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Structural analysis of recombinant human ubiquitin-conjugating enzyme UbcH5c.

ABSTRACT: UbcH5c belongs to the ubiquitin-conjugating enzyme family and plays an important role in catalyzing ubiquitination during TNF-?--triggered NF-?B activation. Therefore, UbcH5c is a potent therapeutic target for the treatment of inflammatory and autoimmune diseases induced by aberrant activation of NF-?B. In this study, we established a stable expression system for recombinant UbcH5c and solved the crystal structure of UbcH5c belonging to space group P22121 with one molecule in the asymmetric unit. This study provides the basis for further study of UbcH5c including the design of UbcH5c inhibitors.

SUBMITTER: Wu F

PROVIDER: S-EPMC5430876 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Structural analysis of recombinant human ubiquitin-conjugating enzyme UbcH5c.

Wu Fangshu F Zhu Junsheng J Li Honglin H Zhu Lili L

Acta pharmaceutica Sinica. B 20170228 3

UbcH5c belongs to the ubiquitin-conjugating enzyme family and plays an important role in catalyzing ubiquitination during TNF-α--triggered NF-κB activation. Therefore, UbcH5c is a potent therapeutic target for the treatment of inflammatory and autoimmune diseases induced by aberrant activation of NF-κB. In this study, we established a stable expression system for recombinant UbcH5c and solved the crystal structure of UbcH5c belonging to space group P22121</su ...[more]

PMID: 28540177

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Targeting E2 ubiquitin-conjugating enzyme UbcH5c by small molecule inhibitor suppresses pancreatic cancer growth and metastasis.

Project description:BackgroundPancreatic cancer is one of the most lethal cancers worldwide. The IAPs function as E3 ubiquitin ligases and contribute to pancreatic cancer initiation, progression, and metastasis. Although IAP-targeted therapies have been developed and shown anticancer efficacy in preclinical settings, none of them has been approved yet.MethodsTranscriptome data from public datasets were used to analyze the correlation of IAPs and E2s, and the biological function of E2 UbcH5c in pancreatic cancer. A structure-based virtual screen was used to identify UbcH5c inhibitor, and surface plasmon resonance analysis and cellular thermal shift assays were employed to evaluate the binding affinity. The anticancer activities were demonstrated through in vitro and in vivo assays, while the related mechanisms were explored through transcriptomic and proteomic analyses and confirmed by western blot, immunofluorescence, and qRT-PCR.ResultsUbcH5c is positively correlated with the expression of IAPs in pancreatic cancer. We further found that UbcH5c is overexpressed and associated with a poor prognosis in pancreatic cancer. We identified a small-molecule UbcH5c inhibitor, termed DHPO, which directly bound to UbcH5c protein. DHPO inhibited cell viability and colony formation, induced apoptosis, and suppressed migration and invasion of pancreatic cancer cells in vitro. The compound inhibited UbcH5c-mediated IκBα degradation and NF-κB activation, which is critical for its anticancer activity. Furthermore, DHPO suppressed the tumor growth and metastasis in two orthotopic pancreatic tumor mouse models.ConclusionsThese results indicated that inhibiting UbcH5c is a novel and effective strategy for treating pancreatic cancer and DHPO represents a new class of UbcH5c inhibitor and may be further developed as an anti-pancreatic cancer therapeutic agent.

| S-EPMC8908661 | biostudies-literature

Ubiquitin in motion: structural studies of the ubiquitin-conjugating enzyme?ubiquitin conjugate.

Project description:Ubiquitination of proteins provides a powerful and versatile post-translational signal in the eukaryotic cell. The formation of a thioester bond between ubiquitin (Ub) and the active site of a ubiquitin-conjugating enzyme (E2) is critical for the transfer of Ub to substrates. Assembly of a functional ubiquitin ligase (E3) complex poised for Ub transfer involves recognition and binding of an E2?Ub conjugate. Therefore, full characterization of the structure and dynamics of E2?Ub conjugates is required for further mechanistic understanding of Ub transfer reactions. Here we present characterization of the dynamic behavior of E2?Ub conjugates of two human enzymes, UbcH5c?Ub and Ubc13?Ub, in solution as determined by nuclear magnetic resonance and small-angle X-ray scattering. Within each conjugate, Ub retains great flexibility with respect to the E2, indicative of highly dynamic species that adopt manifold orientations. The population distribution of Ub conformations is dictated by the identity of the E2: the UbcH5c?Ub conjugate populates an array of extended conformations, and the population of Ubc13?Ub conjugates favors a closed conformation in which the hydrophobic surface of Ub faces helix 2 of Ubc13. We propose that the varied conformations adopted by Ub represent available binding modes of the E2?Ub species and thus provide insight into the diverse E2?Ub protein interactome, particularly with regard to interaction with Ub ligases.

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Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network.

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Recombinant human phenylalanine hydroxylase is a substrate for the ubiquitin-conjugating enzyme system.

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| S-EPMC1217879 | biostudies-other

Molecular basis for the association of human E4B U box ubiquitin ligase with E2-conjugating enzymes UbcH5c and Ubc4.

