scholarly journals Biochemical characterization of Caenorhabditis elegans UBC-1: self-association and auto-ubiquitination of a RAD6-like ubiquitin-conjugating enzyme in vitro

1997 ◽  
Vol 327 (2) ◽  
pp. 357-361 ◽  
Author(s):  
S. David LEGGETT ◽  
M. E. Peter CANDIDO
Keyword(s):  
Caenorhabditis Elegans ◽  
Biochemical Characterization ◽  
Quaternary Structure ◽  
Catalytic Domain ◽  
Site Directed Mutagenesis ◽  
Post Translational Modification ◽  
Ubiquitin Conjugating Enzyme ◽  
Self Association ◽  
Conjugating Enzyme

The Caenorhabditis elegans ubiquitin-conjugating enzyme UBC-1 is distinct from other RAD6 homologues in possessing a C-terminal tail 40 amino acid residues long [Leggett, Jones and Candido (1995) DNA Cell Biol. 14, 883-891]. Such extensions from the core catalytic domain have been found in a subset of known conjugating enzymes, where they have been shown to have diverse roles including target recognition, membrane attachment and sporulation. In the present study we used mutagenesis in vitro to examine the role of the tail in specific aspects of UBC-1 structure and activity. Cross-linking experiments with purified recombinant UBC-1 reveal that it forms dimers and probably tetramers. The acidic tail of UBC-1 has an important role in this interaction because deletions of the tail significantly decrease, but do not abolish, this self-association. Ubiquitin conjugation assays show that, in addition to accepting a thiol-bound ubiquitin at its active site, UBC-1 is stably mono-ubiquitinated. Deletion analysis and site-directed mutagenesis localize the site of ubiquitination to Lys-162 in the tail. These findings demonstrate that the C-terminal tail of UBC-1 is important both for its quaternary structure and post-translational modification in vitro.

scholarly journals Functional CBS modules make IMPDHs octameric

2014 ◽  
Vol 70 (a1) ◽  
pp. C1283-C1283
Author(s):  
Gilles Labesse ◽  
Thomas Alexandre ◽  
Laurène Vaupré ◽  
Isabelle Salard-Arnaud ◽  
Joséphine Lai Kee Him ◽  
...  
Keyword(s):  
Analytical Ultracentrifugation ◽  
Quaternary Structure ◽  
Catalytic Domain ◽  
Structural Data ◽  
Site Directed Mutagenesis ◽  
X Ray Crystallography ◽  
Binding Pockets ◽  
Essential Enzyme ◽  
Cbs Domains

Inosine-5'-monophosphate dehydrogenase (1, 2) (IMPDH) is a major target for antiviral, antiparasitic, antileukemic and immunosuppressive therapies. It is an ubiquitous and essential enzyme for the biosynthesis of guanosine nucleotides. Up to now, IMPDHs have been reported as tetrameric enzymes harbouring a catalytic domain and a tandem of cystathionine-ß-synthase (CBS) modules. The latter had no precise function assigned despite their nearly absolute conservation among IMPDHs and consistent indication of their importance in vivo. The aim of our study was to provide evidence for the role of the CBS modules on the quaternary structure and on the regulation of IMPDHs. A multidisciplinary approach involving enzymology, site-directed mutagenesis, analytical ultracentrifugation, X-ray crystallography, SAXS, cryo-electron microscopy and molecular modelling allowed us to demonstrate that the Pseudomonas aeruginosa IMPDH is functionally active as an octamer and allosterically regulated by MgATP via each CBS module. Revisiting deposited structural data, we found this newly discovered octameric organization conserved in other IMPDH structures. Meanwhile, we demonstrated that the human IMPDH1 formed two distinct octamers that can pile up into isolated fibres in the presence of MgATP while its pathogenic mutant D226N, localised into the CBS domains, appeared to form massively aggregating fibres. The dramatic impact of this mutation could explain the severe retinopathy adRP10. Our data (3) revealed for the first time that IMPDH has an octameric architecture modulated by MgATP binding to the CBS modules, inducing large structural rearrangements. Thus, the regulatory CBS modules in IMPDHs are functional and they can either modulate catalysis or/and macromolecular assembly. Targeting the conserved effector binding pockets identified within the CBS modules might be promising to develop antibacterial compounds or drugs to counter retinopathy onset.

