泛素连接酶E3

蛋白质的泛素化修饰具有高度的特异性,它参与调节细胞内许多的生理活动。蛋白质的泛素化修饰涉及一系列的酶参与反应,包括泛素激活酶E1、结合酶E2以及连接酶E3。而其中泛素连接酶E3对靶蛋白的特异性识别起关键作用。泛素连接酶E3主要由HECT结构域家族、RING结构域家族和U-box结构域家族组成。现对泛素连接酶E3的分类、结构及其对靶蛋白的识别机制等进行综述。     

The E3 Ubiquitin Ligase UBE3C Enhances Proteasome Processivity by Ubiquitinating Partially Proteolyzed Substrates

To maintain protein homeostasis, cells must balance protein synthesis with protein degradation. Accumulation of misfolded or partially degraded proteins can lead to the formation of pathological protein aggregates. Here we report the use of destabilizing domains, proteins whose folding state can be reversibly tuned using a high affinity ligand, as model substrates to interrogate cellular protein quality control mechanisms in mammalian cells using a forward genetic screen. Upon knockdown of UBE3C, an E3 ubiquitin ligase, a reporter protein consisting of a destabilizing domain fused to GFP is degraded more slowly and incompletely by the proteasome. Partial proteolysis is also observed when UBE3C is present but cannot ubiquitinate substrates because its active site has been mutated, it is unable to bind to the proteasome, or the substrate lacks lysine residues. UBE3C knockdown also results in less substrate polyubiquitination. Finally, knockdown renders cells more susceptible to the Hsp90 inhibitor 17-AAG, suggesting that UBE3C protects against the harmful accumulation of protein fragments arising from incompletely degraded proteasome substrates.     

Human ITCH E3 Ubiquitin Ligase Deficiency Causes Syndromic Multisystem Autoimmune Disease

Ubiquitin ligases play an important role in the regulation of the immune system. Absence of Itch E3 ubiquitin ligase in mice has been shown to cause severe autoimmune disease. Using autozygosity mapping in a large Amish kindred, we identified a linkage region on chromosome 20 and selected candidate genes for screening. We describe, in ten patients, identification of a mutation resulting in truncation of ITCH. These patients represent the first reported human phenotype associated with ITCH deficiency. These patients not only have multisystem autoimmune disease but also display morphologic and developmental abnormalities. This disorder underscores the importance of ITCH ubiquitin ligase in many cellular processes.     

Engineering a Ubiquitin Ligase Reveals Conformational Flexibility Required for Ubiquitin Transfer

