Regulation and Consequences of Ubiquitination

泛素化的监管和后果

• 批准号: 8552820
• 负责人: Jonathan Ashwell
• 金额: $ 40.03万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552820
• 关键词: Adult; Affect; Amino Acids; Apoptosis; Apoptosis Inhibitor; Apoptotic; Area; Autophagocytosis; B Cell Proliferation; B-Lymphocytes; BIRC4 gene; Binding; Biochemical Reaction; Bloch Sulzberger syndrome; C-terminal; Cell Nucleus; Cell Survival; Cell physiology; Cells; Cessation of life; Chimeric Proteins; Complex; Coupling; Cytokine Receptors; Cytoplasm; Death Domain; Disease; Dominant-Negative Mutation; Ectodermal Dysplasia; Enzymes; Equilibrium; Exons; Family; Generations; Genetic Transcription; Glaucoma; Goals; Growth Factor; Gut associated lymphoid tissue; Half-Life; Human; Hypergammaglobulinemia; I Kappa B-Alpha; IkappaB kinase; Immune response; Immunoglobulin A; Immunologic Deficiency Syndromes; Infection; Inflammatory; Inflammatory Response; Knock-in Mouse; Knockout Mice; Link; Lung; Lymphocyte; Lysine; MAP Kinase Modules; MAPK8 gene; Mediating; Membrane Microdomains; Mitogen-Activated Protein Kinases; Modification; Molecular; Mus; Mutation; NF-kappa B; Outcome; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Point Mutation; Polyubiquitin; Polyubiquitination; Primary Open Angle Glaucoma; Process; Production; Protein Binding; Proteins; Receptor Signaling; Receptors, Tumor Necrosis Factor, Type II; Regulation; Relative (related person); Role; Salmonella; Series; Signal Pathway; Signal Transduction; Stimulus; T-Lymphocyte; TNF receptor-associated factor 5; TRAF2 gene; Time; Tissues; Toxoplasma gondii; Tumor Necrosis Factor Receptor; Tumor Necrosis Factor-alpha; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligase Complexes; Ubiquitination; Up-Regulation; Withdrawal; Zinc Fingers; cIAP1 protein; covalent bond; cytokine; dimer; human RIPK1 protein; human TNFRSF1A protein; knock-down; loss of function mutation; member; migration; mucosa-associated lymphoid tissue lymphoma; multicatalytic endopeptidase complex; mutant; neuron loss; p65; polypeptide; prevent; protein degradation; receptor; response; retinal neuron; thymocyte; transcription factor; tumor necrosis factor alpha receptor; ubiquitin-protein ligase; upstream kinase

Our involvement in the regulation and consequences of ubiquitination began in the course of studies aimed at understanding why double positive (DP) thymocytes are so sensitive to pro-apoptotic stimuli. We found that induction of DP apoptosis, regardless of the molecular pathway, resulted in the degradation of XIAP and c-IAP1, proteins of the Inhibitor of APoptosis (IAP) family. Importantly, we identified XIAP and c-IAP1 as ubiquitin protein ligases (E3s), enzyme involved in the addition of Ub to target proteins. This activity was dependent upon a motif called the RING domain. In subsequent studies we made the following findings:-Signaling via Tumor Necrosis Factor (TNF) receptor 2 (TNF-R2), but not TNF-R1, results in ubiquitination and degradation of the signaling intermediate TRAF2 (TRAF2 is required for coupling the TNF-R to JNK activation and, with TRAF5, to NF-kappaB).-TNF-R2 -mediated ubiquitination of TRAF2 is mediated by c-IAP1.