SUMO-SIM interactions and their role in substrate recognition and autoregulation of SUMO-dependent ubiquitin ligases

我们和其他实验室对酵母和哺乳动物蛋白质的研究已经确定了相扑相互作用基序(SIMS)，它介导蛋白质与小泛素相关修饰物相扑的非共价相互作用。这种SIMS的存在被发现是一类新的泛素连接酶(ULS)的特征，这种ULS可以特异性地识别SUM化蛋白。我们和其他人将人类RNF4蛋白定性为第一个哺乳动物ULS，并确定了早幼粒细胞白血病蛋白(PML)为其底物。量热分析表明，RNF4通过其多个SIMs优先与相扑链结合。生物信息学分析鉴定了其他几种哺乳动物蛋白质，它们将泛素连接酶结构域与SIMS结合。其中一些连接酶在酵母中表达时会影响相扑结合水平，或者在人类细胞中定位于PML小体，这表明相扑-SIM相互作用与它们的功能有关。对于酵母和人类ULS蛋白，我们都观察到它们自身被苏糖化、泛素化和降解，这表明它们受到自动调节反馈控制的影响。我们将使用突变生化分析的组合来表征单个或组合的SIMS在识别不同类型的相扑修饰底物识别和这些泛素连接酶的自动相扑修饰中的作用。我们将进一步研究不同的应激类型等生理条件如何影响酵母和人类细胞中ULS蛋白的蛋白降解控制。我们已经建立了一个数据库筛选协议，允许识别高概率SIMs，并将该方法应用于预测酵母基因组中编码的相扑相互作用蛋白。我们将验证选定的候选对象，并通过将其应用于人类和其他模式生物的蛋白质组分析来扩展此方法。此外，我们将修改识别非共识SIMs和功能相关基序的方法。

Studies on yeast and mammalian proteins in our as well as other laboratories have identified SUMO interaction motifs (SIMs) that mediate non-covalent interaction of proteins with the small ubiquitin-related modifier SUMO. Presence of such SIMs was found to be a characteristic feature of a novel class of ubiquitin ligases (ULS) that specifically recognize sumoylated proteins. We and others characterized the human RNF4 protein as the first mammalian ULS and identified the Promyelocytic Leukemia Protein (PML) as its substrate. Calorimetric assays revealed that RNF4 via its multiple SIMs preferentially binds to SUMO chains. Bioinformatical analyses identified several other mammalian proteins that combine ubiquitin ligase domains with SIMs. Several of these ligases affect SUMO conjugate levels upon expression in yeast or localized to PML bodies in human cells suggesting that SUMO-SIM interaction is relevant to their function. We observed both for yeast and human ULS proteins that they are themselves sumoylated, ubiquitylated and degraded suggesting that they subject to an auto-regulatory feedback control. We will use a combination of mutational biochemical analyses to characterize the role of individual or combinations of SIMs in the recognition of distinct types of SUMO modification in substrate recognition and auto-sumoylation of these ubiquitin ligases. We will further investigate how physiological conditions such as various stress types affects proteolytic control of ULS proteins in yeast and human cells. We have established a database screening protocol that allows to identify high probability SIMs and applied this method to predict SUMO interacting proteins encoded in the yeast genome. We will validate selected candidates and extend this approach by applying it to the analysis of the proteomes of humans and other model organisms. Furthermore, we will modify the approach to identify non-consensus SIMs und functionally related motifs.