Ubiquitin Specific Protease 11 (USP11): structure and enzymology

组成有机体的单位，细胞，依赖于在不同时间需要的大量蛋白质的存在。为了快速响应环境变化或处理受损的蛋白质，细胞拥有一个有效的蛋白质破坏系统，该系统受到严格调节，因为任何错误都可能对细胞产生严重影响。蛋白质的破坏由一系列事件组成。首先，一种被称为泛素的小蛋白质附着在蛋白质分子上，作为信号引导这些分子进入特定的途径。一种蛋白质上由几个泛素分子组成的特殊链的形成，导致它们被一个大分子机器——蛋白酶体识别，蛋白酶体最终将蛋白质消化成小块。仅将一个泛素片段或其他类型的泛素链附着到蛋白质上也可能导致与蛋白质破坏无关的其他结果。因为正确的时间至关重要，所以有一些机制可以去除或改变这些信号。被称为去泛素化酶的检查点分子可以去除泛素分子，从而改变目标蛋白质的命运。这些去泛素化酶中最丰富的一类是泛素特异性蛋白酶（USPs）。它们通过切断泛素-蛋白键来调节泛素依赖的代谢途径。每个USP特异性地识别有限数量的泛素化蛋白质，它们可以从破坏中挽救这些蛋白质或将其引入细胞内的不同途径。这些分子的结构是复杂的，因为它们需要能够为蛋白质提供结合位点，以便具有特异性，以及附加的泛素链，并在其作用中具有高度选择性。从错误的蛋白质中去除泛素信号可能会对细胞产生灾难性的影响。本研究旨在深入了解一种泛素特异性蛋白酶USP11是如何实现这种特异性的，它是否能够区分不同的泛素链，以及它的分子组成如何影响其功能。为此，我们将确定USP11的离散部分的结构，并确定蛋白质和泛素链的结合表面。由于USP11也与其他蛋白质相互作用，我们将寻找其他结合位点。为了更好地了解这些重要的调节蛋白，第二次尝试将包括观察溶液中整个分子的形状和柔韧性。最终，我们将确定USP11的哪些部分参与识别病毒蛋白HPV-16E7，一旦感染发生，利用USP11的功能来延长其自身的寿命。总之，这将为这些蛋白酶如何工作提供独特的新见解，这将有助于我们更好地理解这个检查点系统在调节正常蛋白质破坏途径、信号事件和病毒感染方面的作用。

The units that make up an organism, the cells, rely on the presence of a large number of proteins that are required at different times. In order to quickly respond to environmental changes or to dispose of damaged proteins, cells harbour an effective system for protein destruction that is tightly regulated as any mistakes can have serious effects for the cell. Protein destruction consists of a series of events. At first a small protein termed ubiquitin is attached to a protein molecule that serves as a signal to direct these molecules into a certain pathway. The formation of a particular type of chain of several ubiquitin molecules on a protein results in them being recognised by a large molecular machine, the proteasome that ultimately digests the protein into small pieces. The attachment of only one ubiquitin moiety or other types of ubiquitin chain to a protein can also lead to other outcomes unrelated to protein destruction. Because the correct timing is essential, there are mechanisms to remove or alter these signals. Check point molecules called deubiquitinating enzymes can remove ubiquitin molecules and consequently alter the fate of a target protein. The most abundant class of these deubiquitinating enzymes in humans are the ubiquitin specific proteases (USPs). They regulate ubiquitin-dependent metabolic pathways by cleaving ubiquitin-protein bonds. Each USP specifically recognizes a limited number of ubiquitinated proteins that they can salvage from destruction or channel into different pathways within cells. The structure of these molecules is complicated in that they need to be able to provide binding sites for the protein, in order to be specific, as well as the attached ubiquitin chain and be highly selective in their action. Removing the ubiquitin signal from the wrong protein could have disastrous effects for the cell. This research aims at gaining insights into how one ubiquitin specific protease, USP11, achieves this specificity, whether it is able to distinguish between different ubiquitin chains and how its molecular make-up influences its function. To this end we will determine structures of discrete parts of USP11 and identify binding surfaces for the protein and the ubiquitin chain. As USP11 also interacts with other proteins we will look for additional binding sites. A second attempt to gain a better understanding of these important regulatory proteins will consist of looking at the shape and flexibility of the whole molecule in solution. Ultimately we will establish which parts of USP11 are involved in the recognition of the viral protein HPV-16E7 that once infection has occurred exploits the function of USP11 in order to extend its own life-span. Together, this will provide unique novel insights into how these proteases work, which will help us to better understand this check-point system in regulating normal protein destruction pathways, signalling events and in viral infection.