Aurora-A遗传变异Phe31Ile与小儿神经母细胞瘤遗传易感性

目的Aurora-A基因过度表达可引起中心粒异常、染色体不稳定和肿瘤形成。本研究探讨Aurora-APhe31Ile多态与神经母细胞瘤（neuroblastoma,NB）遗传易感性的关系。方法以聚合酶链反应（PCR）和限制性片段长度多态性（RFLP）分析方法,检测了19例NB病人和144例正常对照者Aurora-APhe31Ile基因型,比较不同基因型与NB发生和发展的关系。结果Aurora-AIle等位基因频率在NB患者和正常对照中的分布有显著性差异（76.3%vs.49.0%,P=0.04）。Aurora-A三种基因型Phe/Phe、Phe/Ile和Ile/Ile在病例和对照组中的分布有统计学显著性差异（P=0.05,趋势检验）,携带Aurora-AIle/Ile基因型者罹患NB的风险是Phe/Phe或Phe/Ile基因型携带者的2.51倍（95%CI,0.86-7.37）。结论Aurora-A遗传变异Phe31Ile可能是我国神经母细胞瘤的遗传易感因素。     

A novel RNF125 variant associated with Tenorio syndrome alters ubiquitin chain binding

A key signalling pathway required for clearance of viruses from host cells relies on the receptor protein, retinoic acid-inducible gene I (RIG-I). The activity of RIG-I is tightly controlled, and once bound to viral dsRNA, addition of lysine 63-linked ubiquitin chains activates signalling. Meanwhile, the addition of lysine 48-linked ubiquitin chains to RIG-I is required to terminate signalling when the infection has been resolved. Really interesting new gene (RING) finger protein 125 (RNF125) is the E3 ligase responsible for addition of the ubiquitin chains that terminate signalling, with disruption of its function associated with Tenorio syndrome. Here we describe a novel RNF125 gene variant in an individual with clinical symptoms including intellectual disability, macrocephaly and congenital heart disease, consistent with Tenorio syndrome. The newly identified Tenorio syndrome-associated variant [(NM_017831.4):c.670G>C p.Glu224Gln] is the first to be found in the ubiquitin interaction motif (UIM) of RNF125. While the E3 ligase activity of this RNF125 variant is retained, it has an impaired ability to interact with lysine 63-linked ubiquitin chains. The function of the UIM in RNF125 is uncertain; however, this study suggests that the UIM binds lysine 63-linked ubiquitin chains, and that this interaction is required for the normal function of RNF125.     

Mib1 modulates dynamin 2 recruitment via Snx18 to promote Dll1 endocytosis for efficient Notch signaling

Notch signaling regulates normal development and tissue homeostasis. Ligand endocytosis plays critical roles in Notch signaling activation. Endocytic proteins such as epsin and dynamin participate in Notch ligand activity by mediating Notch ligand endocytosis. The ubiquitin ligase Mib1 also plays essential roles in Notch signaling via Notch ligand ubiquitination. However, the molecular links between Mib1 and endocytic proteins have not been fully defined. Here, we show that Mib1 is involved in dynamin 2 recruitment to Dll1 and that Snx18, which interacts with dynamin 2, modestly regulates Dll1 endocytosis. Furthermore, the ubiquitin ligase activity of Mib1 is induced by Notch ligand–receptor interactions. Mib1 promotes the interaction between dynamin 2 and Snx18 in an ubiquitin ligase activity‐dependent manner. These results suggest that Mib1 modulates dynamin recruitment by regulating the interaction between Snx18 and dynamin 2, thereby helping to ensure the efficient signaling activity of Notch ligands.     

Genetic analysis of BRCA1 ubiquitin ligase activity and its relationship to breast cancer susceptibility

The N-terminus of the Breast Cancer-1 predisposition protein (BRCA1) associates with the BRCA1-associated RING domain-1 protein (BARD1) to form a heterodimer, which exhibits ubiquitin ligase activity that is abrogated by known cancer-associated BRCA1 missense mutations. The majority of missense substitutions identified in patients with a personal or a family history of disease have not been followed in pedigrees, nor there is a functional understanding of their impact. We have examined, by extensive missense substitution, the interaction of BRCA1 with components that contribute to its ubiquitin ligase activity, BARD1 and the E2 ubiquitin-conjugating enzyme, UbcH5a. Selection from a randomly generated library of BRCA1 missense mutations for variants that inhibit the interaction with these components identified substitutions in residues found altered in patient DNA, indicating a correlation between loss of component-binding and propensity to disease development. We further show that the BRCA1:E2 interaction is sensitive to substitutions in all structural elements of the BRCA1 N-terminus, whereas the BARD1 interaction is sensitive to a subset of BRCA1 substitutions, which also inhibit E2-binding. Patient variants that inhibit the BRCA1:E2 interaction show loss of ubiquitin ligase activity and correlate with disease susceptibility and theoretical predictions of pathogenicity. These data link the loss of ubiquitin ligase activity, through loss of E2-binding, to the majority of non-polymorphic patient variants described within the N-terminus of BRCA1 and illustrate the likely significant role of BRCA1 ubiquitin ligase activity in tumour suppression.     

