Structural and Functional Analysis of Bacterial and Viral Deubiquitinases

泛素信号传导在许多细胞过程中至关重要。泛素与底物蛋白的共价连接影响其生物学功能。最好的功能是26 S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质连接。泛素还可以通过去泛素化酶（DUB）从底物蛋白质切割。去泛素化活性必须受到高度调节，否则蛋白质将被这些酶非特异性地靶向，导致细胞过程失调。病原生物体有能力操纵宿主生物体中的泛素信号传导，使其对自己有利。这是病原体入侵的关键步骤。 包括细菌和病毒在内的各种病原体都拥有参与遍在蛋白信号传导的蛋白质，包括细菌/病毒E3连接酶和DUB，用于抑制免疫反应以实现感染。 我实验室的长期目标是研究细菌，病毒和人类去泛素化酶的功能，以深入了解它们在细胞过程中的作用。本研究计划的目的是通过阐明它们在宿主生物中的功能、底物和作用机制来表征细菌和几种病毒DUB。我们现在正在扩大我们的研究主题，包括来自各种病原体的DUB。该项目分为几个短期目标： [1]E.大肠杆菌去泛素酶ElaD。对E知之甚少。coli ElaD.我们将确定主机基板和表征ElaD和它的主机基板之间的相互作用的结构基础。 [2]病毒去泛素化酶BPLF 1、ORF 64、UL 36和UL 48的表征。BPLF 1、ORF 64、UL 36和UL 48是来自不同疱疹病毒的去泛素化酶。人们对这些蛋白质的功能知之甚少。例如，对于四种病毒DUB中的三种，已经鉴定出很少的宿主底物蛋白。BPLF 1与PCNA、TRAF 6和NEMO相互作用并使其去泛素化，UL 36使TRAF 3去泛素化，ORF 64使RIG-1去泛素化。 [3]BPLF 1、ORF 64、UL 36和UL 48宿主底物和相互作用蛋白的鉴定。已知用于病毒DUBS的宿主底物非常少。在这部分的建议，我们将确定新的主机基板使用亲和纯化-质谱。我们将通过各种实验在人类细胞中验证所鉴定的病毒DUB底物。 意义：病原体在感染过程中利用多种方法克服免疫反应。拟议的研究将通过鉴定细菌或病毒操纵的底物和途径，增强我们对致病性去泛素化酶在感染过程中的作用的认识。

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.