-Expression of an "E3-dead" c-IAP1 RING point mutant (a dominant negative) prevented TNF-alpha-induced TRAF2 degradation and inhibited apoptosis, demonstrating that c-IAP1 can actually be pro-apoptotic, probably by causing the degradation of TRAF2 and, perhaps, other anti-apoptotic molecules.-Stimulation via TNF-R2 results in the translocation of a c-IAP1/TRAF2 complex to the perinuclear ER, where it encounters a ubiquitin conjugating enzyme (E2), which cooperates with c-IAP1 to cause TRAF2 ubiquitination.-We were the first to characterize mice deficient in c-IAP1, and found that it has an obligate role in the ubiquitination and degradation of another member of the IAP family, c-IAP2.We have also found that ASK1, an important upstream enzyme in the MAP kinase signaling cascade, is a target for c-IAP1 in B cells stimulated with TNF. As a result, MAP kinase signaling is terminated in a timely fashion, moderating B cell responses.Another area in which we have studied ubiquitination is in signaling for activation of the important transcription factor NF-kappaB. NF-kappaB is sequestered in the cytoplasm in a complex with IkappaB. In the "canonical" pathway of activation, signals converge on IkappaB kinase (IKK), which phosphorylates IkappaB resulting in IkappaB K48-linked polyubiquitination, IkappaB degradation by proteasomes, and migration of NF-kappaB to the nucleus. IKK has two enzymatically-active subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma or NEMO. NEMO is essential for NF-kappaB activation, and NEMO mutations or deficiency have been identified as the cause of incontinentia pigmenti (IP) and hypohidrotic ectodermal dysplasia and immunodeficiency (HED-ID). The mechanism by which proximal cytokine receptor signals result in its NEMO-dependent activation remains largely unknown. Among the best-studied of such signaling pathways is that for TNF-alpha. TNF receptor 1 (TNF-R1) occupancy results in receptor trimerization and the serial recruitment of TNF receptor-associated death domain (TRADD), Fas-associated death domain (FADD), receptor-interacting protein (RIP), TRAF2, and c-IAP1 and c-IAP2. RIP in particular is an essential intermediate for downstream activation of NF-kappaB. Upon stimulation with TNF-alpha, RIP binds to NEMO, which brings with it the other IKK components. The RIP that associates with TNF-R1 undergoes polyubiquitination, initially K63-linked, in lipid rafts; the K63-linked polyUb is subsequently removed by the de-ubiquitinating domain of A20 and K48-linked polyUb chains are added by the zinc finger region of A20, resulting in RIP degradation.NF-kappaB can also be activated by an independent, non-canonical pathway, in which the kinase NIK is stabilized, resulting in phosphorylation of p100, its processing to p52, and migration of p52 and RelB dimers to the nucleus where they drive gene transcription.We found that a fusion protein between c-IAP2 and MALT1, which is the major single cause of human MALT lymphoma, activates NF-kappaB by two mechanisms. It activates the canonical pathway by virtue of its paracaspase enzymatic activity (contributed by the MALT1 portion). It also activates non-canonical NF-kappaB, via a paracaspase-independent means, by stabilizing NIK levels. Importantly, E3-inactive c-IAP2 mutants not fused to any other protein do the same thing. We have generated mice in which we "knocked-in" an E3-defective c-IAP2 (it contains a point mutation in the RING domain). We have have found- accumulation of B cells, especially of the marginal zone phenotype, and IgA hypergammaglobulinemia.