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

2018 ◽  
Vol 293 (47) ◽  
pp. 18285-18295 ◽  
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.

scholarly journals Recombinant ubiquitin-conjugating enzyme of Eimeria maxima induces immunogenic maturation in chicken splenic-derived dendritic cells and drives Th1 polarization in-vitro

2020 ◽  
Vol 143 ◽  
pp. 104162
Author(s):  
Shakeel Ahmed Lakho ◽  
Muhammad Haseeb ◽  
Jianmei Huang ◽  
Muhammad Waqqas Hasan ◽  
Muhammad Ali-ul-Husnain Naqvi ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Eimeria Maxima ◽  
Ubiquitin Conjugating Enzyme ◽  
Th1 Polarization ◽  
Conjugating Enzyme

Structural basis of cofactor-mediated stabilization and substrate recognition of the α-tubulin acetyltransferase αTAT1

2015 ◽  
Vol 467 (1) ◽  
pp. 103-113 ◽  
Author(s):  
Satoru Yuzawa ◽  
Sachiko Kamakura ◽  
Junya Hayase ◽  
Hideki Sumimoto
Keyword(s):  
Catalytic Domain ◽  
Substrate Recognition ◽  
Structural Basis ◽  
Stable Complex ◽  
Amino Acid Residues ◽  
Substrate Selection ◽  
Post Translational Modification ◽  
Acetyl Group

The functions of microtubules are controlled in part by tubulin post-translational modification including acetylation of Lys40 in α-tubulin. αTAT1 (α-tubulin acetyltransferase 1), an enzyme evolutionarily conserved among eukaryotes, has recently been identified as the major α-tubulin Lys40 acetyltransferase, in which AcCoA (acetyl-CoA) serves as an acetyl group donor. The regulation and substrate recognition of this enzyme, however, have not been fully understood. In the present study, we show that AcCoA and CoA each form a stable complex with human αTAT1 to maintain the protein integrity both in vivo and in vitro. The invariant residues Arg132 and Ser160 in αTAT1 participate in the stable interaction not only with AcCoA but also with CoA, which is supported by analysis of the present crystal structures of the αTAT1 catalytic domain in complex with CoA. Alanine substitution for Arg132 or Ser160 leads to a drastic misfolding of the isolated αTAT1 catalytic domain in the absence of CoA and AcCoA but not in the presence of excess amounts of either cofactor. A mutant αTAT1 carrying the R132A or S160A substitution is degraded much faster than the wild-type protein when expressed in mammalian Madin–Darby canine kidney cells. Furthermore, alanine-scanning experiments using Lys40-containing peptides reveal that α-tubulin Ser38 is crucial for substrate recognition of αTAT1, whereas Asp39, Ile42, the glycine stretch (amino acid residues 43–45) and Asp46 are also involved. The requirement for substrate selection is totally different from that in various histone acetyltransferases, which appears to be consistent with the inability of αTAT1 to acetylate histones.

scholarly journals Site-directed mutagenesis of the active site of diacylglycerol kinase α: calcium and phosphatidylserine stimulate enzyme activity via distinct mechanisms

2003 ◽  
Vol 375 (3) ◽  
pp. 673-680 ◽  
Author(s):  
Takahiro ABE ◽  
Xiaolan LU ◽  
Ying JIANG ◽  
Clark E. BOCCONE ◽  
Shaomin QIAN ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Regulatory Region ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Kinase C ◽  
Acidic Phospholipids ◽  
Ef Hands ◽  
Mutant Forms

