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Protein aggregation is a hallmark of several neurodegenerative diseases including Huntington's disease and Parkinson's disease. Proteins containing long, homopolymeric stretches of glutamine are especially prone to form aggregates. It has long been known that the small protein modifier, ubiquitin, localizes to these aggregates. In this report, nematode and cell culture models for polyglutamine aggregation are used to investigate the role of the ubiquitin pathway in protein aggregation.
Ubiquitin (E3) ligases interact with specific ubiquitin conjugating (E2) enzymes to ubiquitinate particular substrate proteins. As the combination of E2 and E3 dictates the type and biological consequence of ubiquitination, it is important to understand the basis of specificity in E2:E3 interactions. The E3 ligase CHIP interacts with Hsp70 and Hsp90 and ubiquitinates client proteins that are chaperoned by these heat shock proteins. CHIP interacts with two types of E2 enzymes, UbcH5 and Ubc13-Uev1a. It is unclear, however, why CHIP binds these E2 enzymes rather than others, and whether CHIP interacts preferentially with UbcH5 or Ubc13-Uev1a, which form different types of polyubiquitin chains.
Ubiquitin is a small polypeptide that is conjugated to proteins and commonly serves as a degradation signal. The attachment of ubiquitin (Ub) to a substrate proceeds through a multi-enzyme cascade involving an activating enzyme (E1), a conjugating enzyme (E2), and a protein ligase (E3). We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11. We now show that the import mechanism for UbcM2 and two other human class III E2s (UbcH6 and UBE2E2) uniquely requires the covalent attachment of Ub to the active site cysteine of these enzymes. This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes. Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport.
OTUB1 is a Lys48-specific deubiquitinating enzyme that forms a complex in vivo with E2 ubiquitin (Ub)-conjugating enzymes including UBC13 and UBCH5. OTUB1 binds E2~Ub thioester intermediates and prevents ubiquitin transfer, thereby noncatalytically inhibiting accumulation of polyubiquitin. We report here that a second role of OTUB1-E2 interactions is to stimulate OTUB1 cleavage of Lys48 polyubiquitin. This stimulation is regulated by the ratio of charged to uncharged E2 and by the concentration of Lys48-linked polyubiquitin and free ubiquitin. Structural and biochemical studies of human and worm OTUB1 and UBCH5B show that the E2 enzyme stimulates binding of the Lys48 polyubiquitin substrate by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix. Our results suggest that OTUB1-E2 complexes in the cell are poised to regulate polyubiquitin chain elongation or degradation in response to changing levels of E2 charging and available free ubiquitin.
The eukaryotic ubiquitin-conjugation system sets the turnover rate of many proteins and includes activating enzymes (E1s), conjugating enzymes (UBCs/E2s), and ubiquitin-protein ligases (E3s), which are responsible for activation, covalent attachment and substrate recognition, respectively. There are also ubiquitin-like proteins with distinct functions, which require their own E1s and E2s for attachment. We describe the results of RNA interference (RNAi) experiments on the E1s, UBC/E2s and ubiquitin-like proteins in Caenorhabditis elegans. We also present a phylogenetic analysis of UBCs.
It is widely accepted that ubiquitin-conjugating enzymes contain an active site asparagine that serves as an oxyanion hole, thereby stabilizing a negatively charged transition state intermediate and promoting ubiquitin transfer. Using structural and biochemical approaches to study the role of the conserved asparagine to ubiquitin conjugation by Ubc13-Mms2, we conclude that the importance of this residue stems primarily from its structural role in stabilizing an active site loop.
Mounting evidence implicates chronic oxidative stress as a significant pathogenic factor in the development and progression of retinopathies, including age-related macular degeneration (AMD). The age-dependent toxic accumulation of oxidatively damaged proteins, lipids, and DNA in susceptible cells of the retina arises, at least in part, from a decreased capacity to eliminate these damaged biomolecules. The goal of this study was to determine the expression patterns and function of class III ubiquitin-conjugating enzymes (UbcM3, UBE2E2, and UbcM2) in the retina. These enzymes have been implicated in the ubiquitin-dependent degradation of oxidatively damaged and misfolded proteins.
