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(UBL)的结合系统通常被组织成涉及E1、E2和E3酶的酶级联。在几类泛素E3中，Hect E3在机制上是独一无二的，因为它们形成了一种共价酶-泛素中间体，直接参与了蛋白质泛素化的化学过程。Hect E3在多种疾病状态(宫颈癌、Angelman综合征、利德尔综合征)、疾病相关途径(转化生长因子-β信号、Notch信号、膜蛋白转运)中发挥重要作用，并被多种病毒(人乳头瘤病毒、EBV、逆转录病毒、埃博拉)所占据。此外，单一的Hect E3，Hercs，对于ISG15的结合至关重要，ISG15是一种1型干扰素诱导的UbL，在对病毒和微生物感染的先天性免疫反应中发挥作用。这些例子突出了了解Hect E3的机制、功能和调控的重要性。我们为理解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新的发现。在我们对酵母Rsp5的研究的推动下，一线的调查将集中在Rsp5及其人类同系物的物理相关脱泛素酶(DUBS)的调节上。这代表了一种新的E3调节形式，可能代表了改变人类疾病中Hect E3功能的一个干预点。第二条研究路线将解决Hect E3催化K63连接的多泛素化和催化K48连接的多泛素化的Hect E3之间的区别的机制基础。这是一个重要的问题，因为这两种泛素化的最终后果是不同的。第三条研究线将集中在Hercs Hect E3对ISG15结合系统的机制和调控。我们将确定依赖于Hercs的ISG15结合所需的完整因子集，并确定单个E3如何将结合定向到可能数百个ISG15靶蛋白。深入了解ISG15结合的生物化学对于了解ISG15在先天免疫反应中的作用是至关重要的。公共卫生相关性：泛素和UBLS的蛋白质修饰作用是改变许多关键细胞调节蛋白的稳定性、活性或定位，这些过程的中断或改变在许多疾病状态下都可以看到，特别是癌症。Hect泛素连接酶家族生物化学的阐明将影响我们对致癌机制、信号通路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.