- increased gut-associated lymphoid tissue (GALT) and lymphocyte inflitrates in the lung.- B cell hyperproliferation and relative insensitivity to growth factor-withdrawal apoptosis.- spontaneous B cell NF-kappaB activity via the non-canonical pathway (upregulation of NIK).- the E3-defective c-IAP2 also prevents c-IAP1 from ubiqutinating/degrading NIK, because only one c-IAP molecule can bind TRAF2 (a component of the inhibitory complex that includes NIK) at a time.The phenotype of these B cells is similar to that of human MALT lymphomas. We propose that the loss of c-IAP2 E3 activity, which accompanies the generation of the c-IAP2/MALT1 fusion protein, is a major contributor to disease by activating non-canonical NF-kappaB.- c-IAP2 E3 defective T cells, unlike wild type T cells, are hyperresponsive to TCR occupancy in the absence costimulation.. As a result, infection of these mice with a normally avirulent strain of Toxoplasma gondii led to death via "cytokine storm". These results strongly suggest that non-cannonical NF-kappaB acivation is a costimulation signaling pathway. In the past year we found that T cells from p100 knockout mice, which cannot activate the non-canonical pathway, are also costimulation independent. This is because p100 binds p65 and is a negative regulator of the canonical pathway. Furthermore, p100 levels are decreased in the c-IAP2 knockin mice (due to constitutive processing to the stimulatory p52 form). Therefore, we have identified the balance between p100 and p52 as a key regulator of the ability of T cells to respond to TCR-mediated activation. - c-IAP1 E3-defective c-IAP1 mice have been generated as well. They do not have an overt phenotype. Interestingly, where as non-canonical NF-kB is normal in B cells and B cell proliferation is normal, T cells have modestly eleveated non-canonical NF-kB and increased proliferation (intermediate between wild type and c-IAP2 E3-defective mice). Therefore, there a tissue-specific differences in c-IAP uses.Optineurin is a protein whose mutation is responsible for a subset of adult-onset primary open angle glaucoma. Optineurin contains a motif highly homologous to the Ub-binding motif in NEMO. In fact, we have found the optineurin binds to K63-linked polyUb much better than NEMO, and that it competes with NEMO for ubiquitinated RIP in TNF-stimulated cells. Acquisition of optineurin inhibits NF-kappaB activation, and forced knock-down of optineurin greatly enhances NF-kappaB activation. Given that NF-kappaB greatly enhances excitotoxic neuronal cell death, we have proposed that loss-of-function mutations in optineurin may in fact cause glaucoma due to enhanced retinal neuron cell death. Optineurin also binds Tank-binding kinase 1 (TBK1), a kinase upstream of type 1 inferferon production, in an inducible fasion, and has recently been implicated as a key factor in autophagy to Salmonella. We have generated optineurin knockin mice that lack the first 152 amino acids and cannot bind TBK1 as well as optineurin knockin mice that lace the C-terminal exons and cannont bind polyubiquitin. We are currently studying the effects of these mutations on signaling for inflammatory cytokine production and on autophagy.

