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

点击翻译按钮获取中文摘要

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.