Protein ubiquitination regulates numerous cellular functions in eukaryotes. The prevailing view about the role of RING or U-box ubiquitin ligases (E3) is to provide precise positioning between the attached substrate and the ubiquitin-conjugating enzyme (E2). However, the mechanism of ubiquitin transfer remains obscure. Using the carboxyl terminus of Hsc70-interacting protein as a model E3, we show herein that although U-box binding is required, it is not sufficient to trigger the transfer of ubiquitin onto target substrates. Furthermore, additional regions of the E3 protein that have no direct contact with E2 play critical roles in mediating ubiquitin transfer from E2 to attached substrates. By combining computational structure modeling and protein engineering approaches, we uncovered a conformational flexibility of E3 that is required for substrate ubiquitination. Using an engineered version of the carboxyl terminus of Hsc70-interacting protein ubiquitin ligase as a research tool, we demonstrate a striking flexibility of ubiquitin conjugation that does not affect substrate specificity. Our results not only reveal conformational changes of E3 during ubiquitin transfer but also provide a promising approach to custom-made E3 for targeted proteolysis.Protein modification by ubiquitin and ubiquitin-like proteins is a common mechanism through which numerous cellular pathways are regulated (1). The canonical cascade of ubiquitination involves the action of three enzymes, termed E1, E2, and E3, which activate and then conjugate ubiquitin to its substrates (2, 3). The E3 ligase catalyzes the final step in ubiquitin transfer in a substrate-specific manner. Despite advances in understanding the enzymatic cascade of ubiquitination, the mechanism of ubiquitin transfer to the substrate remains an outstanding issue (4). In particular, the role of E3 ubiquitin ligases and how they adapt to progressively modified substrates to maintain specific ubiquitin chain topology is still a mystery.The known E3s belong to three protein families: HECT, RING, and U-box. HECT domain enzymes form a covalent intermediate with ubiquitin before the final transfer of ubiquitin to substrates. In contrast, RING and U-box E3s have been suggested to function as adaptors that position the substrate in close proximity to the E2-ubiquitin thioester (E2-Ub) (5). It has become common “wisdom” that the substrate has to be precisely positioned to get ubiquitinated (6). The positioning hypothesis originally predicted that E3 substrates would have a specific ubiquitination site. However, the absence of “consensus” ubiquitination sites has become apparent in an increasing list of E3 substrates (7–9). In addition, the crystal structures of several ubiquitination machinery components have revealed a puzzling gap (∼50 Å) between the substrate binding sites and the E2 active sites (10, 11). This raises a fundamental question in ubiquitin transfer. How does the ubiquitin molecule shuttle from the E2 to substrates? Though several interesting models for ubiquitin transfer have been proposed, only limited explicit experimental evidence support these models (4).We used carboxyl terminus of Hsc70-interacting protein (CHIP)³ as a model E3 system to investigate the role of substrate positioning in its ubiquitination. CHIP is a protein quality control E3 that consists of an NH₂-terminal tetratricopeptide repeat (TPR) domain, a helical linker domain, and a COOH-terminal U-box domain (12, 13). The TPR domain of CHIP binds directly to EEVD motifs located at the COOH termini of Hsc/Hsp70 and Hsp90, whereas the U-box domains possess ubiquitin ligase activity. CHIP recruits E2 enzymes of the Ubc4/5 family to ubiquitinate misfolded proteins that occupy the chaperone substrate-binding sites, thus remodeling the chaperones from protein-refolding complexes to complexes that promote degradation (14). Using the chaperone as an adaptor, CHIP targets a variety of substrates for ubiquitination (15). In the absence of substrates, CHIP is also able to ubiquitinate the bound chaperones (16). Thus, there is apparent substrate diversity for CHIP-mediated ubiquitination. Insights into the mechanism of action of CHIP have been provided by an x-ray crystal structure which reveals a remarkable, highly asymmetric dimer (25). Here, we demonstrate the existence of intrinsic structural flexibility in the CHIP homodimer that is required for substrate polyubiquitination. The flexible orientation allows CHIP to accommodate substrates with different sizes and structures. Mutations that restrict the flexibility of CHIP markedly decrease substrate ubiquitination, whereas maintaining flexibility enables us to rebuild a functional ubiquitin ligase with altered substrate specificity. Our results provide evidence for the importance of structural flexibility in E3 ligases, which we propose is of general importance to orchestrate progressive ubiquitin conjugation on substrates.     

The Active Form of E6-associated protein (E6AP)/UBE3A Ubiquitin Ligase Is an Oligomer

Employing ¹²⁵I-polyubiquitin chain formation as a functional readout of ligase activity, biochemical and biophysical evidence demonstrates that catalytically active E6-associated protein (E6AP)/UBE3A is an oligomer. Based on an extant structure previously discounted as an artifact of crystal packing forces, we propose that the fully active form of E6AP is a trimer, analysis of which reveals a buried surface of 7508 Ų and radially symmetric interacting residues that are conserved within the Hect (homologous to E6AP C terminus) ligase superfamily. An absolutely conserved interaction between Phe⁷²⁷ and a hydrophobic pocket present on the adjacent subunit is critical for trimer stabilization because mutation disrupts the oligomer and decreases k_cat 62-fold but fails to affect E2∼ubiquitin binding or subsequent formation of the Hect domain Cys⁸²⁰∼ubiquitin thioester catalytic intermediate. Exogenous N-acetylphenylalanylamide reversibly antagonizes Phe⁷²⁷-dependent trimer formation and catalytic activity (K_i = 12 mm), as does a conserved α-helical peptide corresponding to residues 474–490 of E6AP isoform 1 (K_i = 22 μm) reported to bind the hydrophobic pocket of other Hect ligases, presumably blocking Phe⁷²⁷ intercalation and trimer formation. Conversely, oncogenic human papillomavirus-16/18 E6 protein significantly enhances E6AP catalytic activity by promoting trimer formation (K_activation = 1.5 nm) through the ability of E6 to form homodimers. Recombinant E6 protein additionally rescues the k_cat defect of the Phe⁷²⁷ mutation and that of a specific loss-of-function Angelman syndrome mutation that promotes trimer destabilization. The present findings codify otherwise disparate observations regarding the mechanism of E6AP and related Hect ligases in addition to suggesting therapeutic approaches for modulating ligase activity.     

