Druggable Mechanisms

药物机制

• 批准号: 8532051
• 负责人: YOSHIHIRO NAKATANI
• 金额: $ 33.66万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8532051
• 关键词: Affinity; Binding; Bioinformatics; Biological; Biological Models; Buffers; Complex; Computer software; Consultations; Cysteine; DNA; DNA Sequence; DNA-Protein Interaction; Data; Data Analyses; Data Quality; Detection; Development; Dimensions; Employee Strikes; Ensure; Feedback; Fractionation; Gene Targeting; Generations; Genetic; Genetic Transcription; Genome; Genomics; Goals; Housing; Human Resources; Individual; Informatics; Instruction; Libraries; Mass Spectrum Analysis; Methodology; Methods; Molecular; Nature; Neurons; Online Systems; Pathway interactions; Peptides; Performance; Phase; Plant Resins; Play; Positioning Attribute; Post Translational Modification Analysis; Post-Translational Protein Processing; Preparation; Process; Proteins; Proteomics; Protocols documentation; RNA Sequences; Reading; Research; Research Personnel; Resources; Role; Sampling; Services; Solid; Stable Isotope Labeling; Standardization; Sulfhydryl Compounds; System; Techniques; Technology; Therapeutic; Time; Tissues; Training; Validation; base; chromatin immunoprecipitation; computing resources; data acquisition; drug discovery; experience; flexibility; improved; innovation; instrument; nerve stem cell; neuropathology; novel; protein complex; research study; stoichiometry; therapeutic target; tool; transcription factor

The overriding objective of the Druggable Mechanisms Core (DMC) is to provide state-of-the-art technologies in high-throughput chromatin-immunoprecipitation sequencing (ChlP-seq), RNA sequencing (RNA-seq), and mass spectrometry-based proteomics to each Project for in-depth characterization of transcription factor protein complexes and their respective genetic targets. Data from the DMC will reveal interacting protein partners for Olig1 (Project 1), Runx1 (Project 2), and NPAS4 (Project 3), characterize associated post-translational modifications, identify downstream genetic targets, and finally quantify the coordinate action of these protein-protein and protein-DNA interactions with respect to transcription of respective target genes. The comprehensive view of molecular interactions provided by the DMC will encompass both direct and surrogate therapeutic entry points for neuropathologies associated with Olig1, Runx1, and NPAS4. Over time the synergy established between the DMC and Projects 1-3 may provide an experimental paradigm for transcription factor-directed drug discovery. The specific aims of the DMC are: 1.) Implement innovative mass spectrometry-based proteomics techniques for analysis of protein complexes. We will implement novel proteomics methods to improve dynamic range of protein and peptide identification in conjunction with tandem immunoaffinity purification of protein complexes. These will include solid-phase capture of cysteine-containing peptides combined with stable isotope labeling for quantitative proteomics, and a multidimension separation strategy based on a combination of reversed phase resins (RP/RP). 2.) Develop ChlP-seq and RNA-seq libraries and optimize sequencing on an Applied Biosystems SOLID genome analyzer. The goal of this aim will be to develop reliable ChlP-seq and RNA-seq library construction protocols. Each project will require short read sequencing, which we will perform on our SOLiD genome analyzer. Continuous development of in-house protocols in consultation with AppliedBiosystems will ensure efficient and rapid sequencing on our SOLiD instrument. 3.) Deploy web-based, bioinformatic tools for integrated analysis of ChIP- an RNA-seq data and for comprehensive, user-driven analysis of proteomics data. The goal of this aim is to deploy a web-based system that will provide personnel in Projects 1-3 with an interactive, comprehensive, and dynamic computational platform for interrogation of their genomic and proteomic data, and extension of their observations to the level of pathways and networks.

Druggable Mechanisms Core (DMC) 的首要目标是为每个项目提供最先进的高通量染色质免疫沉淀测序 (ChlP-seq)、RNA 测序 (RNA-seq) 和基于质谱的蛋白质组学技术，以深入表征转录因子蛋白质复合物及其各自的遗传靶标。来自 DMC 的数据将揭示 Olig1（项目 1）、Runx1（项目 2）和 NPAS4（项目 3）的相互作用蛋白质伙伴，表征相关的翻译后修饰，识别下游遗传靶标，并最终量化这些蛋白质-蛋白质和蛋白质-DNA 相互作用相对于各自靶基因转录的协调作用。 DMC 提供的分子相互作用的全面视图将涵盖与 Olig1、Runx1 和 NPAS4 相关的神经病理学的直接和替代治疗切入点。随着时间的推移，DMC 和项目 1-3 之间建立的协同作用可能会为转录因子导向的药物发现提供实验范式。 DMC 的具体目标是： 1.) 实施基于质谱的创新蛋白质组学技术来分析蛋白质复合物。我们将实施新型蛋白质组学方法，结合蛋白质复合物的串联免疫亲和纯化，提高蛋白质和肽鉴定的动态范围。这些将包括含半胱氨酸肽的固相捕获与定量蛋白质组学的稳定同位素标记相结合，以及基于反相树脂（RP/RP）组合的多维分离策略。 2.) 开发 ChlP-seq 和 RNA-seq 文库并在 Applied Biosystems SOLID 基因组分析仪上优化测序。该目标的目标是开发可靠的 ChlP-seq 和 RNA-seq 文库构建方案。每个项目都需要短读测序，我们将在 SOLiD 基因组分析仪上执行该测序。与 AppliedBiosystems 协商不断开发内部方案将确保在我们的 SOLiD 仪器上高效、快速地测序。 3.) 部署基于网络的生物信息学工具，对 ChIP-an RNA-seq 数据进行集成分析，并对蛋白质组数据进行全面的、用户驱动的分析。这一目标是部署一个基于网络的系统，为项目 1-3 的人员提供一个交互式、全面和动态的计算平台，用于询问他们的基因组和蛋白质组数据，并将他们的观察扩展到通路和网络的水平。