Regulation and Consequences of Ubiquitination

泛素化的监管和后果

基本信息

批准号：
9153664
负责人：
Jonathan Ashwell
金额：
$ 32.15万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/9153664
关键词：
Adult Affect Animals Antigens Apoptosis Apoptosis Inhibitor Apoptotic Autophagocytosis B Cell Proliferation B-Lymphocytes BIRC4 gene Binding Biochemical Reaction C-terminal Cell Survival Cell physiology Cells Cessation of life Chimeric Proteins Complex Disease Enzymes Equilibrium Exons Family Generations Goals Growth Factor Gut associated lymphoid tissue Half-Life Human Hypergammaglobulinemia Immune response Immunoglobulin A Infection Inflammatory Response Interferons Knock-in Mouse Knockout Mice Lung Lymphocyte Lymphocytic choriomeningitis virus Lysine Mediating Metabolic Modification Molecular Mus Mutation NF-kappa B Outcome Pathway interactions Phenotype Phosphotransferases Point Mutation Polyubiquitin Polyubiquitination Primary Open Angle Glaucoma Process Production Proteins Regulation Relative (related person)Role Salmonella Series Signal Pathway Signal Transduction Stimulus T memory cell T-Lymphocyte TLR3 gene TLR4 gene TRAF2 gene Time Tissues Toxoplasma gondii Ubiquitin Ubiquitin-Protein Ligase Complexes Ubiquitination Up-Regulation Virus Withdrawal Work cIAP1 protein covalent bond cytokine in vivo mucosa-associated lymphoid tissue lymphoma p65 polypeptide prevent protein degradation response thymocyte ubiquitin-protein ligase upstream kinase

项目摘要

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. Notably, we identified XIAP and c-IAP1 as ubiquitin protein ligases (E3s), enzymes involved in the addition of Ub to target proteins. This activity was dependent upon a motif called the RING domain. We are currently working on the following: We have generated mice in which we "knocked-in" an E3-defective c-IAP2 (it contains a point mutation in the RING domain). We found (1) accumulation of B cells, especially of the marginal zone phenotype, and IgA hypergammaglobulinemia, (2) increased gut-associated lymphoid tissue (GALT) and lymphocyte inflitrates in the lung, (3) B cell hyperproliferation and relative insensitivity to growth factor-withdrawal apoptosis (4) 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. We also 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, whereas 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. In the past year we have found that c-IAP2 knockin mice mount a normal immune response to virus (LCMV) but fail to maintain memory T cells. This is because the antigen-specific T cells die in vivo. We identified defective signaling via 4-1BB, an anti-apoptotic co-stimulatory molecule on activated/memory T cells, as the cause. Currently we are working on the contribution of metabolic abnormalities to the death of memory T cells. 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. 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 C-terminal exons and cannont bind polyubiquitin. The latter express very little optineurin protein. We have found that these mice have defective IFN Type 1 production in response to signaling via TLR3 and TLR4, as well as to infection with virus. Studieson autophagy in cells from these animals are ongoing.

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。值得注意的是，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。我们目前正在研究以下工作：我们已经生成了小鼠，其中我们“敲入”了E3缺陷的C-IAP2（它包含环域中的点突变）。我们发现（1）B细胞的积累，尤其是边缘区表型和IGA高γ-磁蛋白血症，（2）（2）肺中与肠道相关的淋巴组织（GALT）和淋巴细胞屈服增加增加了肺中的淋巴细胞屈服，（3）B细胞超细胞和相对的活性因子（4）sppobspopt sppoptspobthcab spthcab spttobtsissis（4）非规范途径（NIK的上调）。 E3缺陷的C-IAP2还可以防止C-IAP1 ubiqutining/降解NIK，因为只有一个C-IAP分子可以在一次结合TRAF2（包括NIK在内的抑制性复合物的一个组成部分）。这些B细胞的表型与人类麦芽淋巴瘤的表型相似。我们建议，C-IAP2 E3活性的丧失伴随C-IAP2/MALT1融合蛋白的产生，是激活非经典NF-Kappab的主要因素。与野生型T细胞不同，C-IAP2 E3缺陷的T细胞在不存在共刺激的情况下对TCR占用性过高。结果，这些小鼠的感染通常是弓形虫弓形虫的菌株，导致“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。我们还发现，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，尽管非典型的NF-KB在B细胞中是正常的，并且B细胞增殖是正常的，但T细胞具有适度升高的非典型NF-KB，并增加了增殖（中间野生型和C-IAP2 E3检测小鼠）。因此，C-IAP使用存在组织特异性的差异。在过去的一年中，我们发现C-IAP2敲击小鼠对病毒（LCMV）的正常免疫反应安装，但无法维持记忆T细胞。这是因为抗原特异性T细胞在体内死亡。我们通过4-1BB（一种激活/记忆T细胞上的抗凋亡共刺激分子）确定了有缺陷的信号传导，是原因。目前，我们正在研究代谢异常对记忆T细胞死亡的贡献。 Optineurin是一种蛋白质，其突变负责成人发作的原发性敞开角质瘤的子集。 Optineurin含有与Nemo中UB结合基序高度同源的图案。 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C末端外显子和Cannont结合多泛素的Optineurin敲击蛋白小鼠。后者表达很少的Optineurin蛋白。我们发现，这些小鼠通过TLR3和TLR4以及病毒感染而响应信号而响应信号而产生的IFN 1型产生有缺陷。在这些动物的细胞中进行自噬的研究正在进行中。