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

泛素化是细胞内一种主要的翻译后修饰，它包括泛素与其他蛋白质的共价连接。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。随之而来的是，通过泛素结合模式和泛素结合域(UBD)的变化，存在着大量不同种类和范围的蛋白质-蛋白质相互作用，这些泛素结合结构域控制着泛素化蛋白质的命运。由于泛素化与感染等多种生理条件密切相关，复杂的调控和识别网络受到了密切关注。除泛素外，大约还有80种人类蛋白质含有完整的泛素样域(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。除了调节蛋白质之间的相互作用外，完整的UBL的功能还知之甚少。我们的假设是，UBL在其蛋白质序列中显示出特定的变化，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UBD与蛋白酶体穿梭蛋白的UBL特异结合，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的蛋白还与其他UBD相互作用。利用质谱学，我们将鉴定这些UBL-相互作用，然后我们将通过生物分子荧光互补和体外结合实验进一步验证。我们的长期目标是定义人类完整的UBL和UBD之间的蛋白质-蛋白质相互作用网络的格局。利用质谱学，我们将扩大我们的初步研究，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归类到不同的家族。

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.