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

描述(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。线粒体中没有泛素，而线粒体外膜(MOM)蛋白暴露在胞浆中，因此泛素化机制是可以获得的。E3Ub连接酶具有泛素化的特异性。我们最近对人类基因组中的E3进行了注释，并生成了代表几乎整个家族的阵列cDNA和RNAi集合，用于功能基因组学方法。在使用这些收集的筛选中，我们发现了木兰，一个定位于线粒体并激活核因子-B的新的E3。目的/假设：目的是阐明木兰在核因子-B信号转导中的功能和调控。正在测试的假设是，花木兰作为核因子-(B)激活的线粒体调节器，显然作用于ikk和p65的上游，花木兰通过与下游效应器相互作用并激活下游效应器来发挥作用，而花木兰的E3活性可能通过介导蛋白质的自动泛素化和降解而在信号传递中发挥负面作用。本研究的具体目的是进一步研究木兰对核因子-B的激活及其机制，鉴定木兰与核因子-B信号转导相关的蛋白，并研究木兰的E3活性如何调节该蛋白的表达。研究设计：将使用生化和遗传方法来确定木兰的下游效应器。CDNAs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，并将根据上位性对蛋白质进行排序。它们在花木兰介导的核因子-B激活中的作用和机制将进行生化研究。体外和体内E3活性检测将检验木兰的调节作用。将确定响应木兰条件表达而引起的快速生化变化。最后，生化和siRNA分析将调查导致木兰从母体中降解的途径。与癌症的相关性：在黑色素瘤、结肠癌和肝癌中发现了核因子-(B)途径的一些成分的突变，以及该途径在炎症中的关键作用，这突显了该途径与癌症的相关性。靶向该途径的小分子正在临床开发中。拟议研究的结果最终应转化为确定药物发现的新目标。公共卫生相关性通过流行病学和分子遗传学研究，几种用于细胞内交流的生化途径与癌症有关，例如通向一种被称为NF-B的蛋白质活性调节器的途径。我们发现了一种基因，其产物通过一种新的机制控制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。该蛋白的特性及其与核因子-B的连接方式将显著增强我们对核因子-(B)途径的基本了解，并将为肿瘤学以及炎症性疾病等其他适应症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.