PCNA-dependent regulation of p21 ubiquitylation and degradation via the CRL4Cdt2 ubiquitin ligase complex

The DNA polymerase delta processivity factor Proliferating Cell Nuclear Antigen (PCNA) promotes the DNA damage-induced degradation of the replication initiation factor Cdt1 via the CRL4(Cdt2) E3 ubiquitin ligase complex. Here we demonstrate that PCNA promotes the ubiquitylation and degradation of the CDK inhibitor p21 in cells irradiated with low dose of ultraviolet (UV) by a similar mechanism. Human cells that are depleted of Cul4, DDB1 (damage-specific DNA-binding protein-1), or the DCAF Cdt2, are deficient in the UV-induced ubiquitylation and degradation of p21. Depletion of mammalian cells of PCNA by siRNA, or mutations in p21 that abrogate PCNA binding, prevent UV-induced p21 ubiquitylation and degradation, indicating that physical binding with PCNA is necessary for the efficient ubiquitylation of p21 via the CRL4(Cdt2) ubiquitin ligase. Cdt2 functions as the substrate recruiting factor for p21 to the rest of the CRL4 ubiquitin ligase complex. The CRL4(Cdt2) E3 ubiquitin ligase ubiquitylates p21 both in vivo and in vitro, and its activity is dependent on the interaction of p21 with PCNA. Finally, we show that the CRL4(Cdt2) and the SCF(Skp2) ubiquitin ligases are redundant with each other in promoting the degradation of p21 during an unperturbed S phase of the cell cycle.     

有丝分裂激酶Aurora-A活性调节的分子机制

Aurora-A是一种参与有丝分裂的丝氨酸/苏氨酸激酶,其过表达与肿瘤形成有关。Aurora-A激酶活性可以通过磷酸化、去磷酸化以及依赖蛋白酶体的降解等方式进行调节。TPX2(target protein for Xenopus kinesin-like protein 2)、1-2(Inhibitor 2)、Ajuba通过磷酸化Aurora-A将其活化,PP1(type-1 proteinphosphatase)等通过去磷酸化Aurora-A而抑制其活性,而且APC／C-Cdh1依赖的蛋白酶体等可以将其降解。了解和认识Aumra-A激酶活性调节的分子机制将有助于理解它在有丝分裂及肿瘤形成中的作用。     

Midori‐ishi Cyan/monomeric Kusabira‐Orange‐based fluorescence resonance energy transfer assay for characterization of various E3 ligases

Many bacterial pathogens hijack the host ubiquitin system for their own benefit by delivering effectors with ubiquitin ligase (E3) into host cells via the type III secretion system. Therefore, screening for small compounds that selectively inhibit bacterial but not mammalian E3 ligases is a promising strategy for identifying molecules that could substitute for antibiotics. To facilitate high‐throughput screening for bacterial E3 ligase inhibitors, we developed a MiCy/mKO (Midori‐ishi Cyan/monomeric Kusabira‐Orange)‐based FRET (fluorescence resonance energy transfer) assay and validated it on Shigella IpaH E3 ligase effectors. We showed the feasibility of using the MiCy/mKO‐based FRET assay to identify the most appropriate ubiquitin‐conjugating enzymes (E2s) and determine the lysine specificity of a given E3, both hallmarks of E3 activity. Furthermore, we showed the usefulness of the FRET assay in characterizing mammalian E3 ligases, such as TNF receptor‐associated factor 6 (TRAF6) and mouse double minute 2 homologue (MDM2). In addition, we confirmed the feasibility of determining the efficiency of inhibition of E3 ligase activity using inhibitors of E1 ubiquitin‐activating enzymes, such as UBE1‐41, by measuring the IC₅₀. Based on these results, we concluded that the MiCy/mKO‐based FRET assay is useful for characterizing E3 enzyme activity, as well as for high‐throughput E3 inhibitor screening.     

