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

许多细胞调节蛋白的活性是通过泛素的共价修饰来控制的。泛素-连接系统的特异性是通过E3泛素-蛋白连接酶的作用来调节的。值得注意的是，在包括癌症、病毒感染以及心血管、免疫和神经疾病在内的人类疾病中，E3经常被解除管制。E6AP是Hect E3s家族的成员，由UBE3A基因编码，它是这一概念的一个典型例子：人乳头瘤病毒(HPV)的E6癌蛋白对E6AP的E3活性的失调或刺激，决定性地促成了HPV诱导的宫颈癌的发生；UBE3A基因的遗传改变导致E6AP的失活是一种神经发育障碍Angelman综合征的原因；UBEA3基因的扩增导致E6AP的过度表达是Dup15q自闭症谱系障碍的遗传标志。因此，深入了解E6AP的结构-功能关系，不仅可以深入了解HPV E6癌蛋白或UBE3A基因错义突变等相互作用伙伴影响E6AP的E3活性的机制，还可以为设计影响E6AP功能的小分子奠定基础，最终开辟新的治疗策略。然而，我们目前对E6AP的结构的了解主要局限于它的催化Hect结构域。同样，对E6AP的E3活性如何在翻译后水平上进行控制也知之甚少。这个跨学科的项目汇集了一个专注于E6AP生物化学和细胞生物学的世界公认的小组，在分析E6癌蛋白和蛋白质-抑制物复合体的结构方面有着丰富记录的结构生物学家，以及一个专门破译大分子复合体的构象变化和蛋白质-蛋白质界面的质谱组。我们将结合X射线结晶学、冷冻电子显微镜和交联质谱来阐明E6AP单独以及与包括HPV E6癌蛋白和细胞蛋白HERC2在内的相互作用伙伴的高分辨结构，我们已经证明这两种蛋白都可以变构刺激E6AP的E3活性。此外，我们将通过生化和结构手段表征E6AP与我们最近鉴定的刺激性小分子的相互作用，并研究这些小分子效应器是否能够挽救在Angelman综合征患者中发现的E6AP突变的活性。综上所述，拟议的研究将为E6AP的结构以及其活性是如何通过与其他蛋白质和小分子的相互作用而调节的提供深入的见解，最终为小分子策略治疗E6AP相关疾病铺平道路。

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.