Modular CID-Cleavable Cross-Linkers for Proteomic Analysis

用于蛋白质组分析的模块化 CID 可切割交联剂

• 批准号: 1807612
• 负责人: Scott Rychnovsky
• 金额: $ 45万
• 依托单位: University of California-Irvine
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-15 至 2022-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1807612&HistoricalAwards=false
• 关键词: Modular; CID; Cleavable; Cross; Linkers

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Scott Rychnovsky from University of California Irvine to develop new chemical tools to determine the structure of protein complexes. Understanding the function of protein-protein complexes is fundamental to understanding cellular processes. Although there are number of methods to study protein complexes, they all have significant limitations. The method being developed, chemical cross-linking, can be used under the widest variety of conditions and can capture dynamic states of the complexes. The chemical compound, a cross-linker, has two reactive ends that stick to parts of the protein. When the cross-linker sticks to two different proteins, it tells us that those proteins are close together in the complex. The important advance in this project is to make it much easier to identify the precise location the cross-linker sticks to in each protein. This information can be used to build a detailed model of the complex to better understand its behavior. The protein complex studied is the COP9 signalosome, which is important in the function of all cells in the body and mutations of this protein complex have been identified in important neurological disorders such as Alzheimer's disease and Down's syndrome. The program provides training for graduate students and undergraduates in chemistry and chemical biology. Promising undergraduate students are recruited from a local community college to participate in summer research, with the goal of stimulating their interest in scientific research careers.The advent of Collision-Induced Dissociation (CID) cleavable crosslinkers has dramatically simplified the sequencing of cross-linked amino acids by liquid chromatography Mass Spectrometry (LCMS/MS). One objective of this proposal is to develop a family of CID-cleavable cross-linkers to facilitate the analysis of protein complexes. These new cross-linker reagents are broadly useful in the field of structural biology. The second research objective is to map out the dynamic structure of the COP9 signalosome (CSN). CSN is an important regulatory protein that catalyzes the hydrolysis of the Nedd8 protein from the cullin-RING ubiquitin ligase. CSN is found in all eukaryotes; the human form has eight canonical subunits, all of which appear to be required for function. CSN is important in regulating the cell cycle, in neuronal development and in DNA repair. Specific mutations in the CNS subunits have been implicated in a number of brain afflictions, including Alzheimer's disease and Down syndrome. A crystal structure was reported that provides a detailed static model of CSN. A goal of this project is to define the dynamic structure of the complete CSN.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

有了这个奖项，化学部的生命过程化学计划正在资助加州欧文大学的Scott Rychnovsky博士开发新的化学工具来确定蛋白质复合物的结构。 理解蛋白质-蛋白质复合物的功能是理解细胞过程的基础。 虽然有许多方法来研究蛋白质复合物，但它们都有很大的局限性。 正在开发的方法，化学交联，可以在各种各样的条件下使用，并可以捕获复合物的动态状态。 这种化合物是一种交联剂，有两个反应性末端，可以粘在蛋白质的一部分上。 当交联剂粘在两种不同的蛋白质上时，它告诉我们这些蛋白质在复合物中靠得很近。 该项目的重要进展是更容易识别交联剂在每个蛋白质中的精确位置。 这些信息可用于构建复杂的详细模型，以更好地了解其行为。 所研究的蛋白质复合物是COP 9信号体，其在体内所有细胞的功能中是重要的，并且已经在重要的神经系统疾病如阿尔茨海默病和唐氏综合征中鉴定了该蛋白质复合物的突变。 该计划为研究生和本科生提供化学和化学生物学方面的培训。 从当地社区大学招募有前途的本科生参加暑期研究，目的是激发他们对科研事业的兴趣。碰撞诱导解离（CID）可裂解交联剂的出现大大简化了通过液相色谱质谱（LCMS/MS）进行交联氨基酸测序的过程。该提议的一个目标是开发CID可裂解的交联剂家族以促进蛋白质复合物的分析。这些新的交联剂试剂在结构生物学领域中广泛有用。第二个研究目标是绘制出COP 9信号体（CSN）的动态结构。CSN是一种重要的调节蛋白，其催化Cullin-RING泛素连接酶水解Nedd 8蛋白。CSN存在于所有真核生物中;人类形态有八个典型亚基，所有这些亚基似乎都是功能所必需的。CSN在调节细胞周期、神经元发育和DNA修复中是重要的。中枢神经系统亚单位的特定突变与许多脑部疾病有关，包括阿尔茨海默病和唐氏综合征。报道了CSN的晶体结构，提供了详细的静态模型。该项目的目标是确定完整的CSN的动态结构。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

项目成果

Structural dynamics of the human COP9 signalosome revealed by cross-linking mass spectrometry and integrative modeling

• DOI: 10.1073/pnas.1915542117
• 发表时间: 2020-02-25
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Gutierrez, Craig;Chemmama, Ilan E.;Huang, Lan
• 通讯作者: Huang, Lan