme

ABSTRACT The immediate-early 2 (IE2) protein of human herpesvirus 6 is a potent transactivator of cellular and viral promoters. To better understand the biology of IE2, we generated a LexA-IE2 fusion protein and screened, using the yeast two-hybrid system, a Jurkat T-cell cDNA library for proteins that could interact with IE2. The most frequently isolated IE2-interacting protein was the human ubiquitin-conjugating enzyme 9 (Ubc9), a protein involved in the small ubiquitin-like modifier (SUMO) conjugation pathway. Using deletion mutants of IE2, we mapped the IE2-Ubc9-interacting region to residues 989 to 1037 of IE2. The interaction was found to be of functional significance to IE2, as Ubc9 overexpression significantly repressed promoter activation by IE2. The C93S Ubc9 mutant exhibited a similar effect on IE2, indicating that the E2 SUMO-conjugating function of Ubc9 is not required for its repressive action on IE2. No consensus sumoylation sites or evidence of IE2 conjugation to SUMO could be demonstrated under in vivo or in vitro conditions. Moreover, expression levels and nuclear localization of IE2 were not altered by Ubc9 overexpression, suggesting that Ubc9's repressive function likely occurs at the transcriptional complex level. Overall, our results indicate that Ubc9 influences IE2's function and provide new information on the complex interactions that occur between herpesviruses and the sumoylation pathway.

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Inflammation-Induced Alternative Pre-mRNA Splicing in Mouse Alveolar Macrophages

G3 Genes|Genome|Genetics ◽

10.1534/g3.119.400935 ◽

2019 ◽

Vol 10 (2) ◽

pp. 555-567 ◽

Cited By ~ 3

Author(s):

William J. Janssen ◽

Thomas Danhorn ◽

Chelsea Harris ◽

Kara J. Mould ◽

Frank Fang-Yao Lee ◽

...

Keyword(s):

Alveolar Macrophages ◽

Inflammatory Responses ◽

Tricarboxylic Acid Cycle ◽

Mrna Splicing ◽

Dynamic Responses ◽

Chemokine Signaling ◽

A Genome ◽

Wide Scale

Alveolar macrophages serve as central orchestrators of inflammatory responses in the lungs, both initiating their onset and promoting their resolution. However, the mechanisms that program macrophages for these dynamic responses are not fully understood. Over 95% of all mammalian genes undergo alternative pre-mRNA splicing. While alternative splicing has been shown to regulate inflammatory responses in macrophages in vitro, it has not been investigated on a genome-wide scale in vivo. Here we used RNAseq to investigate alternative pre-mRNA splicing in alveolar macrophages isolated from lipopolysaccharide (LPS)-treated mice during the peak of inflammation and during its resolution. We found that lung inflammation induced substantial alternative pre-mRNA splicing in alveolar macrophages. The number of changes in isoform usage was greatest at the peak of inflammation and involved multiple classes of alternative pre-mRNA splicing events. Comparative pathway analysis of inflammation-induced changes in alternative pre-mRNA splicing and differential gene expression revealed overlap of pathways enriched for immune responses such as chemokine signaling and cellular metabolism. Moreover, alternative pre-mRNA splicing of genes in metabolic pathways differed in tissue resident vs. recruited (blood monocyte-derived) alveolar macrophages and corresponded to changes in core metabolism, including a switch to Warburg-like metabolism in recruited macrophages with increased glycolysis and decreased flux through the tricarboxylic acid cycle.

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Genetic and biochemical interactions between an essential kinetochore protein, Cbf2p/Ndc10p, and the CDC34 ubiquitin-conjugating enzyme.

Molecular and Cellular Biology ◽

10.1128/mcb.15.9.4835 ◽

1995 ◽

Vol 15 (9) ◽

pp. 4835-4842 ◽

Cited By ~ 20

Author(s):

H J Yoon ◽

J Carbon

Keyword(s):

