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.
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