Studies of how disordered regions, post-translational processing, and protein interactions affect the structure, dynamics, and activity of ABC transporters

研究无序区域、翻译后加工和蛋白质相互作用如何影响 ABC 转运蛋白的结构、动态和活性

• 批准号: RGPIN-2020-05835
• 负责人: Kanelis, Voula
• 金额: $ 3.64万
• 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: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740860
• 关键词: Studies; how; disordered; regions; post

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 these ATP-using proteins 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 membrane protein superfamily and shares similarity 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 the protein depending on the site modified. Breaking of a specific chemical bond in Ycf1p affects the types of toxins transported. 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 the mechanism of regulation in this important class of proteins. We will conduct studies aimed at determining a high-resolution structures of Ycf1p that will yield information about the organization of the unique region in this protein in relation to the minimum architecture shared by the entire superfamily. We will study how biochemical modifications change the structure and interactions of Ycf1p. Finally, we determine which parts of Ycf1p bind accessory proteins, and how biochemical modifications alter those interactions. 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）。对这些使用 ATP 的蛋白质进行适当的调节至关重要，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 将毒素从细胞的一部分（细胞质）转运到细胞内也存在的膜结合区室。通过这种方式，Ycf1p 通过隔离有毒分子来为细胞解毒。 Ycf1p 是使用 ATP 的膜蛋白超家族的特定亚类的原型，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性。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 中特定化学键的断裂会影响运输的毒素类型。 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 Ycf1p 活性的控制方式，这将适用于超家族的其他成员。迄今为止，很少有研究来解决这一类重要蛋白质的调节机制。我们将进行旨在确定 Ycf1p 高分辨率结构的研究，该结构将产生有关该蛋白质中独特区域的组织与整个超家族共享的最小结构的信息。我们将研究生化修饰如何改变 Ycf1p 的结构和相互作用。最后，我们确定 Ycf1p 的哪些部分结合辅助蛋白，以及生化修饰如何改变这些相互作用。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。