我们参与泛素化的调节和后果始于研究，旨在理解为什么双重阳性（DP）胸腺细胞对促凋亡刺激如此敏感。我们发现，不论分子途径如何，DP凋亡的诱导导致了细胞凋亡抑制剂（IAP）家族的蛋白（IAP）家族的XIAP和C-IAP1降解。重要的是，我们将XIAP和C-IAP1鉴定为泛素蛋白连接酶（E3S），涉及在靶蛋白中添加UB的酶。该活动取决于称为环域的基序。在随后的研究中，我们做出了以下发现： - 通过肿瘤坏死因子（TNF）受体2（TNF-R2）而不是TNF-R1进行信号，导致信号传导中间体TRAF2的泛素化和降解（TRAF2需要TRAF2（TRAF2）（将TRAF2耦合到tnf-R到tnf-r介绍tnf-r至jNF-r-jnk inf-jnk激活和traf-t traf-tf-t。 TRAF2的泛素化是由C-IAP1介导的。表达“ E3 DEAD” C-IAP1环形点突变体（主要负面），阻止了TNF-Alpha诱导的TRAF2降解并抑制凋亡，并抑制了C-IAP1实际上可能是通过DEFOTIC，可能是导致的，可能是导致TRAF的其他可能性，可能是导致的，可能是其他种去的方法。分子通过TNF-R2刺激会导致C-IAP1/TRAF2复合物转移到核周核ER上，在该核ER中遇到了泛素偶联酶（E2），与C-iap1合作，该酶与C-iap1合作，导致TRAF2泛素化。 IAP家族的另一个成员C-AP2的泛素化和降解。我们还发现，MAP激酶信号级联中重要的上游酶Ask1是在用TNF刺激的B细胞中C-IAP1的靶标。结果，MAP激酶信号以及时的方式终止，调节B细胞响应。我们研究的另一个区域是在激活重要转录因子NF-kappab的信号传导中。 NF-kappab在与Ikappab的复合物中隔离在细胞质中。在激活的“规范”途径中，信号在ikappab激酶（IKK）上收敛，这会导致Ikappab磷酸化，从而导致Ikappab K48-链接的多泛素化，Ikappab ikappab by Proteasomes降解，NF-Kappab迁移到核向核。 IKK具有两个酶促的亚基，Ikkalpha和Ikkbeta，以及一个监管亚基，Ikkgamma或Nemo。 NEMO对于NF-kappab的激活至关重要，已确定NEMO突变或缺乏症是导致色素不便的原因（IP）和低蛋白质外胚性外胚层发育异常和免疫缺陷（HED-ID）。近端细胞因子受体信号导致其依赖性激活的机制仍然很大未知。在此类信号通路中最有研究的是TNF-Alpha。 TNF受体1（TNF-R1）占用率会导致受体修剪和TNF受体相关死亡结构域（TRADD），FAS相关死亡结构域（FADD），受体相互作用蛋白（RIP），TRAF2和C-IAP1和C-IAP1和C-IIAP2的受体募集。尤其是RIP是NF-kappab下游激活的必不可少的中间体。用TNF-Alpha刺激后，RIP与Nemo结合，它带来了其他IKK组件。与TNF-R1相关的RIP经历脂质筏中最初是K63连接的多泛素化。 the K63-linked polyUb is subsequently removed by the de-ubiquitinating domain of A20 and K48-linked polyUb chains are added by the zinc finger region of A20, resulting in RIP degradation.NF-kappaB can also be activated by an independent, non-canonical pathway, in which the kinase NIK is stabilized, resulting in phosphorylation of p100, its processing to p52，以及p52和RERB二聚体向核驱动基因转录的细胞核的迁移。我们发现C-IAP2和MALT1之间的融合蛋白是人类麦芽淋巴瘤的主要单一原因，它通过两种机制激活NF-kappab。它通过其paracaspase酶活性（由MALT1部分贡献）激活规范途径。它还通过稳定NIK水平来激活非规范的NF-kappab，通过paracaspase独立的手段来激活非经典的NF-kappab。重要的是，不融合任何其他蛋白质的E3-In活性C-IAP2突变体也做同样的事情。我们已经产生了小鼠，其中“敲入”了E3缺陷的C-IAP2（它包含环域中的点突变）。我们已经发现了B细胞的积累，尤其是边缘区表型和IGA高γ-磁全血征。途径（NIK的上调）.- E3缺陷的C-IAP2还可以防止C-IAP1 ubiqutination/degrading Nik，因为只有一个C-IAP分子可以在某种程度上结合TRAF2（包括NIK在内的抑制性复合物的一个组成部分）。我们建议，伴随C-IAP2/MALT1融合蛋白产生C-IAP2 E3活性的丧失是对疾病的主要促进者，这是激活非经典的NF-KAPPAB.- C-iAP2 E3有缺陷的T细胞，与TCR占用相比，与TCR占用相比，它在某种程度上是一种过度的，并且是一种经常性的捕获。弓形虫Gondii通过“细胞因子风暴”死亡。这些结果强烈表明，非通道的NF-kappab acivation是一种共刺激信号通路。在过去的一年中，我们发现来自无法激活非规范途径的P100敲除小鼠的T细胞也与共同刺激无关。这是因为p100结合了p65，并且是规范途径的负调节剂。此外，C-IAP2敲击蛋白小鼠的P100水平降低（由于构型处理刺激p52形式）。因此，我们已经确定了P100和P52之间的平衡是T细胞对TCR介导的激活反应能力的关键调节剂。 -C-IAP1 E3缺陷的C-IAP1小鼠也已产生。他们没有明显的表型。有趣的是，如果非典型的NF-KB在B细胞中正常并且B细胞增殖是正常的，则T细胞具有适度升高的非典型NF-KB，并增加了增殖并增加（中间野生型和C-IAP2 E3缺乏症小鼠）。因此，C-IAP使用中存在组织特异性的差异。Opineurin是一种蛋白质，其突变是成人发作的原发性开角青光眼的子集。 Optineurin含有与Nemo中UB结合基序高度同源的图案。实际上，我们发现Optineurin与K63连接的Polyub的结合要比Nemo好得多，并且它与Nemo竞争了TNF刺激的细胞中的泛素化RIP。 optineurin的获取抑制了NF-kappab的激活，而强迫敲除optineurin会大大增强NF-kappab激活。鉴于NF-kappab极大地增强了兴奋性神经元细胞死亡，因此我们提出，果胶中的功能丧失突变实际上可能导致视网膜神经元细胞死亡增强引起的青光眼。 Optineurin还结合了储罐结合激酶1（TBK1），这是一种诱导型降低的1型推断产生的激酶，最近被认为是沙门氏菌自噬的关键因素。我们已经产生了缺乏前152个氨基酸的Optineurin敲击蛋白小鼠，无法结合TBK1以及带有C-末端外显子和Cannont结合多泛素的Optineurin敲击蛋白小鼠。我们目前正在研究这些突变对炎症细胞因子产生和自噬的信号传导的影响。