UBA1 is the primary E1 ubiquitin-activating enzyme responsible for generation of activated ubiquitin required for ubiquitination, a process that regulates stability and function of numerous proteins. Decreased or insufficient ubiquitination can cause or drive aging and many diseases. Therefore, a small-molecule enhancing UBA1 activity could have broad therapeutic potential. Here we report that auranofin, a drug approved for the treatment of rheumatoid arthritis, is a potent UBA1 activity enhancer. Auranofin binds to the UBA1's ubiquitin fold domain and conjugates to Cys1039 residue. The binding enhances UBA1 interactions with at least 20 different E2 ubiquitin-conjugating enzymes, facilitating ubiquitin charging to E2 and increasing the activities of seven representative E3s in vitro. Auranofin promotes ubiquitination and degradation of misfolded ER proteins during ER-associated degradation in cells at low nanomolar concentrations. It also facilitates outer mitochondrial membrane-associated degradation. These findings suggest that auranofin can serve as a much-needed tool for UBA1 research and therapeutic exploration.
The ubiquitin-proteasomal degradation mechanism has gained the attention over the past decade. The E2 ubiquitin conjugating enzymes are the crucial part of ubiquitination mechanism and they are believed to hold imperative association for plant development. It accepts ubiquitin from the E1 enzyme and interacts with the E3 ligase to transfer ubiquitin or directly transfers ubiquitin to the substrate. The functional aspects of E2 ubiquitin enzymes in plant systems are unclear. Tomato is being used as a model plant and rarely explored to study E2 ubiquitin enzyme. We have utilized in-silico methods to analyze E2 enzymes in Solanum lycopersicum and 59 genes were identified with UBC family domains. The physio-chemical properties, chromosomal localization, structural organization, gene duplication, promoter analysis, gene ontology and conserved motifs were investigated along with phylogenetic analysis of tomato E2 genes exploring evolutionary relations. The gene expression analysis of RNA sequencing data revealed expression profile of tomato E2 genes in seedling, root, leaf, seed, fruit, and flower tissues. Our study aid in the understanding of distribution, expansion, evolutionary relation and probable participation in plant biological processes of tomato E2 enzymes that will facilitate strong base for future research on ubiquitin-mediated regulations in tomato and other plant systems.
Ubiquitination of a target protein is accomplished through sequential actions of the E1, E2s, and the E3s. E2s dictate the modification topology while E3 ligases confer substrate specificity and recruit the cognate E2. Human genome codes for ~35 different E2 proteins; all of which contain the characteristic ubiquitin-conjugating UBC core domain sufficient for catalysis. Many of these E2 enzymes also have N- or C-terminal extensions; roles of which are not very well understood. We show that the N-terminal extension of Ube2E1 undergoes intramolecular auto-ubiquitination. This self-ubiquitination activity is enhanced in the presence of interacting RING E3 ligases and results in a progressive attenuation of the E2 activity toward substrate/E3 modification. We also find that the N-terminal ubiquitination sites are conserved in all the three Ube2Es and replacing them with arginine renders all three full-length Ube2Es equally active as their core UBC domains. Based on these results, we propose that E3-catalyzed self-ubiquitination acts as a key regulatory mechanism that controls the activity of Ube2E class of ubiquitin E2s.