Reconstruction of an active SOCS3-based E3 ubiquitin ligase complex in vitro: identification of the active components and JAK2 and gp130 as substrates

Abstract

SOCS3 (suppressor of cytokine signaling 3) inhibits the intracellular signaling cascade initiated by exposure of cells to cytokines. SOCS3 regulates signaling via two distinct mechanisms: directly inhibiting the catalytic activity of Janus kinases (JAKs) that initiate the intracellular signaling cascade and catalysing the ubiquitination of signaling components by recruiting components of an E3 ubiquitin ligase complex. Here we investigate the latter mode-of-action biochemically by reconstructing a SOCS3-based E3 ubiquitin ligase complex in vitro using fully purified, recombinant components and examining its ability to promote the ubiquitination of molecules involved in the cytokine signaling cascade. We show that SOCS3 is an active substrate recruitment module for a Cullin5-based E3 ligase and have defined the core protein components required for ubiquitination. SOCS3-induced polyubiquitination was rapid and could proceed through a number of different ubiquitin lysines. SOCS3 catalyzed the ubiquitination of both the IL-6 receptor common chain (gp130) and JAK2.     

Arabidopsis COP10 is a ubiquitin-conjugating enzyme variant that acts together with COP1 and the COP9 signalosome in repressing photomorphogenesis

A group of evolutionarily conserved pleiotropic COP/DET/FUS proteins was initially defined by their ability to repress photomorphogenesis in Arabidopsis. It was proposed that this regulation be mediated by targeting degradation of key cellular regulators that promote photomorphogenesis. Among them, COP1 and the COP9 signalosome have been hypothesized to fulfill the roles as an ubiquitin ligase (E3) and an essential E3 modulator. Here we report that COP10 encodes a protein similar to ubiquitin-conjugating enzyme (E2) variant proteins (UEV). COP10 is part of a nuclear protein complex and capable of directly interacting with both COP1 and the COP9 signalosome. Our data indicates that COP10 defines a possible E2 activity, thus validating the working hypothesis that the pleiotropic COP/DET/FUS group of proteins defined a protein ubiquitination pathway.     

RhoBTB2 is a substrate of the mammalian Cul3 ubiquitin ligase complex

Rhobtb2 is a candidate tumor suppressor located on human chromosome 8p21, a region commonly deleted in cancer. Rhobtb2 is homozygously deleted in 3.5% of primary breast cancers, and gene expression is ablated in approximately 50% of breast and lung cancer cell lines. RhoBTB2 is an 83-kD, atypical Rho GTPase of unknown function, comprising an N-terminal Rho GTPase domain and two tandem BTB domains. In this report, we demonstrate that RhoBTB2 binds to the ubiquitin ligase scaffold, Cul3, via its first BTB domain and show in vitro and in vivo that RhoBTB2 is a substrate for a Cul3-based ubiquitin ligase complex. Moreover, we show that a RhoBTB2 missense mutant identified in a lung cancer cell line is neither able to bind Cul3 nor is it regulated by the ubiquitin/proteasome system, resulting in increased RhoBTB2 protein levels in vivo. We suggest a model in which RhoBTB2 functions as a tumor suppressor by recruiting proteins to a Cul3 ubiquitin ligase complex for degradation.     

The E3 ubiquitin ligase Itch in T cell activation, differentiation, and tolerance

Tagging a small molecule ubiquitin to a protein substrate, or protein ubiquitination, plays an important role in the immune responses. This process is catalyzed by a cascade of enzymatic reactions, with the E3 ubiquitin ligases being the critical enzymes that determine the specificity of substrate recognition. The E3 ligase Itch was identified from a mutant mouse which displays skin scratching and abnormal immune disorders. In the past few years, much progress has been made in our understanding of Itch-promoted protein ubiquitination, modulation of its ligase activity by upstream kinases, and the kinase-ligase interaction in T cell differentiation and tolerance induction.     

BRCA1 : BARD1 induces the formation of conjugated ubiquitin structures, dependent on K6 of ubiquitin, in cells during DNA replication and repair

The N-terminus of the BRCA1 protein bears a RING finger domain that functions as an E3 ubiquitin ligase in vitro where it is able to catalyse the synthesis of monoubiquitin and polyubiquitin targeted proteins. This activity is greatly increased when BRCA1 is in a complex with its N-terminal binding partner BARD1. In this report we use an immunohistochemical approach to demonstrate the association of cellular BRCA1 with the end product of the ubiquitin conjugation and ligation pathway, conjugated ubiquitin. Association is apparent at DNA replication structures in S-phase and following treatment with hydroxyurea and also at sites of double strand break repair after exposure to ionizing radiation. Down-regulation of endogenous, cellular BRCA1 : BARD1 using siRNA results in abrogation of ubiquitin conjugation in these structures, suggesting that heterodimer activity is required for their formation. Conversely, ectopically expressed full-length BRCA1, but not BRCA1 bearing specific N-terminal amino acid substitutions, is able to cooperate with BARD1 to increase ubiquitin conjugation in cells. Conjugation of ubiquitin in foci is inhibited by the expression of ubiquitin bearing a lysine 6 mutation suggesting that the ubiquitin polymers formed at these sites are dependent on lysine-6 for linkage. Together these data demonstrate that BRCA1 directed ligation of ubiquitin occurs during S-phase and in response to replication stress and DNA damage and is therefore likely to be a significant aspect of BRCA1 cellular activity.     