Project description:Human E4B, also called UFD2a, is a U box-containing protein that functions as an E3 ubiquitin ligase and an E4 polyubiquitin chain elongation factor. E4B is thought to participate in the proteasomal degradation of misfolded or damaged proteins through association with chaperones. The U box domain is an anchor site for E2 ubiquitin-conjugating enzymes, but little is known of the binding mechanism. Using X-ray crystallography and NMR spectroscopy, we determined the structures of E4B U box free and bound to UbcH5c and Ubc4 E2s. Whereas previously characterized U box domains are homodimeric, we show that E4B U box is a monomer stabilized by a network of hydrogen bonds identified from scalar coupling measurements. These structural studies, complemented by calorimetry- and NMR-based binding assays, suggest an allosteric regulation of UbcH5c and Ubc4 by E4B U box and provide a molecular basis to understand how the ubiquitylation machinery involving E4B assembles.

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Structure of human ubiquitin-conjugating enzyme E2 G2 (UBE2G2/UBC7).

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| S-EPMC2222581 | biostudies-literature

Solution structure and dynamics of human ubiquitin conjugating enzyme Ube2g2.

Project description:Ube2g2 is an E2 enzyme which functions as part of the endoplasmic reticulum-associated degradation (ERAD) pathway responsible for identification and degradation of misfolded proteins in the endoplasmic reticulum. In tandem with a cognate E3 ligase, Ube2g2 assembles K48-linked polyubiquitin chains and then transfers them to substrate, leading ultimately to proteasomal degradation of the polyubiquitin-tagged substrate. We report here the solution structure and backbone dynamics of Ube2g2 solved by nuclear magnetic resonance spectroscopy. Although the solution structure agrees well with crystallographic structures for the E2 core, catalytically important loops (encompassing residues 95-107 and 130-135) flanking the active site cysteine are poorly defined. (15)N spin relaxation and residual dipolar coupling analysis directly demonstrates that these two loops are highly dynamic in solution. These results suggest that Ube2g2 requires one or more of its protein partners, such as cognate E3, acceptor ubiquitin substrate or thiolester-linked donor ubiquitin, to assume its catalytically relevant conformation. Within the NMR structural ensemble, interactions were observed between His94 and the highly mobile loop residues Asp98 and Asp99, supporting a possible role for His94 as a general base activated by the carboxylate side-chains of Asp98 or Asp99.

| S-EPMC2822102 | biostudies-literature

Dimerization regulates the human APC/C-associated ubiquitin-conjugating enzyme UBE2S.

Project description:At the heart of protein ubiquitination cascades, ubiquitin-conjugating enzymes (E2s) form reactive ubiquitin-thioester intermediates to enable efficient transfer of ubiquitin to cellular substrates. The precise regulation of E2s is thus crucial for cellular homeostasis, and their deregulation is frequently associated with tumorigenesis. In addition to driving substrate ubiquitination together with ubiquitin ligases (E3s), many E2s can also autoubiquitinate, thereby promoting their own proteasomal turnover. To investigate the mechanisms that balance these disparate activities, we dissected the regulatory dynamics of UBE2S, a human APC/C-associated E2 that ensures the faithful ubiquitination of cell cycle regulators during mitosis. We uncovered a dimeric state of UBE2S that confers autoinhibition by blocking a catalytically critical ubiquitin binding site. Dimerization is stimulated by the lysine-rich carboxyl-terminal extension of UBE2S that is also required for the recruitment of this E2 to the APC/C and is autoubiquitinated as substrate abundance becomes limiting. Consistent with this mechanism, we found that dimerization-deficient UBE2S turned over more rapidly in cells and did not promote mitotic slippage during prolonged drug-induced mitotic arrest. We propose that dimerization attenuates the autoubiquitination-induced turnover of UBE2S when the APC/C is not fully active. More broadly, our data illustrate how the use of mutually exclusive macromolecular interfaces enables modulation of both the activities and the abundance of E2s in cells to facilitate precise ubiquitin signaling.

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Identification of the structural and functional human homolog of the yeast ubiquitin conjugating enzyme UBC9.

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| S-EPMC145898 | biostudies-other

Mechanism of polyubiquitin chain recognition by the human ubiquitin conjugating enzyme Ube2g2.

Project description:Ube2g2 is a human ubiquitin conjugating (E2) enzyme involved in the endoplasmic reticulum-associated degradation pathway, which is responsible for the identification and degradation of unfolded and misfolded proteins in the endoplasmic reticulum compartment. The Ube2g2-specific role is the assembly of Lys-48-linked polyubiquitin chains, which constitutes a signal for proteasomal degradation when attached to a substrate protein. NMR chemical shift perturbation and paramagnetic relaxation enhancement approaches were employed to characterize the binding interaction between Ube2g2 and ubiquitin, Lys-48-linked diubiquitin, and Lys-63-linked diubiquitin. Results demonstrate that ubiquitin binds to Ube2g2 with an affinity of 90 ?M in two different orientations that are rotated by 180° in models generated by the RosettaDock modeling suite. The binding of Ube2g2 to Lys-48- and Lys-63-linked diubiquitin is primarily driven by interactions with individual ubiquitin subunits, with a clear preference for the subunit containing the free Lys-48 or Lys-63 side chain (i.e. the distal subunit). This preference is particularly striking in the case of Lys-48-linked diubiquitin, which exhibits an ?3-fold difference in affinities between the two ubiquitin subunits. This difference can be attributed to the partial steric occlusion of the subunit whose Lys-48 side chain is involved in the isopeptide linkage. As such, these results suggest that Lys-48-linked polyubiquitin chains may be designed to bind certain proteins like Ube2g2 such that the terminal ubiquitin subunit carrying the reactive Lys-48 side chain can be positioned properly for chain elongation regardless of chain length.

| S-EPMC3030398 | biostudies-literature

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