scholarly journals Ubiquitin Ligase Activities of Bombyx mori Nucleopolyhedrovirus RING Finger Proteins

2003 ◽  
Vol 77 (2) ◽  
pp. 923-930 ◽  
Author(s):  
Noriko Imai ◽  
Noriyuki Matsuda ◽  
Keiji Tanaka ◽  
Akihiko Nakano ◽  
Shogo Matsumoto ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Ubiquitin Ligase ◽  
Mutational Analysis ◽  
Ring Finger ◽  
Zinc Ion ◽  
Ubiquitin Ligase E3 ◽  
Reticulocyte Lysates ◽  
Ring Finger Proteins ◽  
Ubiquitin Conjugating Enzymes

ABSTRACT The genome of Bombyx mori nucleopolyhedrovirus (BmNPV) is predicted to contain six RING finger proteins: IAP1, ORF35, IAP2, CG30, IE2, and PE38. Several other members of the RING finger family have recently been shown to have the ubiquitin-ligase (E3) activity. We thus examined whether BmNPV RING finger proteins have the E3 activity. In vitro ubiquitination assay with the rabbit reticulocyte lysates and BmNPV RING finger proteins fused with maltose-binding protein (MBP) showed that four of them (IAP2, IE2, PE38, and CG30) were polyubiquitinated in the presence of zinc ion. Furthermore, MBP-IAP2, MBP-IE2, and MBP-PE38 were able to reconstitute ubiquitination activity in cooperation with the Ubc4/5 subfamily of ubiquitin-conjugating enzymes. Mutational analysis also showed that ubiquitination activity of MBP-IAP2, MBP-IE2, and MBP-PE38 were dependent on their RING finger motif. Therefore, these results suggest that IAP2, IE2, and PE38 may function as E3 enzymes during BmNPV infection.

scholarly journals The Yeast GID Complex, a Novel Ubiquitin Ligase (E3) Involved in the Regulation of Carbohydrate Metabolism

2008 ◽  
Vol 19 (8) ◽  
pp. 3323-3333 ◽  
Author(s):  
Olivier Santt ◽  
Thorsten Pfirrmann ◽  
Bernhard Braun ◽  
Jeannette Juretschke ◽  
Philipp Kimmig ◽  
...  
Keyword(s):  
Carbohydrate Metabolism ◽  
Ubiquitin Ligase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Ring Finger ◽  
Vitro Assay ◽  
Fructose 1,6 Bisphosphatase ◽  
Key Enzyme ◽  
Ubiquitin Ligase E3

Glucose-dependent regulation of carbon metabolism is a subject of intensive studies. We have previously shown that the switch from gluconeogenesis to glycolysis is associated with ubiquitin-proteasome linked elimination of the key enzyme fructose-1,6-bisphosphatase. Seven glucose induced degradation deficient (Gid)-proteins found previously in a genomic screen were shown to form a complex that binds FBPase. One of the subunits, Gid2/Rmd5, contains a degenerated RING finger domain. In an in vitro assay, heterologous expression of GST-Gid2 leads to polyubiquitination of proteins. In addition, we show that a mutation in the degenerated RING domain of Gid2/Rmd5 abolishes fructose-1,6-bisphosphatase polyubiquitination and elimination in vivo. Six Gid proteins are present in gluconeogenic cells. A seventh protein, Gid4/Vid24, occurs upon glucose addition to gluconeogenic cells and is afterwards eliminated. Forcing abnormal expression of Gid4/Vid24 in gluconeogenic cells leads to fructose-1,6-bisphosphatase degradation. This suggests that Gid4/Vid24 initiates fructose-1,6-bisphosphatase polyubiquitination by the Gid complex and its subsequent elimination by the proteasome. We also show that an additional gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, is subject to Gid complex-dependent degradation. Our study uncovers a new type of ubiquitin ligase complex composed of novel subunits involved in carbohydrate metabolism and identifies Gid4/Vid24 as a major regulator of this E3.

