STRUCTURAL AND FUNCTIONAL STUDIES OF THE TNF SIGNALING MACHINERIES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们的实验室研究了细胞转导和整合环境信号以影响细胞生存、增殖、分化和死亡等命运选择的分子机制。特别是，我们的工作集中在通过肿瘤坏死因子受体超家族复杂的信号机制来阐明决定细胞命运的分子机制，肿瘤坏死因子受体超家族是哺乳动物生物学的关键调节因子。 我们从生化重组和信号机制的解剖开始，以确定组件和子组件的定义状态。这在很大程度上得益于有限的蛋白质分解以及随后的N-末端测序和质谱分析。使用X射线结晶学来确定它们的详细原子结构是我们用来揭示信号转导的分子基础的主要方法。分离的蛋白质及其络合物的结构测定是通过反常衍射法、同象置换法和分子置换法等多种方法进行的。由于在蛋白质晶体中合理引入反常中心的研究进展，反常衍射法正成为我们研究中最重要的位相方法。这种方法需要像NE-CAT这样的波动束线所提供的高能量分辨率和高通量的X射线束。 结构洞察力对于像这样的复杂系统特别重要，部分原因是它们提供了明确确定给定交互作用的作用所需的特殊性。我们的目标是使用这些结构观点，通过使用细胞生物学实验测试基于结构的假设，帮助揭开复杂的功能问题。最终，通过将我们结构研究中的静态快照转化为对动态信号传递过程的综合理解，我们希望了解细胞存活和细胞死亡的决定规则。由于肿瘤坏死因子信号的失调与许多人类疾病有关，我们的研究将为理解这些疾病的起源提供结构和功能平台。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our laboratory studies the molecular mechanisms by which cells transduce and integrate environmental signals to influence the choices of cell fate such as survival, proliferation, differentiation and death. In particular, our work focuses on elucidating the molecular mechanisms of cell fate determination by the elaborate signaling machineries of the tumor necrosis factor (TNF) receptor superfamily, which are critical regulators of mammalian biology. We begin by biochemical reconstitution and dissection of the signaling machineries to identify defined states of the assemblies and sub-assemblies. This is greatly aided by limited proteolysis followed by N-terminal sequencing and mass spectrometry analysis. Using X-ray crystallography to determine their detailed atomic structures is the primary methodology we use to reveal the molecular basis of signal transduction. Structure determination of isolated proteins and their complexes are performed by various phasing methods such as anomalous diffraction, ismorphous replacement and molecular replacement. Because of the advancement in rational incorporation of anomalous centers into protein crystals, anomalous diffraction is becoming the most important phasing method in our research. This method requires the high energy resolution and high flux X-ray beams as offered by undulator beam lines such as NE-CAT. Structural insights are particularly important for complex systems such as this, in part because they provide the specificity required to determine unambiguously the role of a given interaction. Our aspiration is to use these structural perspectives to help unravel complex functional questions by testing structure-based hypotheses using cell biological experiments. Ultimately, by transforming static snapshots from our structural studies into an integrated understanding of the dynamic signaling process, we hope to understand the rules in this determination of cell survival and cell death. Because dysregulation of TNF signaling is associated with many human diseases, our studies will provide structural and functional platforms for understanding the genesis of these diseases.