Structural Studies Of The FADD-DD And RIP1-DD Complex In The Cell Death Signaling Pathway

Cell death,a normal physiological process for multicellular organisms,plays a crucial role in maintaining the homeostasis of multicellular organisms.Cell death is controlled by a series of strict molecular mechanisms.The most studied molecular mechanisms of cell death is the signaling pathway mediated by death receptors and adaptors such as apoptosis associated factor(Fas),tumor necrosis factor receptor 1(TNFR1)and others.This thesis focused on the structural studies of hFADD-DD and hRIP1-DD complex in the cell death signaling pathway by biochemical and biophysical methods for better understanding of the molecular mechanism of cell death.The recombinant plasmids containing hFADD-DD or hRIPl-DD cDNA were separately constructed for the expression in E.coli.Initial crystals were obtained by extensive screening for conditions.Optimization of crystallization condition led to the crystallographic analysis of a high resolution structure,which however only contained hRIP1-DD(577-669)instead of the complex with hFADD-DD.In order to obtain the crystal of the complex,we tried to express and purify the protein complex by using the co-transformation method and the co-expression method.Finally,various types of crystals were obtained,which did not diffract X-ray.The complex were further analyzed by using the negative staining electron microscopy and liquid chromatography-mass spectrometry.In addition,biolayer Interferometry technology were employed to study the interaction between a hRIPl-DD(552-576)peptide and hFADD-DD.The results showed that there was no interaction between hRIP1-DD(552-576)polypeptide and hFADD-DD.This study layed the important foundation for the further study of hFADD-DD and hRIP1-DD complex and the structural basis for the signaling pathways of cell death.