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In-cell NMR technology to study protein interactions

研究蛋白质相互作用的细胞内核磁共振技术

基本信息

批准号：
8499358
负责人：
ALEXANDER SHEKHTMAN
金额：
$ 21.77万
依托单位：
STATE UNIVERSITY OF NEW YORK AT ALBANY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8499358
关键词：
Affinity Bacteria Binding Biochemistry Biological Biological Process Cells Complex Crystallography Data Databases Development Disease Effectiveness Endocytosis Etiology Eukaryotic Cell HIV HIV-1 Human In Vitro Laboratories Lead Libraries Ligands MAP2K1 gene MAPK1 gene Malignant Neoplasms Maps Methodology Methods Modification Molecular Molecular Structure Monitor Monoubiquitination Multiprotein Complexes NMR Spectroscopy Nuclear Magnetic Resonance Pathway interactions Pharmaceutical Preparations Phosphorylation Plasmids Play Post-Translational Protein Processing Process Protein Kinase Proteins Proteomics RNA Viruses Receptor Protein-Tyrosine Kinases Resolution Role Sampling Series Sorting - Cell Movement Staging Subcellular structure Surface System TSG101 gene Techniques Technology Testing Time Tsg101 protein Ubiquitin Validation Viral Viral Interference Viral Pathogenesis Viral Proteins Virus Diseases Yeasts base biological systems hepatocyte growth factor-regulated tyrosine kinase substrate high throughput screening human disease in vivo interest new technology novel overexpression protein complex protein protein interaction public health relevance receptor mediated endocytosis research study small molecule structural biology tool

项目摘要

DESCRIPTION (provided by applicant): The etiology of many human diseases involves structural changes in multi-component protein complexes caused by aberrant posttranslational modifications (PTM). We propose to develop a novel in-cell NMR-based technology for mapping the STructural changes that accompany protein-protein INTeractions in the cell (STINT-NMR) and use it to analyze molecular interactions of biological significance. STINT-NMR analysis greatly reduces the time required to obtain atomic resolution information on protein complexes compared to traditional in vitro NMR and x-ray crystallography. The resulting data define structural details of the interacting surfaces at atomic resolution. By sequentially expressing enzymatic activities that modify protein-protein interactions, STINT-NMR also allows us to perform "biochemistry inside the cell", where we can monitor the structural consequences of post-translational modifications. The broad objective of this proposal is to develop a novel in vivo atomic resolution technique to understand on structural level how posttranslational modifications regulate protein complexes involved in important biological processes. To fulfill this objective we have three specific aims: 1) Develop STINT-NMR methodology to structurally characterize multiprotein interactions in bacterial cells. As a biological system, we will use the interactions between Ubiquitin and unmodified or posttranslationally modified endocytic proteins Hrs, STAM2, Eps15. For this aim we will create STINT-NMR compatible plasmid constructs capable of overexpressing endocytic proteins, protein kinases, and monoubiquitination machinery in bacteria. We will perform series of STINT-NMR experiments using different combinations of unmodified and modified endocytic proteins to assess the structural changes resulting from PTM's. We will supplement our in- cell NMR experiments with in vitro studies of the modified protein complexes. 2) Develop STINT-NMR for eukaryotic cells to test the influence of intracellular structures on protein-protein interactions. For this aim we will optimize overexpression of Ubiquitin and STAM2 in yeast cells to perform STINT-NMR experiments. 3) Extend STINT-NMR for high-throughput screening of small molecules capable of interfering with the complexes formed between viral proteins required for budding and host endocytic proteins. We will create STINT-NMR compatible plasmid constructs capable of overexpressing viral HIV p6 domain, endocytic proteins TSG101 and Hrs, protein kinase MEK1 and ERK2, and monoubiquitination machinery in bacteria. We will perform a series of STINT-NMR experiments using different combinations of unmodified and modified proteins to establish structural changes associated with complex formation. As a positive control of STINT-NMR HTS methodology, we will screen a small library of known antagonists against TSG101-directed HIV-1 budding. Later, a library of small drug-like molecules (NCI Discover set) will be screened against the p6-TSG101-Hrs complexes using STINT-NMR to identify possible classes of the compounds capable of interfering with viral budding.

描述（由申请人提供）：许多人类疾病的病因涉及由异常翻译后修饰（PTM）引起的多组分蛋白质复合物的结构变化。我们建议开发一种基于细胞内核磁共振的新型技术，用于绘制细胞中蛋白质-蛋白质相互作用伴随的结构变化（STINT-NMR），并用它来分析具有生物学意义的分子相互作用。与传统的体外 NMR 和 X 射线晶体学相比，STINT-NMR 分析大大减少了获得蛋白质复合物原子分辨率信息所需的时间。所得数据以原子分辨率定义相互作用表面的结构细节。通过顺序表达改变蛋白质-蛋白质相互作用的酶活性，STINT-NMR 还允许我们进行“细胞内的生物化学”，我们可以监测翻译后修饰的结构后果。该提案的总体目标是开发一种新颖的体内原子分辨率技术，以在结构水平上了解翻译后修饰如何调节参与重要生物过程的蛋白质复合物。为了实现这一目标，我们有三个具体目标：1) 开发 STINT-NMR 方法来从结构上表征细菌细胞中的多蛋白相互作用。作为一个生物系统，我们将利用泛素与未修饰或翻译后修饰的内吞蛋白 Hrs、STAM2、Eps15 之间的相互作用。为此，我们将创建 STINT-NMR 兼容的质粒构建体，能够在细菌中过度表达内吞蛋白、蛋白激酶和单泛素化机制。我们将使用未修饰和修饰内吞蛋白的不同组合进行一系列 STINT-NMR 实验，以评估 PTM 引起的结构变化。我们将通过修饰蛋白质复合物的体外研究来补充我们的细胞内核磁共振实验。 2) 开发真核细胞的 STINT-NMR，以测试细胞内结构对蛋白质-蛋白质相互作用的影响。为此，我们将优化酵母细胞中泛素和 STAM2 的过度表达，以进行 STINT-NMR 实验。 3) 扩展 STINT-NMR，用于高通量筛选能够干扰出芽所需的病毒蛋白和宿主内吞蛋白之间形成的复合物的小分子。我们将创建 STINT-NMR 兼容的质粒构建体，能够过表达病毒 HIV p6 结构域、内吞蛋白 TSG101 和 Hrs、蛋白激酶 MEK1 和 ERK2 以及细菌中的单泛素化机制。我们将使用未修饰和修饰蛋白质的不同组合进行一系列 STINT-NMR 实验，以确定与复杂形成相关的结构变化。作为 STINT-NMR HTS 方法的阳性对照，我们将筛选一个小型已知拮抗剂库，以对抗 TSG101 定向的 HIV-1 出芽。随后，将使用 STINT-NMR 针对 p6-TSG101-Hrs 复合物筛选类药物小分子库（NCI Discover set），以识别能够干扰病毒出芽的可能化合物类别。