Nuclear Targeting of an Endosomal E3 Ubiquitin Ligase

Ring finger protein 13 (RNF13) is an E3 ubiquitin ligase embedded in endosome membranes. The protein undergoes constitutive post‐translational proteolysis, making its detection difficult unless cells are incubated with a proteasome inhibitor to allow biosynthetic forms to accumulate. When cells were treated with phorbol 12‐myristate 13‐acetate (PMA), RNF13 avoided proteolysis. A similar stabilization was seen on ionomycin treatment of cells. Drug treatment stabilized both the full‐length protein and a membrane‐embedded C‐terminal fragment generated following ectodomain shedding. Immunofluorescence staining revealed that PMA treatment caused the protein to accumulate in recycling endosomes, where it colocalized with transferrin receptor, and on the inner nuclear membrane, where it colocalized with lamin B. Expression of dominant‐negative Rab11 inhibited nuclear localization, suggesting RNF13 was targeted to the inner nuclear membrane through recycling endosomes. New protein synthesis was necessary for this targeting. Nuclear localization was confirmed by immunoelectron microscopy and by purification of the inner nuclear membrane. Stress‐induced transport of an endosomal protein to the inner nuclear membrane is a novel mechanism for introduction of regulatory proteins to the DNA environment. RNF13, with its ubiquitin ligase‐active RING domain, has the potential to turn over key nuclear proteins in response to signals received at the plasma membrane.     

Functions of E3 Ubiquitin Ligase Hyd in Drosophila Tissues

Molecular Biology - The ubiquitin–proteasome system (UPS) is an important regulator of the main cellular processes. The components of the UPS are involved in the regulation of the cell cycle,...     

UHRF2, a Ubiquitin E3 Ligase,Acts as a Small Ubiquitin-like Modifier E3 Ligase for Zinc Finger Protein 131

Small ubiquitin-like modifier (SUMO), a member of the ubiquitin-related protein family, is covalently conjugated to lysine residues of its substrates in a process referred to as SUMOylation. SUMOylation occurs through a series of enzymatic reactions analogous to that of the ubiquitination pathway, resulting in modification of the biochemical and functional properties of substrates. To date, four mammalian SUMO isoforms, a single heterodimeric SUMO-activating E1 enzyme SAE1/SAE2, a single SUMO-conjugating E2 enzyme ubiquitin-conjugating enzyme E2I (UBC9), and a few subgroups of SUMO E3 ligases have been identified. Several SUMO E3 ligases such as topoisomerase I binding, arginine/serine-rich (TOPORS), TNF receptor-associated factor 7 (TRAF7), and tripartite motif containing 27 (TRIM27) have dual functions as ubiquitin E3 ligases. Here, we demonstrate that the ubiquitin E3 ligase UHRF2 also acts as a SUMO E3 ligase. UHRF2 effectively enhances zinc finger protein 131 (ZNF131) SUMOylation but does not enhance ZNF131 ubiquitination. In addition, the SUMO E3 activity of UHRF2 on ZNF131 depends on the presence of SET and RING finger-associated and nuclear localization signal-containing region domains, whereas the critical ubiquitin E3 activity RING domain is dispensable. Our findings suggest that UHRF2 has independent functional domains and regulatory mechanisms for these two distinct enzymatic activities.     

Parkin:一种作用极其广泛的E3泛素连接酶

Parkin,又名PARK2,自发现初始便与帕金森病(Parkinson's disease,PD)密切相关.Parkin被认为是一种神经保护性基因.随着对其结构的深入了解,揭开了作为E3泛素连接酶的面纱.Parkin参与调控细胞周期、线粒体动态平衡和能量代谢等细胞进程,并与许多疾病息息相关,甚至在同一通路中发挥完全相...     

