Studies of the effects of phosphorylation and protein interactions on ATP-binding casette transporter activity

磷酸化和蛋白质相互作用对 ATP 结合盒转运蛋白活性影响的研究

• 批准号: RGPIN-2015-05372
• 负责人: Kanelis, Voula
• 金额: $ 2.19万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=613510
• 关键词: Studies; effects; phosphorylation; protein; interactions

The protein Ycf1p is a member of a large superfamily of membrane proteins found in all organisms. By utilizing the energy of ATP, known as the energy currency of the cell, these proteins actively transport solutes into and out of the cell, regulate the function of other proteins, or open pores or channels to allow charged species to pass through. These ATP-using membrane proteins have a conserved molecular architecture that contains two regions embedded in the cellular envelope (known as the membrane) and two regions located inside the cell (known as the cytoplasm). Proper regulation of this process is essential, as many genetic diseases, including cystic fibrosis and adrenoleukodystrophy, result from dysfunction of different proteins in this ubiquitous superfamily. Ycf1p transports toxins from one part of the cell (cytoplasm) to a membrane-bound compartment that is also found inside the cell. In this way, Ycf1p detoxifies the cell by sequestering poisonous molecules. Ycf1p is a prototype of a specific subclass of the ATP-using protein superfamily and shares similarily with the multidrug-resistance proteins that cause tumors to be resistant to chemotherapy drugs. Ycf1p and Ycf1p-like proteins posses a unique structural region in addition to the minimum architecture shared by the entire superfamily. Ycf1p action is regulated in a complex fashion. Biochemical modification of Ycf1p in the cell, which results in the addition of a charged group to specific sites, can activate or deactivate protein depending on the site modified. Ycf1p also binds a number of other proteins, which regulate Ycf1p transport function or ascribe other functions to Ycf1p. This proposal describes biochemical, biophysical, and cell biology-based experiments designed to gain insights into how Ycf1p activity is controlled, which will be applicable to other members of superfamily. To date, few studies have been done to address understand the mechanism of regulation in this important class of proteins. We will investigate interactions of individual Ycf1p protein segments that are involved in relaying information from the utilization of ATP to transport of toxins, and that bind accessory proteins. We will further address how biochemical modification changes the structure and interactions of Ycf1p. Finally, we will conduct experiments to obtain a 3D picture of Ycf1p, which will yield much sought-after information on the organization of the unique region in Ycf1p with the minimum architecture shared by the entire superfamily. The proposed research will elucidate some of the complex mechanisms by which Ycf1p activity is controlled. Because Ycf1p is part of a large superfamily of proteins that share common structural and mechanistic features, our Ycf1p studies will yield information about this class of proteins, which are conserved throughout biology.

蛋白质YCF1P是在所有生物体中发现的大型膜蛋白的大型超家族的成员。通过利用ATP的能量（称为细胞的能源货币），这些蛋白质会主动将溶质传输到细胞中，调节其他蛋白质的功能，或者开放的孔或通道允许带电的物种通过。这些使用ATP的膜蛋白具有保守的分子结构，其中包含两个嵌入细胞包膜中的区域（称为膜）和位于细胞内部的两个区域（称为细胞质）。适当调节这一过程是必不可少的，因为许多遗传疾病，包括囊性纤维化和肾上腺素肌营养不良，这是由于这种无处不在的超家族中不同蛋白质功能障碍引起的。 YCF1P将毒素从细胞的一部分（细胞质）传输到也发现在细胞内部的膜结合室。这样，YCF1P通过隔离有毒分子来解毒。 YCF1P是使用ATP蛋白质超家族的特定亚类的原型，与导致肿瘤对化学疗法药物具有抗性的多药耐药蛋白相似。除了整个超家族共享的最小结构外，YCF1P和YCF1P样蛋白还具有独特的结构区域。 YCF1P动作以复杂的方式进行调节。细胞中YCF1P的生化修饰，从而导致在特定位点添加带电组的YCF1P可以根据所修饰的位点而激活或停用蛋白质。 YCF1P还结合了许多其他蛋白质，这些蛋白质调节YCF1P传输函数或将其他功能赋予YCF1P。 该提案描述了旨在洞悉YCF1P活性如何控制的生化，生物物理和基于细胞生物学的实验，这将适用于其他超级家族的其他成员。迄今为止，很少进行研究来解决这一重要类别蛋白质中调节的机理。我们将研究各个YCF1P蛋白段的相互作用，这些蛋白段与从ATP利用到毒素的运输以及结合辅助蛋白的信息有关。我们将进一步解决生化修饰如何改变YCF1P的结构和相互作用。最后，我们将进行实验，以获取YCF1P的3D图片，该图片将在YCF1P中提供有关YCF1P独特区域的组织的众多信息，并在整个超级家庭共享的最低限度架构中提供。拟议的研究将阐明控制YCF1P活性的某些复杂机制。由于YCF1P是具有共同结构和机械特征的大型蛋白质超家族的一部分，因此我们的YCF1P研究将产生有关此类蛋白质的信息，这些蛋白质在整个生物学中都保留。