Yeast Cells ◽

Temperature Sensitive ◽

Kinetochore Protein ◽

Ubiquitin Conjugating Enzyme ◽

Endogenous Substrate ◽

Growth Phenotype ◽

Kinetochore Function ◽

Conjugating Enzyme

CBF2/NDC10/CTF14 encodes the 110-kDa subunit of CBF3, a key component of the yeast centromere/kinetochore. Overexpression of yeast CDC34 specifically suppresses the temperature-sensitive growth phenotype of the ndc10-1 mutation. Mutations in CDC34, which specifies a ubiquitin-conjugating enzyme, arrest yeast cells in the G1 phase of the cell cycle, with no intact spindles formed (M. G. Goebl, J. Yochem, S. Jentsch, J. P. McGrath, A. Varshavsky, and B. Byers, Science 241:1331-1335, 1988). The cdc34-2 mutation drastically alters the pattern of Cbf2p modification. Results of experiments using antibodies against Cbf2p and ubiquitin indicate that Cbf2p is ubiquitinated in vivo. Purified Cdc34p catalyzes the formation of Cbf2p-monoubiquitin conjugate in vitro. These data suggest that Cbf2p is an endogenous substrate of the CDC34 ubiquitin-conjugating enzyme and imply that ubiquitination of a kinetochore protein plays a regulatory role in kinetochore function.

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Interaction with a Host Ubiquitin-Conjugating Enzyme Is Required for the Pathogenicity of a Geminiviral DNA β Satellite

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-6-0737 ◽

2009 ◽

Vol 22 (6) ◽

pp. 737-746 ◽

Cited By ~ 50

Author(s):

Omid Eini ◽

Satish Dogra ◽

Luke A. Selth ◽

Ian B. Dry ◽

John W. Randles ◽

...

Keyword(s):

Single Gene ◽

Bimolecular Fluorescence Complementation ◽

Cotton Leaf ◽

Specific Symptom ◽

Ubiquitin Conjugating Enzyme ◽

Ubiquitin Proteasome ◽

Ubiquitin Conjugating Enzymes ◽

Conjugating Enzyme

DNA β is a single-stranded satellite DNA which encodes a single gene, βC1. To better understand the role of βC1 in the pathogenicity of DNA β, a yeast two-hybrid screen of a tomato cDNA library was carried out using βC1 from Cotton leaf curl Multan virus (CLCuMV) DNA β as the bait. A ubiquitin-conjugating enzyme, designated SlUBC3, which functionally complemented a yeast mutant deficient in ubiquitin-conjugating enzymes was identified. The authenticity and specificity of the interaction between βC1 and SlUBC3 was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro, using a protein-binding assay. Analysis of deletion mutants of the βC1 protein showed that a myristoylation-like motif is required both for its interaction with SlUBC3 and the induction of DNA-β-specific symptoms in host plants. The level of polyubiquitinated proteins in transgenic tobacco plants expressing βC1 was found to be reduced compared with wild-type plants. These results are consistent with the hypothesis that interaction of βC1 with SlUBC3 is required for DNA-β-specific symptom induction, and that this is possibly due to downregulation of the host ubiquitin proteasome pathway.

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OTUB1 non-catalytically regulates the stability of the E2 ubiquitin conjugating enzyme UBE2E1

10.1101/359414 ◽

2018 ◽

Author(s):

Nagesh Pasupala ◽

Marie E. Morrow ◽

Lauren T. Que ◽

Barbara A. Malynn ◽

Averil Ma ◽

...

Keyword(s):

Catalytic Mechanism ◽

Polyubiquitin Chains ◽

Protein Ubiquitination ◽

Ubiquitin Conjugating Enzyme ◽

E2 Enzymes ◽

The Stability ◽

In Cells ◽

Conjugating Enzyme

AbstractOTUB1 is a deubiquitinating enzyme that cleaves K48-linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, non-catalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin conjugating enzymes and inhibits their activity by trapping the E2~ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to members of the UBE2D (UBCH5) and UBE2E families, as well as to UBC13 (UBE2N). Cellular roles have been identified for the interaction of OTUB1 with UBC13 and members of the UBE2D family, but not for UBE2E E2 enzymes. We report here a novel role for OTUB1-E2 interactions in modulating E2 protein ubiquitination. We find that depletion of OTUB1 dramatically destabilizes the E2 conjugating enzyme UBE2E1 (UBE2E1) in cells and that this effect is independent of the catalytic activity of OTUB1 but depends on the ability of OTUB1 to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitinationin vitroand in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we have found a new role for OTUB1 in rescuing specific E2s from degradationin vivo.