Protein Microarrays for the Identification of Praja1 E3 Ubiquitin Ligase Substrates

Although they are the primary determinants of substrate specificity, few E3-substrate pairs have been positively identified, and few E3's profiled in a proteomic fashion. Praja1 is an E3 implicated in bone development and highly expressed in brain. Although it has been well studied relative to the majority of E3's, little is known concerning the repertoire of proteins it ubiquitylates. We sought to identify high confidence substrates for Praja1 from an unbiased proteomic profile of thousands of human proteins using protein microarrays. We first profiled Praja1 activity against a panel of E2's to identify its optimal partner in vitro. We then ubiquitylated multiple, identical protein arrays and detected putative substrates with reagents that vary in ubiquitin recognition according to the extent of chain formation. Gene ontology clustering identified putative substrates consistent with information previously known about Praja1 function, and provides clues into novel aspects of this enzyme's function.     

Regulation of the CRL4Cdt2 Ubiquitin Ligase and Cell-Cycle Exit by the SCFFbxo11 Ubiquitin Ligase

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Highlights? FBXO11 targets CDT2, a CRL4 substrate receptor, for proteasomal degradation ? CDK-mediated phosphorylation of CDT2 degron inhibits recognition by FBXO11 ? FBXO11-mediated degradation of CDT2 controls the timing of cell-cycle exit ? FBXO11-CDT2 functional interaction is evolutionary conserved from worms to humans     

The BRCA1 E3 Ubiquitin Ligase Controls Centrosome Dynamics

The breast cancer specific tumor suppressor protein 1, BRCA1, mediates functions for all cells to grow. The puzzle of BRCA1 is that its loss is only associated with tumors in breast and ovarian epithelial cells. In this focused review, we highlight the data linking BRCA1 to the centrosome function, and we suggest that the specificity for breast tumors is due to a loss in restraint on centrosome function. Amplification of centrosome numbers secondary to loss of BRCA1 can drive the cell into the aneuploid state, thus, by this perspective loss of BRCA1 is a mutator phenotype.     

Regulation of LRRK2 Stability by the E3 Ubiquitin Ligase CHIP

Dominantly inherited mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are the most common cause of familial Parkinson''s disease (PD) and have also been identified in individuals with sporadic PD. Although the exact cellular function of LRRK2 remains unknown, most PD-linked mutations appear to be toxic to cells in culture via mechanisms that depend on the kinase activity of LRRK2 or on the formation of cytoplasmic inclusions. Here we show that the E3 ubiquitin ligase CHIP physically associates with LRRK2 and regulates the cellular abundance of LRRK2. We further show that LRRK2 forms a complex with overexpressed and endogenous CHIP and Hsp90. Our data indicates that the destabilization of LRRK2 by CHIP is due to ubiquitination and proteasome-dependent degradation. Hsp90 can attenuate CHIP-mediated degradation and this can be blocked by the Hsp90 inhibitor geldanamycin. These findings provide important insight into the cellular regulation of LRRK2 stability and may lead to the development of therapeutics to treat PD based on controlling LRRK2 stability.     

Itch WW Domains Inhibit Its E3 Ubiquitin Ligase Activity by Blocking E2-E3 Ligase Trans-thiolation

Nedd4-family E3 ubiquitin ligases regulate an array of biologic processes. Autoinhibition maintains these catalytic ligases in an inactive state through several mechanisms. However, although some Nedd4 family members are activated by binding to Nedd4 family-interacting proteins (Ndfips), how binding activates E3 function remains unclear. Our data reveal how these two regulatory processes are linked functionally. In the absence of Ndfip1, the Nedd4 family member Itch can bind an E2 but cannot accept ubiquitin onto its catalytic cysteine. This is because Itch is autoinhibited by an intramolecular interaction between its HECT (homologous to the E6-AP carboxy terminus domain) and two central WW domains. Ndfip1 binds these WW domains to release the HECT, allowing trans-thiolation and Itch catalytic activity. This molecular switch also regulates the closely related family member WWP2. Importantly, multiple PY motifs are required for Ndfip1 to activate Itch, functionally distinguishing Ndfips from single PY-containing substrates. These data establish a novel mechanism for control of the function of a subfamily of Nedd4 E3 ligases at the level of E2-E3 trans-thiolation.     

