Mechanism and Function of HECT E3s in Ubiquitin and ISG15 Conjugation

描述（由申请人提供）：泛素和泛素样蛋白（Ubls）的缀合系统通常组织成涉及E1、E2和E3酶的酶级联。在几类泛素E3中，HECT E3在机制上是独特的，因为它们形成共价酶-泛素中间体并直接参与蛋白质泛素化的化学过程。HECT E3在几种疾病状态（宫颈癌、Angelman综合征、Liddle综合征）、疾病相关途径（TGF-β信号传导、Notch信号传导、膜蛋白运输）中发挥重要作用，并被几种类型的病毒（人乳头瘤病毒、EBV、逆转录病毒、埃博拉病毒）征用。此外，单个HECT E3 HercS对于ISG 15的缀合是关键的，ISG 15是1型干扰素诱导的Ubl，其在对病毒和微生物感染的先天免疫应答中起作用。这些例子强调了理解HECT E3的机制、功能和调节的重要性。我们对理解HECT E3的功能和机制做出了重要贡献，并建议通过三条主要的研究路线来扩展最近的发现。第一条线的调查，由我们对酵母Rsp 5的调查驱动，将集中在Rsp 5及其人类同源物的物理相关的去泛素化酶（DUBs）的调节。这代表了E3调节的一种新形式，其可能代表在人类疾病中改变HECT E3功能的干预点。研究的第二条线将解决催化K63连接的多聚泛素化的HECT E3和催化K48连接的多聚泛素化的HECT E3之间的区别的机制基础。这是一个重要的问题，因为这两种类型的泛素化的最终结果是不同的。第三条研究路线将集中于HercS HECT E3对ISG 15缀合系统的机制和调节。我们将确定HercS依赖性ISG 15缀合所需的一整套因子，并确定这个单一的E3如何指导与数百种ISG 15靶蛋白的缀合。深入了解ISG 15结合的生物化学对于理解ISG 15在先天免疫应答中的功能至关重要。公共卫生相关性：通过泛素和Ubls的蛋白质修饰用于修饰许多关键细胞调节蛋白的稳定性、活性或定位，并且在许多疾病状态（特别是癌症）中观察到这些过程的破坏或改变。HECT家族泛素连接酶的生物化学的阐明将影响我们对致癌机制、信号传导途径以及对病毒和微生物感染的反应的理解。

DESCRIPTION (provided by applicant): Conjugation systems for ubiquitin and Ubiquitin-like proteins (Ubls) are generally organized into enzyme cascades involving E1, E2, and, E3 enzymes. Among the several classes of ubiquitin E3s, the HECT E3s are mechanistically unique in that they form a covalent enzyme-ubiquitin intermediate and participate directly in the chemistry of protein ubiquitination. HECT E3s play important roles in several disease states (cervical cancer, Angelman syndrome, Liddle's syndrome), disease-related pathways (TGF-beta signaling, Notch signaling, trafficking of membrane proteins), and are commandeered by several types of viruses (human papillomaviruses, EBV, retroviruses, Ebola). In addition, a single HECT E3, HercS, is critical for conjugation of ISG15, a type 1 interferon-induced Ubl that plays a role in the innate immune response to viral and microbial infections. These examples highlight the importance of understanding the mechanism, function, and regulation of HECT E3s. We have made important contributions toward understanding HECT E3 functions and mechanisms and propose to extend recent discoveries through three major lines of investigation. The first line of investigation, driven by our investigations on yeast Rsp5, will focus on the regulation of Rsp5 and its human homologs by physically associated deubiquitinating enzymes (DUBs). This represents a novel form of E3 regulation that may represent a point of intervention in altering HECT E3 function in human disease. The second line of investigation will address the mechanistic basis for the distinction between HECT E3s that catalyze K63-linked polyubiquitination and those that catalyze K48- linked polyubiquitination. This is an important problem because the ultimate consequences of these two types of ubiquitination are distinct. The third line of investigation will focus on the mechanism and regulation of ISG15 conjugation system by the HercS HECT E3. We will identify the complete set of factors required for HercS-dependent ISG15 conjugation and determine how this single E3 directs conjugation to perhaps hundreds of ISG15 target proteins. A thorough understanding of the biochemistry of ISG15 conjugation is essential for understanding the function of ISG15 in innate immune responses. Public health relevance: Protein modification by ubiquitin and Ubls serves to modify the stability, activity, or localization of many key cell regulatory proteins, and disruptions or alterations in these processes are seen in many disease states, particularly cancer. The elucidation of the biochemistry of the HECT family of ubiquitin ligases will impact our understanding of mechanisms of carcinogenesis, signaling pathways, and the response to viral and microbial infections.