GLYCOPROTEIN HORMONE FOLDING

糖蛋白激素折叠

• 批准号: 6042777
• 负责人: WILLIAM R MOYLE
• 金额: $ 18.4万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-15 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6042777
• 关键词: CHO cells; chemical models; chimeric proteins; chorionic gonadotropin; crosslink; cystine; fluorescence resonance energy transfer; follicle stimulating hormone; gene mutation; glycoproteins; intermolecular interaction; oligosaccharides; peptide hormone biosynthesis; protein folding; protein sequence; transfection

Reproduction and thyroid function are controlled by the glycoprotein hormones, heterodimeric proteins composed of an alpha-subunit common to all and a hormone-specific beta-subunit. These subunits are structurally similar and the heterodimer is stabilized by a portion of the beta-subunit that is wrapped around the alpha-subunit and latched by a disulfide to the beta-subunit core. While this arrangement has obvious implications for the stabilities of these proteins, it raises questions as to how they are synthesized. Some studies in vivo suggest the subunits might combine before the seatbelt is latched. However, it is clear that subunit combination in vitro occurs efficiently even when the seatbelt remains latched. Glycoprotein hormone synthesis remains poorly understood in spite of its potential importance as a source of hCG metabolites observed during ectopic pregnancy, Down's syndrome, and even some cancers. Here we describe studies designed to learn how hCG is folded and assembled in vivo and to determine how the subunits combine in vitro. During the latter studies we will test a novel model of subunit combination that explains how one part of the alpha-subunit can pass through the small opening created by attachment of the beta-subunit seatbelt to the subunit core. The following specific aims are planned. In Aim 1 we will identify parts of the beta-subunit that influence its rate of folding in transfected CHO cells. These studies rely on the technique of homolog scanning and will employ analogs created by combining parts of the alpha- and beta-subunits and by combining parts of the hCG and hFSH beta-subunits. In Aim 2 we will determine if subunit combination occurs before or after the seatbelt is latched in cells and learn if this process is influenced by the alpha-subunit. These studies will employ an antibody that can separate heterodimers in which the seatbelt is latched from those in which it yet to form. In Aim 3 we will determine the mechanism of subunit combination in vitro, a process that occurs while the seatbelt latch remains closed. These studies will involve measurements of the distance between the seatbelt and other parts of the free beta-subunit. They will also involve careful measurement of the rates of association and dissociation of subunit analogs, some of which lack seatbelts or contain seatbelts that are unable to be latched.

生殖和甲状腺功能由糖蛋白激素控制，糖蛋白激素是由所有人共有的 α 亚基和激素特异性 β 亚基组成的异二聚体蛋白质。这些亚基结构相似，异二聚体通过包裹 α 亚基的一部分 β 亚基稳定，并通过二硫键锁定到 β 亚基核心。 虽然这种排列对这些蛋白质的稳定性有明显的影响，但它提出了它们是如何合成的问题。 一些体内研究表明，这些亚基可能会在安全带锁紧之前结合在一起。 然而，很明显，即使安全带保持扣紧状态，体外亚基组合也会有效发生。尽管糖蛋白激素合成作为异位妊娠、唐氏综合症甚至某些癌症期间观察到的 hCG 代谢物的来源具有潜在的重要性，但人们对它的了解仍然知之甚少。 在这里，我们描述了旨在了解 hCG 如何在体内折叠和组装以及确定亚基如何在体外结合的研究。 在后面的研究中，我们将测试一种新的亚基组合模型，该模型解释了α亚基的一部分如何通过将β亚基安全带连接到亚基核心而形成的小开口。 规划了以下具体目标。 在目标 1 中，我们将鉴定影响转染 CHO 细胞中折叠速率的 β 亚基部分。 这些研究依赖于同源扫描技术，并将采用通过组合部分 α 和 β 亚基以及组合部分 hCG 和 hFSH β 亚基创建的类似物。 在目标 2 中，我们将确定亚基组合是否发生在安全带锁入细胞之前或之后，并了解该过程是否受到 α 亚基的影响。 这些研究将使用一种抗体，可以将安全带锁住的异二聚体与尚未形成的异二聚体分开。 在目标 3 中，我们将确定体外亚基组合的机制，这是在安全带锁保持关闭时发生的过程。 这些研究将涉及测量安全带与游离β亚基其他部分之间的距离。 它们还将涉及仔细测量亚基类似物的结合和解离速率，其中一些亚基类似物没有安全带或包含无法锁紧的安全带。