Druggable Mechanisms

可药物机制核心(DMC)的首要目标是提供最先进的 高通量嗜铬蛋白免疫沉淀测序(ChLP-SEQ)、RNA测序技术 (RNA-seq)和基于质谱学的蛋白质组学到每个项目，以深入表征 转录因子蛋白复合体及其各自的遗传靶点。来自DMC的数据将揭示 寡聚(项目1)、Runx1(项目2)和NPAS4(项目3)的相互作用蛋白质伙伴，特征 相关的翻译后修饰，识别下游基因靶点，并最终量化 这些蛋白质-蛋白质和蛋白质-DNA相互作用与转录的协调作用 各自的靶基因。DMC提供的分子相互作用的综合观点将 包括与寡聚相关的神经病理的直接和替代治疗切入点， Runx1和NPAS4。在FIME上，在DMC和项目1-3之间建立的协同作用可以提供 转录因子导向药物发现的实验范式。公契的具体目标是： 1)实施创新的基于质谱学的蛋白质组学技术用于蛋白质分析 复合体。我们将应用新的蛋白质组学方法来提高蛋白质和多肽的动态范围 蛋白质复合体的串联免疫亲和纯化联用鉴定。这些措施包括 固相捕获结合稳定同位素标记技术定量检测含半胱氨酸多肽 蛋白质组学，以及基于反相树脂组合的多维分离策略 (RP/RP)。 2.)建立ChlP-seq和RNA-seq文库并优化应用生物系统的测序 固体基因组分析仪。这个目标将是开发可靠的ChlP-seq和RNA-seq文库 构造协议。每个项目都需要短读测序，我们将在我们的固体上执行 基因组分析仪。在与应用的生物系统磋商中持续开发内部协议 将确保在我们的固体仪器上进行高效和快速的测序。 3.)部署基于网络的生物信息学工具，用于集成分析芯片-RNA-SEQ数据和 对蛋白质组学数据进行全面的、用户驱动的分析。这一目标的目标是部署基于Web的 该系统将为项目1-3中的人员提供交互式、全面和动态的 用于查询它们的基因组和蛋白质组数据的计算平台，以及它们的扩展 观察到路径和网络的水平。

The overriding objective of the Druggable Mechanisms Core (DMC) is to provide state-of-the-art technologies in high-throughput chromafin-immunoprecipitafion 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 Oligl (Project 1), Runxl (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 Oligl, Runxl, and NPAS4. Over fime 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 pepfide identificafion in conjuncfion with tandem immunoaffinity purificafion 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 construcfion protocols. Each project will require short read sequencing, which we will perform on our SOLiD genome analyzer. Continuous development of in-house protocols in consultafion 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.