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Effect of Arabidopsis COP10 ubiquitin E2 enhancement activity across E2 families and functional conservation among its canonical homologues

Biochemical Journal ◽

10.1042/bj20081943 ◽

2009 ◽

Vol 418 (3) ◽

pp. 683-690 ◽

Cited By ~ 18

Author(s):

On Sun Lau ◽

Xing Wang Deng

Keyword(s):

Dna Damage ◽

Arabidopsis Thaliana ◽

Plant Development ◽

Functional Conservation ◽

Human Ubiquitin ◽

Ubiquitin Conjugating Enzyme ◽

Variant Protein ◽

Conjugating Enzyme

Arabidopsis thaliana COP10 (constitutive photomorphogenic 10) is a UEV [Ub (ubiquitin)-conjugating enzyme (E2) variant protein] that is required for repression of seedling photomorphogenesis in darkness. COP10 forms a complex {the CDD complex [COP10–DET1 (de-etiolated 1)–DDB1 (DNA damage binding protein 1) complex]} with DET1 and DDB1a in vivo and can enhance the activity of Ub-conjugating enzyme (E2) in vitro. To investigate whether COP10 might act as a general regulator of E2s, we tested the specificity of COP10 E2 enhancement activity across E2 families of Arabidopsis. We found that COP10 is capable of enhancing members of four E2 subgroups significantly, while having a milder effect on another. Surprisingly, we found that close canonical E2 homologues of COP10, such as UbcH5a (human ubiquitin-conjugating enzyme 5), are also capable of enhancing E2s. Furthermore, we detected direct interactions between COP10 and three of the enhanced E2s, hinting at a possible mechanism for the enhancements. The present study suggests that some E2s, including the generic Ubc4/5p families involved in many processes, might possess dual activities: the formation of the classic E2–Ub thiol ester and the previously unknown E2 enhancement activity. Therefore COP10, despite being a catalytically inactive E2, might still enhance a variety of E2s and regulate numerous aspects of plant development.

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Distinct activation of an E2 ubiquitin-conjugating enzyme by its cognate E3 ligases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1415621112 ◽

2015 ◽

Vol 112 (7) ◽

pp. E625-E632 ◽

Cited By ~ 11

Author(s):

Itamar Cohen ◽

Reuven Wiener ◽

Yuval Reiss ◽

Tommer Ravid

Keyword(s):

Transition State ◽

Protein Quality ◽

Noncovalent Interactions ◽

Cellular Protein ◽

Ring Domain ◽

E3 Ligases ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzyme

A significant portion of ubiquitin (Ub)-dependent cellular protein quality control takes place at the endoplasmic reticulum (ER) in a process termed “ER-associated degradation” (ERAD). Yeast ERAD employs two integral ER membrane E3 Ub ligases: Hrd1 (also termed “Der3”) and Doa10, which recognize a distinct set of substrates. However, both E3s bind to and activate a common E2-conjugating enzyme, Ubc7. Here we describe a novel feature of the ERAD system that entails differential activation of Ubc7 by its cognate E3s. We found that residues within helix α2 of Ubc7 that interact with donor Ub were essential for polyUb conjugation. Mutagenesis of these residues inhibited the in vitro activity of Ubc7 by preventing the conjugation of donor Ub to the acceptor. Unexpectedly, Ub chain formation by mutant Ubc7 was restored selectively by the Hrd1 RING domain but not by the Doa10 RING domain. In agreement with the in vitro data, Ubc7 α2 helix mutations selectively impaired the in vivo degradation of Doa10 substrates but had no apparent effect on the degradation of Hrd1 substrates. To our knowledge, this is the first example of distinct activation requirements of a single E2 by two E3s. We propose a model in which the RING domain activates Ub transfer by stabilizing a transition state determined by noncovalent interactions between the α2 helix of Ubc7 and Ub and that this transition state may be stabilized further by some E3 ligases, such as Hrd1, through additional interactions outside the RING domain.

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Stu2p binds tubulin and undergoes an open-to-closed conformational change

The Journal of Cell Biology ◽

10.1083/jcb.200511010 ◽

2006 ◽

Vol 172 (7) ◽

pp. 1009-1022 ◽

Cited By ~ 87

Author(s):

Jawdat Al-Bassam ◽

Mark van Breugel ◽

Stephen C. Harrison ◽

Anthony Hyman

Keyword(s):

Conformational Change ◽

Conformational Transition ◽

Budding Yeast ◽

Microtubule Dynamics ◽

Open Structure ◽

Microtubule Associated Proteins ◽

Associated Proteins ◽

The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

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