Vascular endothelial growth factor receptor 2 (VEGFR2, encoded by KDR) regulates endothelial function and angiogenesis. VEGFR2 undergoes ubiquitination that programs this receptor for trafficking and proteolysis, but the ubiquitin-modifying enzymes involved are ill-defined. Herein, we used a reverse genetics screen for the human E2 family of ubiquitin-conjugating enzymes to identify gene products that regulate VEGFR2 ubiquitination and proteolysis. We found that depletion of either UBE2D1 or UBE2D2 in endothelial cells caused a rise in steady-state VEGFR2 levels. This rise in plasma membrane VEGFR2 levels impacted on VEGF-A-stimulated signalling, with increased activation of canonical MAPK, phospholipase Cγ1 and Akt pathways. Analysis of biosynthetic VEGFR2 is consistent with a role for UBE2D enzymes in influencing plasma membrane VEGFR2 levels. Cell-surface-specific biotinylation and recycling studies showed an increase in VEGFR2 recycling to the plasma membrane upon reduction in UBE2D levels. Depletion of either UBE2D1 or UBE2D2 stimulated endothelial tubulogenesis, which is consistent with increased VEGFR2 plasma membrane levels promoting the cellular response to exogenous VEGF-A. Our studies identify a key role for UBE2D1 and UBE2D2 in regulating VEGFR2 function in angiogenesis.
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.
UBTD1 is a previously uncharacterized ubiquitin-like (UbL) domain containing protein with high homology to the mitochondrial Dc-UbP/UBTD2 protein. Here we show that UBTD1 and UBTD2 belong to a family of proteins that is conserved through evolution and found in metazoa, funghi, and plants. To gain further insight into the function of UBTD1, we screened for interacting proteins. In a yeast-2-hybrid (Y2H) screen, we identified several proteins involved in the ubiquitylation pathway, including the UBE2D family of E2 ubiquitin conjugating enzymes. An affinity capture screen for UBTD1 interacting proteins in whole cell extracts also identified members of the UBE2D family. Biochemical characterization of recombinant UBTD1 and UBE2D demonstrated that the two proteins form a stable, stoichiometric complex that can be purified to near homogeneity. We discuss the implications of these findings in light of the ubiquitin proteasome system (UPS).
Ripening affects the quality and nutritional contents of fleshy fruits and is a crucial process of fruit development. Although several studies have suggested that ubiquitin-conjugating enzyme (E2s or UBC enzymes) are involved in the regulation of fruit ripening, little is known about the function of E2s in papaya (Carica papaya).
Polyubiquitination by E2 and E3 enzymes is crucial to cell cycle control, epigenetic regulation, and development. The hallmark of the E2 family is the ubiquitin (Ub)-conjugating (UBC) domain that forms a dynamic thioester conjugate with ubiquitin (E2~Ub). Numerous studies have focused on E2 surfaces, such as the N-terminal and crossover helices, that directly interact with an E3 or the conjugated ubiquitin to stabilize the active, "closed" state of the E2~Ub. However, it remains unclear how other E2 surfaces regulate ubiquitin transfer. Here, we demonstrate the helix-turn-helix (HTH) motif of the UBC tunes the intrinsic polyubiquitination activity through distinct functions in different E2s. Interestingly, the E2HTH motif is repurposed in UBE2S and UBE2R2 to interact with the conjugated or acceptor ubiquitin, respectively, modulating ubiquitin transfer. Furthermore, we propose that Anaphase-Promoting Complex/Cyclosome binding to the UBE2SHTH reduces the conformational space of the flexible E2~Ub, demonstrating an atypical E3-dependent activation mechanism. Altogether, we postulate the E2HTH motif evolved to provide new functionalities that can be harnessed by E3s and permits additional regulation to facilitate specific E2-E3-mediated polyubiquitination.
