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

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.

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 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.

scholarly journals Current state of and need for enzyme engineering of 2-deoxy-D-ribose 5-phosphate aldolases and its impact

2021 ◽  
Author(s):  
Juha Rouvinen ◽  
Martina Andberg ◽  
Johan Pääkkönen ◽  
Nina Hakulinen ◽  
Anu Koivula
Keyword(s):  
Protein Engineering ◽  
Catalytic Mechanism ◽  
Cascade Reactions ◽  
Single Step ◽  
Enzyme Engineering ◽  
Enzymatic Reactions ◽  
Tim Barrel ◽  
The Stability

Abstract Deoxyribose-5-phosphate aldolases (DERAs, EC 4.1.2.4) are acetaldehyde-dependent, Class I aldolases catalyzing in nature a reversible aldol reaction between an acetaldehyde donor (C2 compound) and glyceraldehyde-3-phosphate acceptor (C3 compound, C3P) to generate deoxyribose-5-phosphate (C5 compound, DR5P). DERA enzymes have been found to accept also other types of aldehydes as their donor, and in particular as acceptor molecules. Consequently, DERA enzymes can be applied in C–C bond formation reactions to produce novel compounds, thus offering a versatile biocatalytic alternative for synthesis. DERA enzymes, found in all kingdoms of life, share a common TIM barrel fold despite the low overall sequence identity. The catalytic mechanism is well-studied and involves formation of a covalent enzyme-substrate intermediate. A number of protein engineering studies to optimize substrate specificity, enzyme efficiency, and stability of DERA aldolases have been published. These have employed various engineering strategies including structure-based design, directed evolution, and recently also machine learning–guided protein engineering. For application purposes, enzyme immobilization and usage of whole cell catalysis are preferred methods as they improve the overall performance of the biocatalytic processes, including often also the stability of the enzyme. Besides single-step enzymatic reactions, DERA aldolases have also been applied in multi-enzyme cascade reactions both in vitro and in vivo. The DERA-based applications range from synthesis of commodity chemicals and flavours to more complicated and high-value pharmaceutical compounds. Key points • DERA aldolases are versatile biocatalysts able to make new C–C bonds. • Synthetic utility of DERAs has been improved by protein engineering approaches. • Computational methods are expected to speed up the future DERA engineering efforts. Graphical abstract

scholarly journals Characterization of OTUB1 activation and inhibition by different E2 enzymes

2019 ◽  
Author(s):  
Lauren T. Que ◽  
Marie E. Morrow ◽  
Cynthia Wolberger
Keyword(s):  
Deubiquitinating Enzyme ◽  
Polyubiquitin Chains ◽  
Kinetics And Thermodynamics ◽  
Conjugating Enzymes ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
Stimulation Of

AbstractOTUB1 is a highly expressed cysteine protease that specifically cleaves K48-linked polyubiquitin chains. This unique deubiquitinating enzyme (DUB) can bind to a subset of E2 ubiquitin conjugating enzymes, forming complexes in which the two enzymes can regulate one another’s activity. OTUB1 can non-catalytically suppress the ubiquitin conjugating activity of its E2 partners by sequestering the charged E2~Ub thioester and preventing ubiquitin transfer. The same E2 enzymes, when uncharged, can stimulate the DUB activity of OTUB1 in vitro, although the importance of OTUB1 stimulation in vivo remains unclear. In order to assess the potential balance between these activities that might occur in cells, we characterized the kinetics and thermodynamics governing the formation and activity of OTUB1:E2 complexes. We show that both stimulation of OTUB1 by E2 enzymes and noncatalytic inhibition of E2 enzymes by OTUB1 occur at physiologically relevant concentrations of both partners. Whereas E2 partners differ in their ability to stimulate OTUB1 activity, we find that this variability is not correlated with the affinity of each E2 for OTUB1. In addition to UBE2N and the UBE2D isoforms, we find that OTUB1 inhibits polyubiquitination activity of all three UBE2E enzymes, UBE2E1, UBE2E2, and UBE2E3. Interestingly, although OTUB1 also inhibits the autoubiquitination activity of UBE2E1 and UBE2E2, it is unable to suppress autoubiquitination by UBE2E3.

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

1995 ◽  
Vol 15 (9) ◽  
pp. 4835-4842 ◽  
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.

scholarly journals Involvement of the DNA Repair Protein hHR23 inp53Degradation

2003 ◽  
Vol 23 (24) ◽  
pp. 8960-8969 ◽  
Author(s):  
Sandra Glockzin ◽  
Francois-Xavier Ogi ◽  
Arnd Hengstermann ◽  
Martin Scheffner ◽  
Christine Blattner
Keyword(s):  
Dna Repair ◽  
26S Proteasome ◽  
Polyubiquitin Chains ◽  
Proteolytic Pathway ◽  
Repair Protein ◽  
Uba Domain ◽  
Dna Repair Protein ◽  
The Stability

ABSTRACT The stability of the tumor suppressor protein p53 is regulated via the ubiquitin-proteasome-dependent proteolytic pathway. Like most substrates of this pathway, p53 is modified by the attachment of polyubiquitin chains prior to proteasome-mediated degradation. However, the mechanism(s) involved in the delivery of polyubiquitylated p53 molecules to the proteasome are currently unclear. Here, we show that the human DNA repair protein hHR23 binds to polyubiquitylated p53 via its carboxyl-terminal ubiquitin-associated (Uba) domain shielding p53 from deubiquitylation in vitro and in vivo. In addition, downregulation of hHR23 expression within cells by RNA interference results in accumulation of p53. Since the Ubl domain of hHR23 has been shown to interact with the 26S proteasome, we propose that hHR23 is intrinsically involved in the delivery of polyubiquitylated p53 molecules to the proteasome. In this model, the Uba domain of hHR23 binds to polyubiquitin chains formed on p53 and protects them from deubiquitylation, while the Ubl domain delivers the polyubiquitylated p53 molecules to the proteasome.

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

2009 ◽  
Vol 22 (6) ◽  
pp. 737-746 ◽  
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.

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

2009 ◽  
Vol 418 (3) ◽  
pp. 683-690 ◽  
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.

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

2008 ◽  
Vol 82 (14) ◽  
pp. 6911-6926 ◽  
Author(s):  
Zhenghe Li ◽  
Daniel Barajas ◽  
Tadas Panavas ◽  
David A. Herbst ◽  
Peter D. Nagy
Keyword(s):  
Replication Protein ◽  
Host Proteins ◽  
A Genome ◽  
Associated Proteins ◽  
Ubiquitin Conjugating Enzyme ◽  
Wide Scale ◽  
Split Ubiquitin ◽  
Conjugating Enzyme

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.

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

2015 ◽  
Vol 112 (7) ◽  
pp. E625-E632 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document