Rma1, a novel type of RING finger protein conserved from Arabidopsis to human, is a membrane-bound ubiquitin ligase

2001 ◽  
Vol 114 (10) ◽  
pp. 1949-1957 ◽  
Author(s):  
N. Matsuda ◽  
T. Suzuki ◽  
K. Tanaka ◽  
A. Nakano
Keyword(s):  
Ubiquitin Ligase ◽  
Mutational Analysis ◽  
Ring Finger ◽  
Ring Finger Protein ◽  
Finger Protein ◽  
Ubiquitin Ligase E3 ◽  
Membrane Bound ◽  
E2 Enzymes ◽  
Membrane Anchoring ◽  
Ring Finger Motif

Rma1 is a protein with a RING finger motif and a C-terminal membrane-anchoring domain and is well conserved among higher eukaryotes. We show that fusion proteins between maltose binding protein (MBP) and human or Arabidopsis Rma1 are polyubiquitinated, when incubated with the rabbit reticulocyte or the wheat germ lysate, respectively. The polyubiquitination of MBP-Rma1 has been reconstituted by incubation with purified ubiquitin, the ubiquitin-activating enzyme E1, and one of the two ubiquitin-conjugating E2 enzymes (Ubc4 or UbcH5a). Other E2 enzymes tested, E2-20k, E2-25k, Ubc3 and Ubc8, are not able to confer this modification. Mutational analysis shows that the RING finger motif of Rma1 is necessary for the auto-ubiquitination of MBP-Rma1. Thus, Rma1 represents a novel, membrane-bound type of ubiquitin ligase E3, which probably functions with the Ubc4/5 subfamily of E2. The MBP moiety but not Rma1 itself is ubiquitinated in the auto-ubiquitination reaction of MBP-Rma1. Free MBP in solution is not a substrate of Rma1. These observations indicate that bringing the substrate into its physical vicinity is very important for the action of ubiquitin ligase.

In Vitro Ubiquitination Platform Identifies Methyl Ellipticiniums as Ubiquitin Ligase Inhibitors

2021 ◽  
pp. 247255522110006
Author(s):  
Brice A. P. Wilson ◽  
Donna Voeller ◽  
Emily A. Smith ◽  
Antony Wamiru ◽  
Ekaterina I. Goncharova ◽  
...  
Keyword(s):  
Growth Factor ◽  
Ubiquitin Ligase ◽  
Protein Function ◽  
Antitumor Immunity ◽  
Ring Finger ◽  
Growth Factor Receptor ◽  
Ubiquitin Ligases ◽  
Cell Receptor ◽  
Ubiquitin Ligase E3

The transfer of the small protein ubiquitin to a target protein is an intricately orchestrated process called ubiquitination that results in modulation of protein function or stability. Proper regulation of ubiquitination is essential, and dysregulation of this process is implicated in several human diseases. An example of a ubiquitination cascade that is a central signaling node in important disease-associated pathways is that of CBLB [a human homolog of a viral oncogene Casitas B-lineage lymphoma (CBL) from the Cas NS-1 murine retrovirus], a RING finger ubiquitin ligase (E3) whose substrates include a number of important cell-signaling kinases. These include kinases important in immune function that act in the T cell receptor and costimulatory pathways, the Tyro/Axl/MerTK (TAM) receptor family in natural killer (NK) cells, as well as growth factor receptor kinases like epidermal growth factor receptor (EGFR). Loss of CBLB has been shown to increase innate and adaptive antitumor immunity. This suggests that small-molecule modulation of CBLB E3 activity could enhance antitumor immunity in patients. To explore the hypothesis that enzymatic inhibition of E3s may result in modulation of disease-related signaling pathways, we established a high-throughput screen of >70,000 chemical entities for inhibition of CBLB activity. Although CBLB was chosen as a proof-of-principle target for inhibitor discovery, we demonstrate that our assay is generalizable to monitoring the activity of other ubiquitin ligases. We further extended our observed in vitro inhibition with additional cell-based models of CBLB activity. From these studies, we demonstrate that a class of natural product–based alkaloids, known as methyl ellipticiniums (MEs), is capable of inhibiting ubiquitin ligases intracellularly.

scholarly journals Mammalian DET1 Regulates Cul4A Activity and Forms Stable Complexes with E2 Ubiquitin-Conjugating Enzymes

2007 ◽  
Vol 27 (13) ◽  
pp. 4708-4719 ◽  
Author(s):  
Elah Pick ◽  
On-Sun Lau ◽  
Tomohiko Tsuge ◽  
Suchithra Menon ◽  
Yingchun Tong ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Associated Factors ◽  
Cultured Cells ◽  
Negative Regulator ◽  
Polyubiquitin Chain ◽  
Light Responses ◽  
E3 Ligases ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes

