Characterization of the ubiquititn-like domains of the proteasome shuttle proteins & defining the landscape of the ubiquitin-like proteins interactome

泛素化是细胞中一个主要的翻译后修饰，它由泛素与其他蛋白质的共价附着组成。令人惊讶的是，人类基因组中大约有600种不同的E3泛素连接酶。同时，通过泛素结合模式和控制泛素化蛋白“命运”的泛素结合结构域（UBD）的变化，蛋白质与蛋白质之间的相互作用种类繁多。由于泛素化与感染等多种生理条件密切相关，因此复杂的调控和识别网络受到密切关注。/除泛素外，大约有80种人类蛋白质含有完整泛素样结构域（UBL），它们与泛素具有相似的序列和结构，但不与其他蛋白质结合。除了介导蛋白质之间的相互作用外，人们对整合ubl的功能知之甚少。我们的假设是，ubl在其蛋白质序列中显示出特异性变化，从而改变了它们与含有UBD的特定结合伙伴的亲和力。我们的项目使用蛋白质组学解决与蛋白质结合特异性相关的基本问题。我们的短期目标是发现哪些UBDs与蛋白酶体穿梭蛋白的UBL特异性结合，并确定这些UBL序列的微小变化如何影响结合。至少有9种人类穿梭蛋白与蛋白酶体亚基S5a相互作用，介导蛋白酶体底物识别。此外，对于S5a，这些含有ubl的蛋白也与其他ubd相互作用。我们将使用质谱法鉴定这些ubl相互作用，然后我们将使用生物分子荧光互补和体外结合实验进一步验证这些相互作用。/我们的长期目标是定义人类整体ubl和ubd之间蛋白质-蛋白质相互作用网络的景观。使用质谱法，我们将扩展我们的初步研究，以揭示哪些蛋白质与其他显示不同特征的完整ubl相互作用，并根据它们的结合伙伴将它们分类为不同的家族。

Ubiquitylation is a major post-translation modification in the cell, which consists of the covalent attachment of ubiquitin to other proteins. Astoundingly, there are around 600 different E3 ubiquitin-ligase enzymes in the human genome. Concomitantly, there is an enormous variety and range of protein-protein interactions, via variations in the ubiquitin conjugation pattern and the ubiquitin binding domains (UBD) that control the "fate" of the ubiquitylated proteins. The intricate regulatory and recognition network is under intense scrutiny due to the fact that ubiquitylation is intimately involved in a multitude of physiological conditions like infection. / In addition to ubiquitin, there are approximately 80 human proteins that contain integral ubiquitin-like domains (UBL), which share similar sequence and structure with ubiquitin, but are not conjugated to other proteins. Beyond mediating protein-protein interactions, the function of the integral UBLs is very poorly understood. Our hypothesis is that UBLs display specific changes in their protein sequences that alter their affinities to specific binding partners that contain UBD. Our project addresses fundamental questions related to protein binding specificity using proteomics. / Our short-term goal is to uncover which UBDs bind specifically to the UBLs of the proteasome shuttle proteins and determine how small changes in these UBL sequences effect binding. There are at least nine human shuttle proteins that interact with the proteasome subunit S5a to mediate proteasome substrate recognition. In addition, to S5a, these UBL-containing proteins also interact with other UBDs. Using mass spectrometry we will identify these UBL-interactors that we will then further validate using biomolecular fluorescence complementation and in vitro binding experiments. / Our long-term goal is to define the landscape of the protein-protein interaction network between human integral UBLs & UBDs. Using mass spectrometry, we will expend our initial study to uncover which proteins interact with the other integral UBLs that display different characteristics and classify them in different families based on their binding partners.