Decoding E6AP/UBE3A function: Structural revelations and small molecule modulators

许多细胞调节蛋白的活性通过与泛素的共价修饰来控制。泛素缀合系统的特异性由E3泛素蛋白连接酶的作用介导。值得注意的是，E3在人类疾病中经常失调，包括癌症、病毒感染和心血管、免疫学以及神经系统疾病。E6 AP是HECT E3家族的成员，由UBE 3A基因编码，代表了这一观点的一个主要例子：人乳头瘤病毒（HPV）的E6癌蛋白对E6 AP的E3活性的失调或刺激决定性地促进HPV诱导的宫颈癌发生;由UBE 3A基因的遗传改变引起的E6 AP失活是Angelman综合征（一种神经发育障碍）的原因; UBEA 3基因扩增导致E6 AP过表达是Dup 15 q自闭症谱系障碍的遗传标志。因此，深入了解E6 AP的结构-功能关系，既可以深入了解相互作用伙伴（如HPV E6癌蛋白或UBE 3A基因中的错义突变）影响E6 AP的E3活性的机制，也可以为设计影响E6 AP功能的小分子奠定基础，并最终开辟新的治疗策略。然而，我们目前对E6 AP结构的了解主要限于其催化HECT结构域。同样，关于E6 AP的E3活性如何在翻译后水平上被控制的知之甚少。这个跨学科的项目汇集了一个世界公认的专注于E6 AP生物化学和细胞生物学的小组，在分析E6癌蛋白和蛋白抑制剂复合物的结构方面有着良好记录的结构生物学家，以及一个质谱分析小组，专门研究大分子复合物中的构象变化和蛋白质-蛋白质界面。我们将结合联合收割机X射线晶体学，冷冻电子显微镜和交联质谱来阐明E6 AP单独和与包括HPV E6癌蛋白和细胞蛋白HERC 2在内的相互作用伙伴的高分辨率结构，这两者都已被我们证明可以变构刺激E6 AP的E3活性。此外，我们将表征E6 AP与刺激性小分子的相互作用，这是我们最近发现的，通过生物化学和结构手段，并调查是否这样的小分子效应器可以挽救在个人与Angelman综合征中发现的E6 AP突变体的活性。总而言之，拟议的研究将深入了解E6 AP的结构以及如何通过与其他蛋白质和小分子的相互作用来调节其活性，最终为治疗E6 AP相关疾病的小分子策略铺平道路。

The activity of many cell regulatory proteins is controlled by covalent modification with ubiquitin. The specificity of the ubiquitin-conjugation system is mediated by the action of E3 ubiquitin-protein ligases. Notably, E3s are frequently deregulated in human diseases including cancer, viral infections, and cardiovascular, immunological as well as neurological disorders. E6AP, which is a member of the family of HECT E3s and encoded by the UBE3A gene, represents a prime example for this notion: Dysregulation or stimulation of E6AP's E3 activity by the E6 oncoprotein of human papillomaviruses (HPVs) decisively contributes to HPV-induced cervical carcinogenesis; inactivation of E6AP by genetic alterations of the UBE3A gene is the cause of the Angelman syndrome, a neurodevelopmental disorder; UBEA3 gene amplification resulting in E6AP overexpression is the genetic hallmark of the Dup15q autism spectrum disorder. Thus, a thorough understanding of structure-function relationships of E6AP should both provide intimate insights into the mechanisms, by which interaction partners such as the HPV E6 oncoprotein or missense mutations in the UBE3A gene affect the E3 activity of E6AP, and lay the foundation for the design of small molecules that affect E6AP function and eventually open up new therapeutic strategies. However, our current knowledge about the structure of E6AP is mainly limited to its catalytic HECT domain. Similarly, only little is known about how the E3 activity of E6AP is controlled at the posttranslational level.This interdisciplinary project brings together a world-recognized group focusing on E6AP biochemistry and cell biology, structural biologists with a strong record in analyzing structures of E6 oncoproteins and protein-inhibitor complexes, and a mass spectrometry team specialized in deciphering conformational changes and protein-protein interfaces in macromolecular complexes. We will combine X-ray crystallography, cryo-electron microscopy and cross-linking mass spectrometry to elucidate the high-resolution structure of E6AP alone and in complex with interaction partners including the HPV E6 oncoprotein and the cellular protein HERC2, both of which have been shown by us to allosterically stimulate the E3 activity of E6AP. Furthermore, we will characterize the interaction of E6AP with stimulatory small molecules, which were recently identified by us, by biochemical and structural means and investigate whether such small molecule effectors can rescue the activity of E6AP mutants found in individuals with Angelman syndrome. Taken together, the proposed studies will provide intimate insights into the structure of E6AP and how its activity is modulated by interaction with other proteins and with small molecules, eventually paving the way for small molecule strategies in the treatment of E6AP-associated disorders.