Druggable Mechanisms

Druggable Mechanisms Core（DMC）的首要目标是为每个项目提供高通量染色质免疫沉淀测序（ChIP-seq）、RNA测序（RNA-seq）和基于质谱的蛋白质组学方面的最新技术，以深入表征转录因子蛋白质复合物及其各自的遗传靶点。来自DMC的数据将揭示Olig 1（项目1）、Runx 1（项目2）和NPAS 4（项目3）的相互作用蛋白伴侣，表征相关的翻译后修饰，鉴定下游遗传靶点，并最终定量这些蛋白质-蛋白质和蛋白质-DNA相互作用对相应靶基因转录的协调作用。DMC提供的分子相互作用的综合观点将涵盖与Olig 1、Runx 1和NPAS 4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为转录因子指导的药物发现提供实验范式。DMC的具体目标是：1.）实施创新的基于质谱的蛋白质组学技术，用于分析蛋白质复合物。我们将实施新的蛋白质组学方法，以提高蛋白质和肽识别的动态范围，结合蛋白质复合物的串联免疫亲和纯化。这些将包括固相捕获含半胱氨酸的肽结合稳定同位素标记定量蛋白质组学，和基于反相树脂（RP/RP）的组合的多维分离策略。2.）在Applied Biosystems SOLID基因组分析仪上开发ChIP-seq和RNA-seq文库并优化测序。该目标的目标将是开发可靠的ChIP-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基因组分析仪上进行。在咨询了我们的SOLiD仪器后，我们将持续开发内部方案，以确保在我们的SOLiD仪器上进行高效快速的测序。3.）第三章部署基于网络的生物信息学工具，用于ChIP（RNA测序数据）的综合分析，并用于蛋白质组学数据的全面、用户驱动的分析。这个目标的目标是部署一个基于网络的系统，将提供项目1-3的人员与他们的基因组和蛋白质组数据的询问，并延长他们的观察到的通路和网络的水平的交互式，全面和动态的计算平台。

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.