ABSTRACT DET1 (de-etiolated 1) is an essential negative regulator of plant light responses, and it is a component of the Arabidopsis thaliana CDD complex containing DDB1 and COP10 ubiquitin E2 variant. Human DET1 has recently been isolated as one of the DDB1- and Cul4A-associated factors, along with an array of WD40-containing substrate receptors of the Cul4A-DDB1 ubiquitin ligase. However, DET1 differs from conventional substrate receptors of cullin E3 ligases in both biochemical behavior and activity. Here we report that mammalian DET1 forms stable DDD-E2 complexes, consisting of DDB1, DDA1 (DET1, DDB1 associated 1), and a member of the UBE2E group of canonical ubiquitin-conjugating enzymes. DDD-E2 complexes interact with multiple ubiquitin E3 ligases. We show that the E2 component cannot maintain the ubiquitin thioester linkage once bound to the DDD core, rendering mammalian DDD-E2 equivalent to the Arabidopsis CDD complex. While free UBE2E-3 is active and able to enhance UbcH5/Cul4A activity, the DDD core specifically inhibits Cul4A-dependent polyubiquitin chain assembly in vitro. Overexpression of DET1 inhibits UV-induced CDT1 degradation in cultured cells. These findings demonstrate that the conserved DET1 complex modulates Cul4A functions by a novel mechanism.

Comparative analysis of testis transcriptomes associated with male infertility in triploid cyprinid fish

2019 ◽  
Vol 31 (2) ◽  
pp. 248 ◽  
Author(s):  
Wuhui Li ◽  
Hui Tan ◽  
Junmei Liu ◽  
Jie Hu ◽  
Jialin Cui ◽  
...  
Keyword(s):  
Male Infertility ◽  
B Cell ◽  
Ring Finger ◽  
B Cell Lymphoma ◽  
Diploid Parent ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Caspase 7 ◽  
Ring Finger Proteins ◽  
Ubiquitin Conjugating Enzymes

Spermatogenesis involves a series of cellular transformations and thousands of regulated genes. Previously, we showed that the triploid fish (3nBY) cannot produce mature spermatozoa. In the present study, evaluation of the testis microstructure revealed that germ cells of 3nBY could develop into round spermatids, but then degenerated, resulting in male infertility. In this study we comparatively analysed the testis transcriptomes from 3nBY and its diploid parent YB and identified a series of differentially expressed genes (DEGs) that were enriched in the Wnt signalling pathway and the apoptotic and ubiquitin-mediated proteolysis processes in 3nBY. Gene ontology functional analyses revealed that some DEGs in 3nBY were directly associated with the process of gamete generation, development and sperm flagellum assembly. In addition, the expression of a number of genes related to meiosis (Inhibitor Of DNA Binding 2 (ID2), Ovo Like Transcriptional Repressor 1 (OVOL1)), mitochondria (ATP1b (ATPase Na+/K+ Transporting Subunit Beta 1), ATP2a (ATPase, Ca++ Transporting, Cardiac Muscle, Slow Twitch 2), ATP5a (ATP Synthase F1 Subunit Alpha), Mitochondrially Encoded Cytochrome C Oxidase I (COX1), NADH Dehydrogenase Subunit 4 (ND4)) and chromatin structure (Histone 1 (H1), Histone 2a (H2A), Histone 2b (H2B), Histone 3 (H3), Histone 4 (H4)) was lower in the testes of 3nBY, whereas the expression of genes encoding ubiquitin (Ubiquitin Conjugating Enzymes (UBEs), Ring Finger Proteins (RNFs)) and apoptosis (CASPs (Caspase 3, Caspase 7,Caspase 8), BCLs (B-Cell Lymphoma 3, B-Cell CLL/Lymphoma 2, B Cell CLL/Lymphoma 10)) proteins involved in spermatid degeneration was higher. These data suggest that the disrupted expression of genes associated with spermatogenesis and the increased expression of mitochondrial ubiquitin, which initiates cell apoptosis, may result in spermatid degeneration in male 3nBY. This study provides information regarding the potential molecular regulatory mechanisms underlying male infertility in polyploid fish.