The von Hippel-Lindau tumor suppressor protein is a component of an E3 ubiquitin-protein ligase activity.

pVHL, the product of the VHL tumor suppressor gene, plays an important role in the regulation of cell growth and differentiation of human kidney cells, and inactivation of the VHL gene is the most frequent genetic event in human kidney cancer. The biochemical function of pVHL is unknown. Here we report that pVHL exists in vivo in a complex that displays ubiquitination-promoting activity in conjunction with the universally required components E1, E2, and ubiquitin. pVHL-associated ubiquitination activity requires, at a minimum, pVHL to bind elongin C and Cul-2, relatives of core components of SCF (Skp1-Cdc53/Cul-1-F-box protein) E3 ligase complexes. Notably, certain tumor-derived mutants of pVHL demonstrate loss of associated ubiquitination promoting activity. These results identify pVHL as a component of a potential SCF-like E3 ubiquitin-protein ligase complex and suggest a direct link between pVHL tumor suppressor and the process of ubiquitination.     

The COP9 signalosome regulates the Neurospora circadian clock by controlling the stability of the SCFFWD-1 complex

The COP9 signalosome (CSN) promotes the function of SCF-type cullin-based ubiquitin ligase complexes in vivo. Paradoxically, removal of the Nedd8 modification of cullins by CSN inhibits the ubiquitin ligase activity of SCF complexes in vitro. Ubiquitination-mediated degradation of the Neurospora circadian clock protein FREQUENCY (FRQ) is critical for clock function. Ubiquitination of FRQ requires FWD-1, the substrate-recruiting subunit of an SCF complex. Here we show that disruption of a subunit of CSN (csn-2) impairs the degradation of FRQ and compromises its normal circadian expression. A FRQ-independent oscillator drives conidiation in the csn-2 mutant, resulting in a 2-d conidiation rhythm that persists in constant darkness (DD), constant light (LL), light-to-dark (LD) transitions, and temperature cycles. Strikingly, the levels of FWD-1 are drastically reduced in csn-2 mutant, explaining the impaired degradation of FRQ. Reduction of FWD-1 levels in the mutant requires its F-box, suggesting that its degradation is due to autoubiquitination. In addition, SKP-1 and CUL-1 of the SCF(FWD-1) complex are also unstable in the mutant. Therefore, our results establish an important role of CSN in the circadian clock of Neurospora. Our findings also reconcile the CSN paradox and suggest that a major function of CSN is to maintain the stability of SCF ubiquitin ligases in vivo.     

Nonredundant and complementary functions of TRAF2 and TRAF3 in a ubiquitination cascade that activates NIK-dependent alternative NF-kappaB signaling

The adaptor and signaling proteins TRAF2, TRAF3, cIAP1 and cIAP2 may inhibit alternative nuclear factor-kappaB (NF-kappaB) signaling in resting cells by targeting NF-kappaB-inducing kinase (NIK) for ubiquitin-dependent degradation, thus preventing processing of the NF-kappaB2 precursor protein p100 to release p52. However, the respective functions of TRAF2 and TRAF3 in NIK degradation and activation of alternative NF-kappaB signaling have remained elusive. We now show that CD40 or BAFF receptor activation result in TRAF3 degradation in a cIAP1-cIAP2- and TRAF2-dependent way owing to enhanced cIAP1, cIAP2 TRAF3-directed ubiquitin ligase activity. Receptor-induced activation of cIAP1 and cIAP2 correlated with their K63-linked ubiquitination by TRAF2. Degradation of TRAF3 prevented association of NIK with the cIAP1-cIAP2-TRAF2 ubiquitin ligase complex, which resulted in NIK stabilization and NF-kappaB2-p100 processing. Constitutive activation of this pathway causes perinatal lethality and lymphoid defects.     

Proteolysis-independent regulation of PI3K by Cbl-b-mediated ubiquitination in T cells

Cbl-b, a ring-type E3 ubiquitin protein ligase, is implicated in setting the threshold of T lymphocyte activation. The p85 regulatory subunit of phosphatidylinositol 3 kinase (PI3K) was identified as a substrate for Cbl-b. We have shown that Cbl-b negatively regulated p85 in a proteolysis-independent manner. Cbl-b is involved in the recruitment of p85 to CD28 and T cell antigen receptor zeta through its E3 ubiquitin ligase activity. The enhanced activation of Cbl-b(-/-) T cells was suppressed by the inhibition of PI3K. The results suggest a proteolysis-independent function for Cbl-b in the modification of protein recruitment.