Diacylglycerol kinases (DAGKs) catalyse ATP-dependent phosphorylation of sn-1,2-diacylglycerol that arises during stimulated phosphatidylinositol turnover. DAGKα is activated in vitro by Ca2+ and by acidic phospholipids. The regulatory region of DAGKα includes an N-terminal RVH motif and EF hands that mediate Ca2+-dependent activation. DAGKα also contains tandem C1 protein kinase C homology domains. We utilized yeast, Saccharomyces cerevisiae, which lacks an endogenous DAGK, to express DAGKα and to determine the enzymic activities of different mutant forms of pig DAGKα in vitro. Six aspartate residues conserved in all DAGKs were individually examined by site-directed mutagenesis. Five of these aspartate residues reside in conserved blocks that correspond to sequences in the catalytic site of phosphofructokinases. Mutation of D434 (Asp434) or D650 abolished all DAGKα activity, whereas substitution of one among D465, D497, D529 and D697 decreased the activity to 6% or less of that for wild-type DAGKα. Roles of homologous residues in phosphofructokinases suggested that the N-terminal half of the DAGK catalytic domain binds Mg-ATP and the C-terminal half binds diacylglycerol. A DAGKα mutant with its entire regulatory region deleted showed a much decreased activity that was not activated by Ca2+, but still exhibited PS (phosphatidylserine)-dependent activation. Moreover, mutations of aspartate residues at the catalytic domain had differential effects on activation by Ca2+ and PS. These results indicate that Ca2+ and PS stimulate DAGKα via distinct mechanisms.

scholarly journals Mutations in RAD6, a yeast gene encoding a ubiquitin-conjugating enzyme, stimulate retrotransposition.

1990 ◽  
Vol 10 (3) ◽  
pp. 1017-1022 ◽  
Author(s):  
S Picologlou ◽  
N Brown ◽  
S W Liebman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fold Increase ◽  
Yeast Gene ◽  
Gene Encoding ◽  
Repair Gene ◽  
Dna Repair Gene ◽  
Ubiquitin Conjugating Enzyme ◽  
First Time ◽  
Conjugating Enzyme

The Saccharomyces cerevisiae DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme which polyubiquitinates histones in vitro. Here we show that mutations in rad6 increase the frequency of transposition of the retrotransposon Ty into the CAN1 and URA3 loci. Using isogenic RAD6 and rad6 strains, we measured a more than 100-fold increase in the spontaneous rate of retrotransposition due to rad6, although there was no increase in the Ty message level. This is the first time that a mutation in a host gene has been shown to result in an increased rate of retrotransposition.

scholarly journals Creation of a Pluripotent Ubiquitin-Conjugating Enzyme

2001 ◽  
Vol 21 (19) ◽  
pp. 6537-6548 ◽  
Author(s):  
Christopher Ptak ◽  
Chantelle Gwozd ◽  
J. Torin Huzil ◽  
Todd J. Gwozd ◽  
Grace Garen ◽  
...  
Keyword(s):  
Dna Repair ◽  
Amino Acid ◽  
Catalytic Domain ◽  
Ring Domain ◽  
Single Amino Acid ◽  
Functional Region ◽  
Ubiquitin Conjugating Enzyme ◽  
Carboxy Terminal ◽  
Protein Ligases ◽  
Conjugating Enzyme

ABSTRACT We describe the creation of a pluripotent ubiquitin-conjugating enzyme (E2) generated through a single amino acid substitution within the catalytic domain of RAD6 (UBC2). This RAD6 derivative carries out the stress-related function of UBC4 and the cell cycle function of CDC34 while maintaining its own DNA repair function. Furthermore, it carries out CDC34's function in the absence of the CDC34 carboxy-terminal extension. By using sequence and structural comparisons, the residues that define the unique functions of these three E2s were found on the E2 catalytic face partitioned to either side by a conserved divide. One of these patches corresponds to a binding site for both HECT and RING domain proteins, suggesting that a single substitution in the catalytic domain of RAD6 confers upon it the ability to interact with multiple ubiquitin protein ligases (E3s). Other amino acid substitutions made within the catalytic domain of RAD6 either caused loss of its DNA repair function or modified its ability to carry out multiple E2 functions. These observations suggest that while HECT and RING domain binding may generally be localized to a specific patch on the E2 surface, other regions of the functional E2 face also play a role in specificity. Finally, these data also indicate that RAD6 uses a different functional region than either UBC4 or CDC34, allowing it to acquire the functions of these E2s while maintaining its own. The pluripotent RAD6 derivative, coupled with sequence, structural, and phylogenetic data, suggests that E2s have diverged from a common multifunctional progenitor.

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

2006 ◽  
Vol 80 (20) ◽  
pp. 10218-10228 ◽  
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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