MODULATION OF NF-kB ACTIVATION BY A NOVEL MITOCHONDRIAL E3 UBIQUITIN LIGASE

描述（由申请人提供）：泛素化是一种翻译后修饰，因其在蛋白质降解中的作用而闻名。虽然线粒体中不存在泛素，但线粒体外膜（ORM）蛋白暴露于胞质溶胶，因此可接近泛素化机制。E3 Ub连接酶赋予泛素化特异性。我们最近注释了人类基因组中的E3，并产生了代表几乎整个家族的阵列cDNA和RNAi集合，用于功能基因组方法。在使用这些收集物的筛选中，我们发现了MULAN，一种定位于线粒体并激活NF-（B.目的/假设：阐明MULAN在NF-（B）信号转导中的作用和调控。正在测试的假设是MULAN作为NF-β B活化的线粒体调节剂，显然作用于IKK和p65的上游，MULAN通过与下游效应物相互作用并活化下游效应物而起作用，并且MULAN的E3活性可以通过介导蛋白质的自动泛素化和降解而在信号传导中起负面作用。具体目的是进一步表征MULAN对NF-β B的激活并研究相关机制，以鉴定参与NF-β B信号传导的MULAN相互作用蛋白，并研究MULAN的E3活性如何调节蛋白的表达。研究设计：将使用生化和遗传方法来鉴定MULAN的下游效应物。cDNA表达和siRNA介导的功能丧失分析将测试它们与NF-β B活化的相关性，并将基于上位性在途径中排列蛋白质。它们在MULAN介导的NF-（B）活化中的作用和机制将被生物化学研究。E3活性的体外和体内测定将检查MULAN的调节。将确定响应于MULAN的条件表达而引起的快速生化变化。最后，生物化学和siRNA分析将研究导致MULAN从大肠杆菌降解的途径。癌症相关性：NF-β B通路与癌症的相关性通过在黑素瘤、结肠癌和肝癌中发现其某些通路组分的突变以及该通路在炎症中的关键作用而得到强调。靶向该途径的小分子正在临床开发中。拟议研究的结果最终应转化为药物发现新靶点的确定。通过流行病学和分子遗传学研究，已经发现用于细胞内通讯的几种生物化学途径与癌症有关，例如导致基因活性的蛋白质调节剂NF-（B）的途径。我们发现了一个基因，其产物通过一种新的机制控制NF-β B的激活，并且其在癌症中的作用也通过在黑色素瘤和胶质母细胞瘤细胞中明显升高的水平而被提出。对这种蛋白质的表征以及它如何与NF-β B连接将显著增强我们对NF-β B通路的基本理解，并且应该为肿瘤学以及其他适应症如炎性疾病中的药物发现提供新的靶点和机会。

DESCRIPTION (provided by applicant): Ubiquitylation is a post-translational modification best known for its role in protein degradation. While ubiquitin is absent from mitochondria, mitochondrial outer membrane (MOM) proteins are exposed to cytosol and are thus accessible to the ubiquitylation machinery. E3 Ub ligases confer specificity to ubiquitylation. We recently annotated the E3s in the human genome and generated collections of arrayed cDNA and RNAi representing nearly the entire family, for functional genomic approaches. In a screen using these collections, we uncovered MULAN, a novel E3 that localizes to mitochondria and activates NF-(B. Objective/Hypothesis: The goals are to elucidate the function and regulation of MULAN in NF-(B signaling. The hypotheses being tested are that MULAN acts as a mitochondrial regulator of NF-(B activation, apparently acting upstream of IKK and p65, that MULAN functions by interacting with and activating a downstream effector, and that MULAN's E3 activity can play a negative role in signaling by mediating the protein's auto- ubiquitylation and degradation. The Specific Aims are to further characterize the activation of NF-(B by MULAN and investigate mechanisms involved, to identify MULAN-interacting proteins involved in signaling to NF-(B, and to investigate how MULAN's E3 activity regulates the protein's expression. Study design: Biochemical and genetic approaches will be used to identify MULAN's downstream effectors. cDNA expression and siRNA-mediated loss-of-function analyses will test their relevance for NF-(B activation and will order the proteins in a pathway based on epistasis. Their role and mechanisms in MULAN-mediated NF-(B activation will be investigated biochemically. In vitro and in vivo assays for E3 activity will examine MULAN's regulation. Rapid biochemical changes elicited in response to the conditional expression of MULAN will be determined. Lastly, biochemical and siRNA analyses will investigate the pathway leading to MULAN's degradation from the MOM. Cancer relevance: The relevance of the NF-(B pathway for cancer is underscored by the finding of mutations in some of its pathway components in melanoma, colon and liver cancers as well as by the pathway's critical role in inflammation. Small molecules targeting the pathway are in clinical development. The results of the proposed studies should ultimately translate into the identification of new targets for drug discovery. PUBLIC HEALTH RELEVANCE Several biochemical pathways used for communication within cells have been implicated in cancer through epidemiology and molecular genetic studies, such as the pathway leading to a protein regulator of gene activity known as NF-(B. We discovered a gene whose product controls the activation of NF-(B by a novel mechanism, and its role in cancer is also suggested by apparently elevated levels in melanoma and glioblastoma cells. Characterization of this protein and how it connects to NF-(B will significantly enhance our basic understanding of the NF-(B pathway, and should provide new targets and opportunities for drug discovery in Oncology, as well as other indications such as inflammatory diseases.