Development of H/D exchange technology for the study of large protein complexes

开发用于研究大型蛋白质复合物的 H/D 交换技术

基本信息

批准号：
7741193
负责人：
Patrick Robert Griffin
金额：
$ 39.47万
依托单位：
SCRIPPS FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-12-01 至 2012-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7741193
关键词：
Binding Binding Sites Biochemical Biological Biological Models Cardiovascular system Collection Communication Complement Complex Data Deuterium Development Disease Epitopes Family G protein coupled receptor kinase G-Protein-Coupled Receptors Hydrogen Immune Immune system Inflammation JUN gene Ligand Binding Domain Ligands MAP Kinase Signaling Pathways MAPK10 gene Malignant Neoplasms Maps Metabolic Diseases Methodology Methods Mitogen-Activated Protein Kinases Molecular Nerve Degeneration Nuclear Orphan Receptor Nuclear Receptors Orphan Osteoporosis Parkinson Disease Peroxisome Proliferator-Activated Receptors Pharmacology Phosphotransferases Protein Binding Proteins Proteomics Publishing RXR Regulation Research Institute Research Personnel Rhodopsin Screening procedure Signal Pathway Signal Transduction Pathway Software Tools Sphingosine-1-Phosphate Receptor Substrate Interaction System Technology Therapeutic Agents Time Work base common cellular transcription factor ATF complex biological systems data acquisition data management high throughput screening human GPRC5C protein immunoregulation improved inhibitor/antagonist interest kinase inhibitor member mitogen-activated protein kinase p38 protein complex public health relevance receptor relational database sphingosine 1-phosphate tool vascular bed

项目摘要

DESCRIPTION (provided by applicant): In this proposal we seek to optimize and apply HDX as an enabling technology for the functional analysis of complex biological systems, such as transcriptional complexes and G-protein coupled receptors. We will study nuclear receptor complexes, GPCRs, and the MAP kinase signaling pathway. These biological targets have been implicated in metabolic disorders, cancer, osteoporosis, immune regulation, and neurodegeneration. Although implicated in a wide range of disorders, our group and our collaborators have specific expertise in metabolic disorders, immune regulation, and Parkinson's disease. A better understanding of the mechanism of action of modulators of NRs, GPCRs and kinase signaling pathways will enable the development of more functionally selective and safer therapeutic agents. The technological aspects of this work are to optimize and apply hydrogen/deuterium exchange (HDX) to enable characterization of complex proteins such as GPCRs and large multi-protein complexes involved in the regulation of signal transduction pathways. Recently we have made several significant advances in HDX technology. Here we will build from this work and optimize methodology for the characterization of complex systems. These methods will improve on the current limits of dynamic range, protein size, and throughput. In addition, our automated system for data acquisition will be integrated with a relational database and a new suite of software tools will be further developed to facilitate seamless integration of HDX data acquisition with data management, analysis, and display. We will apply this technology to the functional analysis of nuclear receptor transcriptional complexes, S1P family receptors, and the MAP kinase JNK3. We will also demonstrate the utility of HDX technology in the context of a fully integrated high throughput screening center. Combined, these studies will raise the impact of this enabling biophysical technology on structural studies of complex biological systems. PUBLIC HEALTH RELEVANCE: In this proposal we seek to optimize and apply Hydrogen/Deuterium Exchange (HDX) as an enabling technology for the functional analysis of complex biological systems such as transcriptional complexes and G-protein coupled receptors. We will study nuclear receptor complexes, GPCRs, and the MAP kinase signaling pathway. Although these targets have been implicated in a wide range of disorders, we will focus on application of the technology to metabolic disorders, immune regulation and Parkinson's disease where a better understanding of the mechanism of action of modulators of NRs, GPCRs and kinase signaling pathways will enable the development of more functionally selective and safer therapeutic agents.

描述（由申请人提供）：在本提案中，我们寻求优化和应用 HDX 作为复杂生物系统功能分析的使能技术，例如转录复合物和 G 蛋白偶联受体。我们将研究核受体复合物、GPCR 和 MAP 激酶信号通路。这些生物靶点与代谢紊乱、癌症、骨质疏松症、免疫调节和神经退行性疾病有关。尽管涉及多种疾病，但我们的团队和合作者在代谢紊乱、免疫调节和帕金森病方面拥有特定的专业知识。更好地了解 NR、GPCR 和激酶信号传导途径调节剂的作用机制将有助于开发功能更具选择性和更安全的治疗药物。这项工作的技术方面是优化和应用氢/氘交换 (HDX)，以实现复杂蛋白质的表征，例如 GPCR 和参与信号转导途径调节的大型多蛋白质复合物。最近，我们在 HDX 技术方面取得了几项重大进展。在这里，我们将从这项工作中构建并优化复杂系统表征的方法。这些方法将改善动态范围、蛋白质大小和通量的当前限制。此外，我们的数据采集自动化系统将与关系数据库集成，并将进一步开发一套新的软件工具，以促进 HDX 数据采集与数据管理、分析和显示的无缝集成。我们将将该技术应用于核受体转录复合物、S1P家族受体和MAP激酶JNK3的功能分析。我们还将展示 HDX 技术在完全集成的高通量筛选中心中的实用性。结合起来，这些研究将提高这种生物物理技术对复杂生物系统结构研究的影响。公共健康相关性：在本提案中，我们寻求优化和应用氢/氘交换 (HDX)，作为对转录复合物和 G 蛋白偶联受体等复杂生物系统进行功能分析的支持技术。我们将研究核受体复合物、GPCR 和 MAP 激酶信号通路。尽管这些靶点与多种疾病有关，但我们将重点关注该技术在代谢紊乱、免疫调节和帕金森病方面的应用，更好地了解 NR、GPCR 和激酶信号通路调节剂的作用机制将有助于使开发更具功能选择性和更安全的治疗剂成为可能。