Molecular Interactions in Signaling and Vesicle Sorting

信号传导和囊泡分选中的分子相互作用

• 批准号: 6731791
• 负责人: JAMES D BALEJA
• 金额: $ 25.85万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6731791
• 关键词: calcium ion; conformation; electrospray ionization mass spectrometry; immunoprecipitation; intracellular transport; magnesium ion; metals; nuclear magnetic resonance spectroscopy; protein binding; protein protein interaction; protein signal sequence; protein structure function; stoichiometry; tissue /cell culture; vesicle /vacuole

DESCRIPTION (provided by applicant): Proteins containing EH domains and proteins containing Asn-Pro-Phe (NPF) sequences are ubiquitously involved in fundamental molecular sorting mechanisms. Reps1, an EH domain-containing protein, and FIP2, an NPF-containing protein, are downstream targets of Ras and Ral GTPases and the EGF receptor, suggesting that they are involved in both signal transduction and vesicle-mediated trafficking. Despite the importance of the EH domain:NPF interaction in building the protein networks responsible for vesicle-mediated transport and signal transduction, the factors that regulate their association are largely unknown. Knowing the basic principles that govern their regulation will lead to strategies to control these proteins and a better understanding of their biological roles. The EH domain of Reps1 binds directly to the NPF sequence of FIP2. Using Reps1:FIP2 as model system, the objective of this proposal is to evaluate the factors that modulate EH domain:NPF interaction. EH domains bind Ca2+ using the EF-hand fold. In Aim 1, EH domain affinity for Ca2+ and Mg2+ will be measured to test a new regulatory hypothesis operating in vesicle sorting. To examine the conformational changes that accompany metal ion binding, the structures of the apo and Mg2+-bound domains will be determined using NMR and compared to the Ca2+-bound structure. FIP2 and Reps1 both oligomerize through a coiled-coil domain. In Aim 2, we will determine the structure of the FIP2 coiled-coil domain and how oligomerization governs the strength of the interaction between these proteins. To understand the mechanism of binding, the structures from a series of unbound peptides as well as an EH domain-NPF complex will be determined in Aim 3. An inhibitory peptide, designed to be stable in cell-based assays, will also be optimized and tested as a tool to understand the roles of EH domains in vesicle-mediated internalization and secretion. Ultimately, the results of these experiments will provide insight into the vesicle sorting mechanisms important for diverse processes such as fertilization, neurotransmission, and cell growth. These investigations on the EH domain:NPF interaction are also expected to advance the application of NMR in understanding molecular recognition.

描述（由申请人提供）：含有EH结构域和包含ASN-PRO-PHE（NPF）序列的蛋白质的蛋白质与基本分子分类机制无关紧要。 REPS1是含EH结构域的蛋白质，FIP2（一种含NPF的蛋白）是RAS和RAL GTPase和EGF受体的下游靶标，表明它们参与信号转导和小囊泡介导的运输。尽管EH结构域的重要性：NPF相互作用在构建负责囊泡介导的运输和信号转导的蛋白质网络中，但调节其关联的因素在很大程度上是未知的。了解管理其法规的基本原则将导致控制这些蛋白质的策略，并更好地了解其生物学作用。 REPS1的EH结构域直接与FIP2的NPF序列结合。使用REPS1：FIP2作为模型系统，该建议的目的是评估调节EH域的因素：NPF相互作用。 EH域使用EF手折叠Ca2+。在AIM 1中，将测量对Ca2+和Mg2+的EH结构域的亲和力，以测试在囊泡排序中运行的新调节假设。为了检查金属离子结合伴随的构象变化，将使用NMR确定APO和MG2+结合域的结构，并将其与Ca2+结构结构进行比较。 FIP2和REPS1都通过盘绕螺旋域进行了寡聚。在AIM 2中，我们将确定FIP2盘绕螺旋结构域的结构，以及如何控制这些蛋白质之间相互作用的强度。为了了解结合的机制，将在AIM 3中确定一系列未结合肽以及EH结构域NPF复合物的结构。一种抑制性肽在基于细胞的测定中的抑制性肽也将被优化和测试，作为了解EH结构域在vesicle介导的内部化和分泌中的作用的工具。最终，这些实验的结果将提供对囊泡分选机制对于诸如施肥，神经传递和细胞生长等多种过程至关重要的囊泡分选机制。对EH结构域：NPF相互作用的这些研究也有望提高NMR在理解分子识别中的应用。