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。     

Human melanocortin 1 receptor-mediated ubiquitination of nonvisual arrestins. Role of Mahogunin Ring Finger 1 E3 ligase

Signaling from the melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor (GPCR) crucial for melanocyte proliferation and differentiation, is regulated by cytosolic β-arrestins (ARRBs). MC1R signaling is also negatively modulated by the E3-ubiquitin ligase Mahogunin Ring Finger-1 (MGRN1), whose mutation causes hyperpigmentation, congenital heart defects and neurodegeneration in mice. We showed previously that although MC1R interacts stably with human ARRB1 or ARRB2, only ARRB2 mediates receptor desensitization and internalization. We analyzed MC1R-dependent ARRB ubiquitination, and the possible role of MGRN1. ARRB1 expressed in heterologous cells or human melanoma cells migrated in SDS-PAGE as a 55 kDa protein whereas ARRB2 migrated as two major bands of apparent molecular weight near 45 and 55 kDa, with an intermediate mobility band occasionally detected. These forms were related by post-translational modification rather than by proteolysis. Presence of MC1R favored expression of the 45 kDa protein, the form that interacted preferentially with MC1R. MC1R also mediated poly- or multimonoubiquitination of ARRB2. Ubiquitination was agonist-independent, but required a native MC1R conformation and/or normal receptor trafficking to the plasma membrane, as it was not observed for loss-of-function MC1R variants. In a heterologous expression system, MC1R-dependent ARRB ubiquitination was enhanced by overexpression of MGRN1 and was impaired by siRNA-mediated MGRN1 knockdown thus pointing to MGRN1 as the responsible E3-ligase. Co-immunoprecipitation experiments demonstrated interaction of MGRN1 and ARRBs in the presence of MC1R, suggesting a scaffolding role for the GPCR that may determine the selectivity of E3-ubiquitin ligase engagement and the functional outcome of ARRB ubiquitination.     

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.     

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.     

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.     

HTLV-1 Tax Stimulates Ubiquitin E3 Ligase,Ring Finger Protein 8, to Assemble Lysine 63-Linked Polyubiquitin Chains for TAK1 and IKK Activation

Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of cellular processes, including I-κB kinase (IKK)/NF-κB signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in the development of adult T-cell leukemia (ATL) in HTLV-1-infected individuals, but the underlying mechanisms remain obscure. IKK and its upstream kinase, TGFβ-activated kinase 1 (TAK1), contain ubiquitin-binding subunits, NEMO and TAB2/3 respectively, which interact with K63-linked polyubiquitin (K63-pUb) chains. Recruitment to K63-pUb allows cross auto-phosphorylation and activation of TAK1 to occur, followed by TAK1-catalyzed IKK phosphorylation and activation. Using cytosolic extracts of HeLa and Jurkat T cells supplemented with purified proteins we have identified ubiquitin E3 ligase, ring finger protein 8 (RNF8), and E2 conjugating enzymes, Ubc13:Uev1A and Ubc13:Uev2, to be the cellular factors utilized by Tax for TAK1 and IKK activation. In vitro, the combination of Tax and RNF8 greatly stimulated TAK1, IKK, IκBα and JNK phosphorylation. In vivo, RNF8 over-expression augmented while RNF8 ablation drastically reduced canonical NF-κB activation by Tax. Activation of the non-canonical NF-κB pathway by Tax, however, is unaffected by the loss of RNF8. Using purified components, we further demonstrated biochemically that Tax greatly stimulated RNF8 and Ubc13:Uev1A/Uev2 to assemble long K63-pUb chains. Finally, co-transfection of Tax with increasing amounts of RNF8 greatly induced K63-pUb assembly in a dose-dependent manner. Thus, Tax targets RNF8 and Ubc13:Uev1A/Uev2 to promote the assembly of K63-pUb chains, which signal the activation of TAK1 and multiple downstream kinases including IKK and JNK. Because of the roles RNF8 and K63-pUb chains play in DNA damage repair and cytokinesis, this mechanism may also explain the genomic instability of HTLV-1-transformed T cells and ATL cells.     

E3泛素连接酶接头蛋白Keap1的研究进展

Kelch样ECH关联蛋白1(Kelch-like ECH-associated protein 1,Keap1)是E3泛素连接酶的底物识别亚单位,在蛋白质的泛素化修饰中起重要作用.蛋白质的泛素化修饰作为一种重要且复杂的蛋白质翻译后修饰,在自噬和蛋白酶体系统中作为降解信号而被利用.野生型Keap1能够识别、结合多种底物...     

Targeting the Ubiquitin E3 Ligase MuRF1 to Inhibit Muscle Atrophy

Progressive muscle wasting, also known as myopathy or muscle atrophy is a debilitating and life-threatening disorder. Myopathy is a pathological condition of many diseases including cancer, diabetes, COPD, and AIDS and is a natural consequence of inactivity and aging (sarcopenia). Muscle atrophy occurs when there is a net loss of muscle mass resulting in a change in the balance between protein synthesis and protein degradation. The ubiquitin pathway and specific ubiquitin pathway enzymes have been directly implicated in the progression of atrophy. The ubiquitin E3 ligase Muscle-specific RING Finger E3 ligase (MuRF1) is upregulated and increases protein degradation and muscle wasting in numerous muscle atrophy models. The inhibition of MuRF1 could be a novel mechanism to prevent or reverse muscle wasting associated with various pathologies. We screened a small molecule library for inhibitors to MuRF1 activity and identified P013222, an inhibitor of MuRF1 autoubiquitylation. Further, P013222 was shown to inhibit MuRF1-dependent substrate ubiquitylation, and was active in inhibiting MuRF1 in a cellular atrophy model. Thus MuRF1 can be targeted in a specific manner and produce positive results in cellular atrophy models.     

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.     

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.     

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...     

E3 Ubiquitin Ligase NEDD4 Promotes Influenza Virus Infection by Decreasing Levels of the Antiviral Protein IFITM3

Interferon (IFN)-induced transmembrane protein 3 (IFITM3) is a cell-intrinsic factor that limits influenza virus infections. We previously showed that IFITM3 degradation is increased by its ubiquitination, though the ubiquitin ligase responsible for this modification remained elusive. Here, we demonstrate that the E3 ubiquitin ligase NEDD4 ubiquitinates IFITM3 in cells and in vitro. This IFITM3 ubiquitination is dependent upon the presence of a PPxY motif within IFITM3 and the WW domain-containing region of NEDD4. In NEDD4 knockout mouse embryonic fibroblasts, we observed defective IFITM3 ubiquitination and accumulation of high levels of basal IFITM3 as compared to wild type cells. Heightened IFITM3 levels significantly protected NEDD4 knockout cells from infection by influenza A and B viruses. Similarly, knockdown of NEDD4 in human lung cells resulted in an increase in steady state IFITM3 and a decrease in influenza virus infection, demonstrating a conservation of this NEDD4-dependent IFITM3 regulatory mechanism in mouse and human cells. Consistent with the known association of NEDD4 with lysosomes, we demonstrate for the first time that steady state turnover of IFITM3 occurs through the lysosomal degradation pathway. Overall, this work identifies the enzyme NEDD4 as a new therapeutic target for the prevention of influenza virus infections, and introduces a new paradigm for up-regulating cellular levels of IFITM3 independently of IFN or infection.     

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,...     

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.     

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.