scholarly journals The APC11 RING-H2 Finger Mediates E2-Dependent Ubiquitination

2000 ◽  
Vol 11 (7) ◽  
pp. 2315-2325 ◽  
Author(s):  
Joel D. Leverson ◽  
Claudio A.P. Joazeiro ◽  
Andrew M. Page ◽  
Han-kuei Huang ◽  
Philip Hieter ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
26S Proteasome ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Protein Substrates ◽  
Ubiquitin Ligase Activity ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitin Conjugating Enzymes

Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that theSaccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.

scholarly journals Formation of Bombyx mori nucleopolyhedrovirus IE2 nuclear foci is regulated by the functional domains for oligomerization and ubiquitin ligase activity

2005 ◽  
Vol 86 (3) ◽  
pp. 637-644 ◽  
Author(s):  
Noriko Imai ◽  
Shogo Matsumoto ◽  
WonKyung Kang
Keyword(s):  
Bombyx Mori ◽  
Ubiquitin Ligase ◽  
Coiled Coil ◽  
Ring Finger ◽  
Functional Domains ◽  
Focus Formation ◽  
Ring Finger Domain ◽  
Coiled Coil Region ◽  
Ubiquitin Ligase Activity ◽  
Nuclear Foci

Baculovirus IE2 functions as a transregulator and is also involved in viral DNA replication. However, the mechanism for these functions remains unknown. It has previously been reported that Bombyx mori nucleopolyhedrovirus (BmNPV) IE2 has a ubiquitin ligase activity that is dependent on the RING finger domain and that IE2 can oligomerize through its C-terminal coiled-coil region. Here, confocal microscopy analysis demonstrated that IE2 formed nuclear foci only during the early phase of infection (2–6 h post-infection). Therefore, it was determined whether the IE2 functional regions described above could affect this characteristic distribution. Transient expression of ie2 also showed focus formation, suggesting that IE2 does not require any other viral factors. IE2 mutants lacking the C-terminal coiled-coil region did not form foci, while a mutant of the RING finger domain showed nuclear foci that appeared larger and brighter than those formed by wild-type IE2. In addition, IE2 exhibited enlarged foci in infected cells following treatment with a proteasome inhibitor, suggesting that foci enlargement resulted from accumulation of IE2 due to inhibition of the ubiquitin-proteasome pathway. These results suggest that BmNPV IE2 oligomerization and ubiquitin ligase activity functional domains regulate nuclear foci formation.

scholarly journals The CUL1 C-Terminal Sequence and ROC1 Are Required for Efficient Nuclear Accumulation, NEDD8 Modification, and Ubiquitin Ligase Activity of CUL1

2000 ◽  
Vol 20 (21) ◽  
pp. 8185-8197 ◽  
Author(s):  
Manabu Furukawa ◽  
Yanping Zhang ◽  
Joseph McCarville ◽  
Tomohiko Ohta ◽  
Yue Xiong
Keyword(s):  
Nuclear Localization ◽  
Ubiquitin Ligase ◽  
Nuclear Import ◽  
Ring Finger ◽  
Nuclear Accumulation ◽  
Protein Families ◽  
Terminal Sequence ◽  
Ubiquitin Ligase Activity

ABSTRACT Members of the cullin and RING finger ROC protein families form heterodimeric complexes to constitute a potentially large number of distinct E3 ubiquitin ligases. We report here that the highly conserved C-terminal sequence in CUL1 is dually required, both for nuclear localization and for modification by NEDD8. Disruption of ROC1 binding impaired nuclear accumulation of CUL1 and decreased NEDD8 modification in vivo but had no effect on NEDD8 modification of CUL1 in vitro, suggesting that ROC1 promotes CUL1 nuclear accumulation to facilitate its NEDD8 modification. Disruption of NEDD8 binding had no effect on ROC1 binding, nor did it affect nuclear localization of CUL1, suggesting that nuclear localization and NEDD8 modification of CUL1 are two separable steps, with nuclear import preceding and required for NEDD8 modification. Disrupting NEDD8 modification diminishes the IκBα ubiquitin ligase activity of CUL1. These results identify a pathway for regulation of CUL1 activity—ROC1 and the CUL1 C-terminal sequence collaboratively mediate nuclear accumulation and NEDD8 modification, facilitating assembly of active CUL1 ubiquitin ligase. This pathway may be commonly utilized for the assembly of other cullin ligases.

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