Isomer-Specific Interactions of X-Pro Motifs: Investigating Fundamental Mechanisms of Signaling by Pro-Rich Sequences

X-Pro 基序的异构体特异性相互作用：研究 Pro-Rich 序列信号传导的基本机制

• 批准号: 1157806
• 负责人: Linda Nicholson
• 金额: $ 41.51万
• 依托单位: Cornell University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1157806&HistoricalAwards=false
• 关键词: Isomer; Specific; Interactions; Pro; Motifs

This project addresses a novel hypothesis regarding how a particular "on/off" switch inside a cell is regulated. The switch of interest involves a biological signaling interaction between two specific proteins that connect innate immunity signaling (a cell's first line of defense) to cell migration (the directed movement of a cell). This work builds on the experimental observation that IRAK1 (a key signaling protein in innate immunity) adopts two very different and slowly interconverting structures (cis and trans isomers of a specific Trp-Pro peptide bond), exactly in the region where it binds to VASP (a protein that regulates cell migration). VASP has two distinct domains (EVH1 and EVH2). Only the VASP-EVH1 domain directly binds to IRAK1, and this interaction is selective for only the trans isomer of IRAK1. Since IRAK1 has nearly equal populations of cis and trans structures before VASP enters the picture, the initial IRAK1:VASP binding utilizes only about half of the total IRAK1 that is present. This fast initial binding step is followed by a slow binding phase, the rate of which depends on the rate of flipping back and forth of the cis and trans isomers. This slow rate can be greatly accelerated by an enzyme (cyclophilin A, or CypA) that catalyzes the flipping process. Hence, CypA effectively catalyzes the IRAK1-VASP binding interaction by facilitating rapid conversion of cis to trans to replace the trans that has bound to VASP. The EVH2 domain of VASP self-associates as a cluster of four (a tetramer); consequently, full-length VASP is a tetramer that displays four EVH1 domains in close proximity. Interestingly, recent discoveries suggest that when innate immunity signaling is triggered, activated IRAK1 is also a tetramer. When placed into the context of a tetramer:tetramer interaction (the "on" state of the switch), there is far less IRAK1 tetramer in which all 4 IRAK1 components are in the VASP-binding competent trans state. In this case, enzyme-catalyzed acceleration of the cis/trans flipping (isomerization) rate is even more critical for achieving a binary "on/off" switch, in which the time constants for turning the switch on or off are short enough to be effective in the fast paced dynamics of the inner workings of a cell. This project will provide 1) a kinetic and thermodynamic description of the monomer:monomer IRAK1:VASP interaction, and 2) a quantitative analysis of the binding enhancement associated with the specific clustering of IRAK1 and the role of cis-trans isomerization in achieving this enhancement on a biologically relevant timescale. Together, these results will reveal how a key biological signaling interaction works to rapidly connect innate immunity signaling to cell migration. This work synergistically couples to cell biologists who study the participation of IRAK1 and VASP in biological processes such as innate immunity and cell migration, and to systems biologists who mathematically model signaling pathways and who require thermodynamic and kinetic data for critical steps in such pathways.The research in this project will be integrated in several ways into the teaching of a large (70-80 students) graduate level 3-credit course on Protein Structure, Function and Dynamics (BioMG 6310). As part of this class, a team-based project is assigned in which students write a proposal to "save the world" using proteins (with focus on energy and the environment), and projects are presented at the end of the semester at a public poster session. This activity has fostered connections with Cornell's Atkinson Center for a Sustainable Future (ACSF), including display of the posters at ACSF to a diverse audience of visitors, and the appointment of the PI as an ACSF Faculty Fellow. This NSF-sponsored research will be incorporated into BioMG 6310 lectures to illustrate fundamental concepts. Outreach to underrepresented groups includes giving lab tours for The Learning Web (a local youth development agency that serves a diversity of rural and city children), and bringing excitement for science to the 7th and 9th Wards of New Orleans during annual rebuilding trips with GEEKS (Graduates Employing Empathy, Knowledge and Service), a Cornell graduate student organization that the PI advises. The PI serves as research mentor for undergraduate (independent study and summer research) and graduate students in her lab. Students are trained in an interdisciplinary set of techniques that include NMR, biophysical methods, mathematical modeling, biochemistry, and cell biology.

