Biological functions of melanoma antigen (MAGE) proteins

蛋白质的丰度和亚细胞定位有助于细胞内发生的活动。蛋白质的稳定性和定位在一定程度上取决于翻译后修饰。提出的研究特别考虑了76残基泛素蛋白与底物蛋白可逆附着的作用，这动态地改变了泛素化蛋白的稳定性、活性或亚细胞定位。例如，细胞周期蛋白经历高度调控的泛素化和去泛素化，微调其稳定性、活性和定位。然而，执行可逆泛素化/去泛素化过程的蛋白质，以及修饰这些泛素连接酶和去泛素酶活性的其他蛋白质，人们知之甚少。我们之前的研究已经证明，黑色素瘤抗原基因（MAGE）蛋白调节对多种生理过程重要的蛋白质的泛素化和去泛素化。特别是，MAGE蛋白家族的L2成员（MAGEL2）调节其他蛋白的细胞内定位和稳定性。我们的团队发现MAGEL2与E3泛素连接酶（如RBX1-SCF）和去泛素酶（如USP7）形成功能复合物，从而通过对主时钟组件的活性和稳定性的影响来调节昼夜节律过程。我们发现MAGEL2调节BBS蛋白的稳定性，BBS蛋白对中心体和纤毛的功能很重要，纤毛是在细胞外环境中感知和响应信号的亚细胞细胞器。我们发现MAGEL2和另一种MAGE蛋白，即necdin，通过依赖于ESCRT（运输所需的内体分选复合物）机制的过程调节瘦素受体的细胞内穿梭。我们的研究目标是探索蛋白质泛素化对细胞内穿梭和蛋白质稳定性的调节机制。MAGE蛋白在这一过程中的重要性从以下观察中可以明显看出：编码MAGE蛋白的基因突变导致人类疾病（Schaaf-Yang综合征的MAGEL2、产前Bartter综合征的MAGED2、染色体断裂综合征的MAGEG1），以及小鼠MAGE基因敲除的表型。我们将使用分子和细胞模型来确定MAGE蛋白的作用，它是泛素化（Ub）复合物的组成部分。具体来说，我们将使用蛋白质-蛋白质相互作用技术来鉴定mage -泛素连接酶-去泛素酶复合物的成分；确定MAGE蛋白如何在培养细胞系中微调细胞过程；3)研究组织中泛素化如何调节细胞过程中重要的基因和蛋白质。最终，本研究将探索通过泛素化和去泛素化修饰蛋白质如何精细调节细胞中蛋白质的活性。

The abundance and subcellular localization of proteins contributes to the ctivities that take place inside cells. Both protein stability and localization are determined in part by post-translational modification. The proposed research specifically considers the role of the reversible attachment of a 76 residue ubiquitin protein to substrate proteins, which dynamically changes the stability, activity, or subcellular localization of the ubiquitinated protein. For example, cell cycle proteins undergo highly regulated ubiquitination and deubiquitination, fine tuning their stability, activity and localization. However, the proteins that perform the reversible ubiquitination / deubiquitination processes, and other proteins that modify the activity of these ubiquitin ligases and deubiquitinases are poorly understood. Our previous research has demonstrated that Melanoma Antigen Gene (MAGE) proteins regulate the ubiquitination and deubiquitination of proteins important for a variety of physiological processes. In particular, the L2 member of the MAGE family of proteins (MAGEL2) regulates the intracellular localization and stability of other proteins. Our team found that MAGEL2 forms a functional complex with E3 ubiquitin ligases (e.g. RBX1-SCF) and deubiquitinases (e.g. USP7), thereby regulating circadian rhythm processes through effects on the activity and stability of master clock components. We found that MAGEL2 regulates the stability of BBS proteins important for the function of centrosomes and of cilia, subcellular organelles that sense and respond to signals in the extracellular environment. We found that MAGEL2 and another MAGE protein, namely necdin, regulate the intracellular shuttling of the leptin receptor through processes dependent on ESCRT (endosomal sorting complexes required for transport) machinery. Our research program objective is to explore the mechanisms for regulation of intracellular shuttling and stability of proteins by protein ubiquitination. The importance of MAGE proteins in this process is evident from the observations that mutation in the genes encoding MAGE proteins cause disease in humans (MAGEL2 in Schaaf-Yang syndrome, MAGED2 in antenatal Bartter syndrome, MAGEG1 in a chromosome breakage syndrome), with phenotypes in mouse MAGE gene knockouts as well. We will use molecular and cellular models to determine the role of MAGE proteins, which are components of ubiquitination (Ub) complexes. Specifically, we will use protein-protein interaction techniques to identify components of the MAGE-ubiquitin ligase-deubiquitinase complexes; determine how MAGE proteins fine tune cellular processes using cell lines in culture and 3) examine how genes and proteins important in cellular processes are regulated by ubiquitination in tissues. Ultimately, this research will explore how modification of proteins by ubiquitination and deubiquitination fine tunes the activity of proteins in the cell.