Structural and Functional Analysis of Bacterial and Viral Deubiquitinases

泛素信号在许多细胞过程中都是至关重要的。泛素与底物蛋白的共价结合影响其生物学功能。最具代表性的功能是26S介导的底物蛋白的蛋白酶体降解。泛素通过由E1、E2和E3酶组成的酶级联反应与底物蛋白相连。泛素也可以被脱泛素酶(DUBS)从底物蛋白中切割出来。去泛素化活性必须受到高度调控，否则蛋白质将成为这些酶的非特异性靶标，导致细胞过程失控。病原生物体有能力操纵寄主生物体中的泛素信号，从而使其受益。这是病原体入侵的关键一步。包括细菌和病毒在内的各种致病生物都含有与泛素信号传递有关的蛋白质，包括细菌/病毒E3连接酶和DUBS，它们被用来抑制免疫反应，从而使感染成为可能。 我的实验室的长期目标是研究细菌、病毒和人类去泛素酶的功能，以深入了解它们在细胞过程中的作用。这项研究的目标是通过阐明它们的功能、底物和在宿主生物体中的作用机制来表征一种细菌和几种病毒的特征。我们现在正在扩大我们的研究主题，以包括来自各种病原体的配音。该项目分为几个短期目标： [1]大肠杆菌脱泛素酶Elad的结构和功能特征。人们对E.ColiElad知之甚少。我们将识别宿主底物，并表征ELAD与其宿主底物相互作用的结构基础。 [2]病毒脱泛素酶BPLF1、ORF64、UL36和UL48的特性。BPLF1、ORF64、UL36和UL48是来源于不同疱疹病毒的脱泛素酶。人们对这些蛋白质的功能知之甚少。例如，在四个病毒复制片段中的三个中，几乎没有发现宿主底物蛋白。BPLF1与增殖细胞核抗原、TRAF6和NEMO相互作用并去泛素化，UL36去泛素化TRAF3和ORF64去泛素化RIG-1。 [3]BPLF1、ORF64、UL36和UL48寄主底物(S)及其相互作用蛋白的鉴定。已知的病毒复制的宿主底物很少。在提案的这一部分，我们将使用亲和纯化-质谱法鉴定新的宿主底物。我们将通过各种实验在人类细胞中验证已识别的病毒复制底物。 意义：病原体利用各种方法来克服感染过程中的免疫反应。拟议的研究将通过确定细菌或病毒操纵的底物和途径，加强我们对致病去泛素酶在感染过程中作用的了解。

Ubiquitin signalling is critical in many cellular processes. The covalent attachment of ubiquitin to substrate proteins affects their biological functions. The best characterized function is 26S mediated proteasomal degradation of the substrate protein. Ubiquitin is attached to substrate proteins through an enzymatic cascade consisting of E1, E2 and E3 enzymes. Ubiquitin can also be cleaved from substrate proteins by deubiquitinases (DUBs). Deubiquitination activity must be highly regulated otherwise proteins would be targeted by these enzymes non-specifically resulting in deregulated cellular processes. Pathogenic organisms have the ability to manipulate ubiquitin signalling in host organisms to their advantage. This is a critical step in invasion by the pathogen. A variety of pathogenic organisms including bacteria and viruses possess proteins involved in ubiquitin signalling including bacterial/viral E3 ligases and DUBs that are used to dampen the immune response to enable infection. The long-term goals of my lab are to investigate the function of bacterial, viral and human deubiquitinases to provide in-depth understanding of their roles in cellular processes. The objective of this research program is to characterize a bacterial and several viral DUBs by elucidating their function, substrates and mechanism of action in host organisms. We are now expanding our research theme to include DUBs from various pathogenic organisms. The project is divided into several short term objectives: [1] Structural and Functional Characterization of the E. coli deubiquitinase ElaD. Very little is known about E. coli ElaD. We will identify host substrates and characterize the structural basis of interaction between ElaD and its host substrates. [2] Characterization of viral deubiquitinases BPLF1, ORF64, UL36 and UL48. BPLF1, ORF64, UL36 and UL48 are deubiquitinases from different herpesviruses. There is very little known about the function of these proteins. For instance, few host substrate proteins have been identified for three of the four viral DUBs. BPLF1 interacts with and deubiquitinates PCNA, TRAF6 and NEMO, UL36 deubiquitinates TRAF3 and ORF64 deubiquitinates RIG-1. [3] Identification of BPLF1, ORF64, UL36 and UL48 host substrate(s) and interacting proteins. There are very few host substrates known for viral DUBS. In this part of the proposal we will identify novel host substrates using affinity purification-mass spectrometry. We will follow up by validating the identified viral DUB substrates in human cells with a variety of experiments. Significance: Pathogenic organisms utilize a variety of methods to overcome the immune response during infection. The proposed studies will enhance our knowledge of the role of pathogenic deubiquitinases during infection by identifying substrates and pathways that are manipulated by the bacteria or viruses.