RING-between RING (RBR)-type ubiquitin (Ub) ligases (E3s) such as Parkin receive Ub from Ub-conjugating enzymes (E2s) in response to ligase activation. However, the specific E2s that transfer Ub to each RBR-type ligase are largely unknown because of insufficient methods for monitoring their interaction. To address this problem, we have developed a method that detects intracellular interactions between E2s and activated Parkin. Fluorescent homotetramer Azami-Green fused with E2 and oligomeric Ash (Assembly helper) fused with Parkin form a liquid-liquid phase separation (LLPS) in cells only when E2 and Parkin interact. Using this method, we identified multiple E2s interacting with activated Parkin on damaged mitochondria during mitophagy. Combined with in vitro ubiquitination assays and bioinformatics, these findings revealed an underlying consensus sequence for E2 interactions with activated Parkin. Application of this method to other RBR-type E3s including HOIP, HHARI, and TRIAD1 revealed that HOIP forms an LLPS with its substrate NEMO in response to a proinflammatory cytokine and that HHARI and TRIAD1 form a cytosolic LLPS independent of Ub-like protein NEDD8. Since an E2-E3 interaction is a prerequisite for RBR-type E3 activation and subsequent substrate ubiquitination, the method we have established here can be an in-cell tool to elucidate the potentially novel mechanisms involved in RBR-type E3s.
Rice, a monocot model crop, contains at least 48 putative E2 ubiquitin (Ub)-conjugating enzymes. Based on homology comparisons with 40 Arabidopsis E2 proteins and 35 human E2s, 48 rice E2s were classified into 15 different groups. Yeast two-hybrid analyses using the U-box-domain regions of armadillo (ARM)-U-box E3 Ub-ligases and the Ub-conjugating (UBC) domains of E2s showed that, among 40 rice E2s, 11 E2s accounted for 70% of the interactions with 17 ARM-U-box E3s. Thus, a single E2 could interact with multiple ARM-U-box E3s, suggesting the presence of E2 hubs for E2-E3 interactions in rice. Rice SPL11 ARM-U-box E3 displayed distinct self-ubiquitination patterns, including poly-ubiquitination, mono-ubiquitination, or no ubiquitination, depending on different E2 partners. This suggests that the mode of ubiquitination of SPL11 E3 is critically influenced by individual E2s.
Fruits are unique to flowering plants and play a central role in seed maturation and dispersal. Molecular dissection of fruit ripening has received considerable interest because of the biological and dietary significance of fruit. To better understand the regulatory mechanisms underlying fruit ripening, we report here the first comprehensive analysis of the nuclear proteome in tomato fruits.
Cholesterol is an essential lipid for cellular function and membrane integrity, and hence its cellular levels and distribution must be tightly regulated. Biosynthesis of cholesterol is ramped when its cellular levels are low. Herein, the ER-resident and rate-limiting enzymes 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and squalene monooxygenase (SQLE) play a prominent role. We have recently reported that MARCH6, an E3 ubiquitin ligase, specifically promotes cholesterol-stimulated ubiquitylation and subsequent proteasomal degradation of SQLE, but not of HMGCR. To further delineate how post-translational regulation of SQLE and HMGCR is differentially achieved, we hypothesized that their sterol-dependent degradation machinery makes use of distinct E2 ubiquitin conjugating enzymes.
Protein phosphorylation is a modification that offers a dynamic and reversible mechanism to regulate the majority of cellular processes. Numerous diseases are associated with aberrant regulation of phosphorylation-induced switches. Phosphorylation is emerging as a mechanism to modulate ubiquitination by regulating key enzymes in this pathway. The molecular mechanisms underpinning how phosphorylation regulates ubiquitinating enzymes, however, are elusive. Here, we show the high conservation of a functional site in E2 ubiquitin-conjugating enzymes. In catalytically active E2s, this site contains aspartate or a phosphorylatable serine and we refer to it as the conserved E2 serine/aspartate (CES/D) site. Molecular simulations of substrate-bound and -unbound forms of wild type, mutant and phosphorylated E2s, provide atomistic insight into the role of the CES/D residue for optimal E2 activity. Both the size and charge of the side group at the site play a central role in aligning the substrate lysine toward E2 catalytic cysteine to control ubiquitination efficiency. The CES/D site contributes to the fingerprint of the E2 superfamily. We propose that E2 enzymes can be divided into constitutively active or regulated families. E2s characterized by an aspartate at the CES/D site signify constitutively active E2s, whereas those containing a serine can be regulated by phosphorylation.
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