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

• 批准号: 8102717
• 负责人: ALEXANDER SHEKHTMAN
• 金额: $ 26.98万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 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/8102717
• 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

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. PUBLIC HEALTH RELEVANCE: Many human diseases are caused by aberrant post-translational modifications which, it turn, result in changes in protein-protein interactions. We developed a new technology, STINT-NMR, which allows us to study STructural INTeractions between post- translationally modified proteins inside the cell with atomic resolution by using Nuclear Magnetic Resonance (NMR) spectroscopy. We will apply this technology to study aberrant structures and the molecular processes that may lead to disease states. We will also develop this technology to search for small drug-like molecules that may regulate these molecular processes.

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）引起的多组分蛋白复合物的结构变化。我们建议开发一种新的基于细胞内核磁共振的技术，用于绘制细胞中蛋白质-蛋白质相互作用的结构变化（STINT-NMR），并使用它来分析具有生物学意义的分子相互作用。与传统的体外核磁共振和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。得到的数据在原子分辨率上定义了相互作用表面的结构细节。通过顺序表达修饰蛋白-蛋白相互作用的酶活性，stnt - nmr还允许我们执行“细胞内生物化学”，在那里我们可以监测翻译后修饰的结构后果。本提案的总体目标是开发一种新的体内原子分辨率技术，以了解在结构水平上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stnt - nmr方法来结构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，我们将创建能够在细菌中过表达内吞蛋白、蛋白激酶和单泛素化机制的STINT-NMR兼容质粒结构。我们将使用未修饰和修饰的内吞蛋白的不同组合进行一系列stnt - nmr实验，以评估PTM引起的结构变化。我们将用修饰蛋白复合物的体外研究来补充细胞内核磁共振实验。2)对真核细胞进行STINT-NMR，检测胞内结构对蛋白-蛋白相互作用的影响。为此，我们将优化酵母细胞中泛素和STAM2的过表达，进行STINT-NMR实验。3)扩展stnt - nmr，用于高通量筛选能够干扰出芽所需病毒蛋白与宿主内噬蛋白之间形成复合物的小分子。我们将构建能够在细菌中过表达HIV p6结构域、内吞蛋白TSG101和Hrs、蛋白激酶MEK1和ERK2以及单泛素化机制的stnt - nmr兼容质粒。我们将使用未修饰和修饰蛋白质的不同组合进行一系列stnt - nmr实验，以确定与复合物形成相关的结构变化。作为STINT-NMR HTS方法的阳性对照，我们将筛选一小部分已知的抗tsg101诱导HIV-1出芽的拮抗剂。随后，一个小药物样分子文库（NCI Discover set）将针对p6-TSG101-Hrs复合物使用stnt - nmr筛选，以确定能够干扰病毒出芽的化合物的可能类别。