这个项目提出了一个新的假设，关于细胞内一个特定的“开/关”开关是如何调节的。 感兴趣的开关涉及两种特定蛋白质之间的生物信号相互作用，将先天免疫信号传导（细胞的第一道防线）与细胞迁移（细胞的定向运动）联系起来。 这项工作建立在实验观察的基础上，即IRAK 1（先天免疫中的关键信号蛋白）采用两种非常不同且缓慢相互转化的结构（特定Trp-Pro肽键的顺式和反式异构体），正好在它与VASP（一种调节细胞迁移的蛋白质）结合的区域。 VASP有两个不同的结构域（EVH1和EVH2）。只有VASP-EVH1结构域直接结合IRAK1，并且这种相互作用仅对IRAK1的反式异构体具有选择性。 由于在VASP进入之前IRAK1具有几乎相等的顺式和反式结构群体，因此初始IRAK1：VASP结合仅利用存在的总IRAK1的约一半。 这个快速的初始结合步骤之后是缓慢的结合阶段，其速率取决于顺式和反式异构体来回翻转的速率。这种缓慢的速度可以通过催化翻转过程的酶（亲环素A或CypA）大大加速。因此，CypA通过促进顺式到反式的快速转化以替换已经结合到VASP的反式，有效地催化IRAK1-VASP结合相互作用。 VASP的EVH2结构域自缔合为四个的簇（四聚体）;因此，全长VASP是四聚体，其显示紧密接近的四个EVH1结构域。 有趣的是，最近的发现表明，当先天免疫信号被触发时，激活的IRAK1也是一种四聚体。 当置于四聚体：四聚体相互作用（开关的"开启"状态）的背景下时，存在少得多的IRAK 1四聚体，其中所有4种IRAK 1组分都处于VASP结合活性反式状态。 在这种情况下，酶催化的顺式/反式翻转（异构化）速率的加速对于实现二元"开/关"开关甚至更关键，其中用于打开或关闭开关的时间常数足够短以在细胞内部工作的快节奏动态中有效。 该项目将提供1）单体：单体IRAK1：VASP相互作用的动力学和热力学描述，以及2）与IRAK1特异性聚集相关的结合增强的定量分析，以及顺反异构化在生物学相关时间尺度上实现这种增强的作用。总之，这些结果将揭示一个关键的生物信号相互作用如何快速将先天免疫信号与细胞迁移联系起来。这项工作与研究IRAK1和VASP参与先天免疫和细胞迁移等生物过程的细胞生物学家协同合作，和系统生物学家谁数学模型信号通路和谁需要热力学和动力学数据的关键步骤，这些途径。在这个项目的研究将在几个方面整合到一个大的教学（70 - 80名学生）蛋白质结构，功能和动力学（BioMG 6310）研究生水平3学分课程。 作为这门课的一部分，一个基于团队的项目被分配给学生写一个使用蛋白质“拯救世界”的提案（重点是能源和环境），并在学期结束时在公共海报会议上展示项目。 这项活动促进了与康奈尔大学阿特金森可持续未来中心（ACSF）的联系，包括在ACSF向不同的参观者展示海报，并任命PI为ACSF教职研究员。 这项NSF赞助的研究将被纳入BioMG 6310讲座，以说明基本概念。对代表性不足的群体的宣传包括为学习网（一个为农村和城市儿童提供服务的当地青年发展机构）提供实验室图尔斯，并在与GEEKS（毕业生就业同情，知识和服务）的年度重建旅行中为新奥尔良的第七和第九区带来科学的兴奋，PI建议的康奈尔大学研究生组织。PI担任本科生（独立学习和夏季研究）和研究生在她的实验室的研究导师。 学生接受跨学科的技术培训，包括NMR，生物物理方法，数学建模，生物化学和细胞生物学。