Dissection of the HIV Vif Interaction with Human E3 Ubiquitin Ligase

The human immunodeficiency virus type 1 (HIV-1) protein Vif recruits the host E3 ubiquitin ligase, composed of cullin 5 (Cul5), Rbx2, Elongin B, and Elongin C (EloBC), to polyubiquitinate the antiviral protein APOBEC3G. Multiple regions in the C-terminal half of Vif interact with the E3 ligase. We have purified individual regions of Vif and investigated their thermodynamic contributions to the ligase assembly in vitro using isothermal titration calorimetry and fluorescence anisotropy. Our results quantify the high-affinity interactions between the Vif BC box and EloBC and between the Vif zinc finger and Cul5, as well as the modest interaction between the Vif cullin box and Cul5. Our purified Vif constructs also provide direct biochemical evidence that the Vif cullin box, containing the PPLP region, leads to the dimerization of Vif-EloBC complexes but not Cul5-Vif-EloBC complexes.HIV Vif antagonizes the human antiviral protein APOBEC3G by hijacking the human Elongin B/C (EloBC)-cullin-SOCS box (ECS)-type E3 ubiquitin ligase, resulting in the polyubiquitination of APOBEC3G and subsequently its proteasomal degradation. Canonical ECS-type ubiquitin ligases consist of a cullin scaffold protein to which adaptor and substrate receptor proteins bind at the N terminus. HIV Vif serves as a substrate receptor protein—its N terminus recruits APOBEC3G, while multiple C-terminal regions assemble with the E3 ligase (9, 13, 24). The E3 ligase interacting regions include a zinc finger (residues 100 to 140), a BC box (residues 141 to 154), and a cullin box (residues 155 to 176) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.(A) A sequence schematic of Vif showing the regions that interact with A3G, A3F, EloBC, and Cul5. (B) An illustration of the assembly of the Vif-E3 ubiquitin ligase. (C) A homology model of Vif-Cul5-EloBC, where the Vif BC box-EloBC is actual structural data (PDB ID 3DCG).Vif binds the cullin adaptor proteins EloB and EloC through the BC-box region (24). The BC box is a loop-helix motif with the consensus sequence (T/S)LxxxCxxx(V/L/I) (7), and it also exists in cellular proteins that interact with EloBC. While Vif does not fit this consensus perfectly, it still binds EloBC with high affinity, and this interaction is lost upon mutation or deletion of consensus BC-box residues (10, 24, 25). This interaction has been described previously for the cellular proteins VHL (15), SOCS2 (3), SOCS3 (1), SOCS4 (4), and recently HIV Vif (14).Both the Vif zinc finger and cullin box interact with the E3 ligase scaffold protein cullin 5 (Cul5) (11, 12, 20, 21). It has been established that the zinc finger is required for Vif to bind Cul5. Mutation of critical histidine or cysteine residues in this region or the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) abolishes the Vif-Cul5 interaction (8, 11, 25). The sequence of the Vif cullin box is not as conserved as those of cellular SOCS-box proteins, which have a defined structure and determine the specificities of their respective cullins (6). The role of the Vif cullin box is not clear, but it has been suggested to promote dimerization of Vif, involving the conserved PPLP region (22, 23), and has recently been implicated in APOBEC3G binding (5, 17). While its importance in Cul5 binding has been demonstrated in coimmunoprecipitation experiments (14), experimental data also exist showing that the Vif zinc finger alone still immunoprecipitates Cul5 (11, 21).To dissect the assembly of the Vif-E3 ubiquitin ligase, we quantified the binding interactions between various C-terminal Vif constructs, EloBC, and Cul5 by isothermal titration calorimetry (ITC) and fluorescence polarization (FP). We additionally probed the effects of the cullin box on Vif dimerization.