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，以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（MOM）蛋白暴露于细胞质中，因此泛素化机制可以接近。E3 Ub连接酶赋予泛素化特异性。我们最近对人类基因组中的E3s进行了注释，并生成了几乎代表整个家族的cDNA和RNAi序列，用于功能基因组方法。在使用这些集合的筛选中，我们发现了MULAN，一种定位于线粒体并激活NF-(B)的新型E3。目的/假设：目的是阐明MULAN在NF-(B)信号传导中的作用及其调控。目前正在测试的假设是MULAN作为线粒体NF- B激活的调节剂，明显作用于IKK和p65的上游，MULAN通过与下游效应物相互作用并激活其功能，MULAN的E3活性可以通过介导蛋白质的自泛素化和降解在信号传导中发挥负面作用。具体目的是进一步表征MULAN对NF-(B)的激活并研究其机制，鉴定参与NF-(B)信号传导的MULAN相互作用蛋白，并研究MULAN的E3活性如何调节该蛋白的表达。研究设计：生化和遗传方法将用于鉴定木兰的下游效应。cDNA表达和sirna介导的功能丧失分析将测试它们与NF- B激活的相关性，并将在基于上位性的途径中排序蛋白质。它们在木兰介导的NF- B活化中的作用和机制将进行生化研究。体外和体内的E3活性测定将检验MULAN的调控作用。MULAN条件表达引起的快速生化变化将被确定。最后，生化和siRNA分析将研究导致MULAN从MOM降解的途径。癌症相关性：在黑色素瘤、结肠癌和肝癌中发现了NF- B通路的一些通路成分的突变，以及该通路在炎症中的关键作用，这一点强调了NF- B通路与癌症的相关性。靶向该途径的小分子正在临床开发中。拟议研究的结果应该最终转化为药物发现的新靶点的确定。通过流行病学和分子遗传学研究发现，细胞内用于通讯的几种生化途径与癌症有关，例如通往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，其产物通过一种新机制控制NF-(B)的激活，其在癌症中的作用也被黑色素瘤和胶质母细胞瘤细胞中明显升高的水平所暗示。该蛋白的表征及其与NF-(B)的连接方式将显著增强我们对NF-(B)通路的基本理解，并为肿瘤学以及其他适应症（如炎症性疾病）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.