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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=683648
• 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.**

Ycf1p 蛋白是所有生物体中发现的膜蛋白大超家族的成员。通过利用被称为细胞能量货币的 ATP 能量，这些蛋白质主动将溶质转运进出细胞，调节其他蛋白质的功能，或打开孔或通道以允许带电物质通过。这些使用 ATP 的膜蛋白具有保守的分子结构，其中包含嵌入细胞包膜的两个区域（称为膜）和位于细胞内部的两个区域（称为细胞质）。正确调节这一过程至关重要，因为许多遗传性疾病，包括囊性纤维化和肾上腺脑白质营养不良，都是由这个普遍存在的超家族中不同蛋白质的功能障碍引起的。******Ycf1p 将毒素从细胞的一个部分（细胞质）转运到另一部分细胞内也存在膜结合区室。通过这种方式，Ycf1p 通过隔离有毒分子来为细胞解毒。 Ycf1p 是 ATP 使用蛋白超家族特定亚类的原型，与导致肿瘤对化疗药物产生耐药性的多药耐药蛋白具有相似之处。除了整个超家族共有的最小结构之外，Ycf1p 和 Ycf1p 样蛋白还具有独特的结构区域。 Ycf1p 的作用以复杂的方式受到调节。细胞中 Ycf1p 的生化修饰会导致在特定位点添加带电基团，可以根据修饰的位点激活或失活蛋白质。 Ycf1p 还结合许多其他蛋白质，这些蛋白质调节 Ycf1p 转运功能或将其他功能归因于 Ycf1p。******该提案描述了基于生物化学、生物物理和细胞生物学的实验，旨在深入了解如何控制 Ycf1p 活性，这将适用于超家族的其他成员。迄今为止，很少有研究来了解这一类重要蛋白质的调节机制。我们将研究单个 Ycf1p 蛋白片段的相互作用，这些片段参与从 ATP 的利用到毒素运输的信息传递，并结合辅助蛋白。我们将进一步讨论生化修饰如何改变 Ycf1p 的结构和相互作用。最后，我们将进行实验以获得 Ycf1p 的 3D 图片，这将产生有关 Ycf1p 中独特区域的组织的许多抢手信息，以及整个超家族共享的最小架构。拟议的研究将阐明控制 Ycf1p 活性的一些复杂机制。由于 Ycf1p 是一个具有共同结构和机制特征的蛋白质大超家族的一部分，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是保守的。 **