Structural Studies Of Pelota And P53 Upregulated Modulator Of Apoptosis

1 Introduction to macromolecular X-ray crystallographyStructural biology is a discipline that investigates macromolecular structure to atomic resolution and the associated functions.X-ray crystallography,NMR and Cryo-electron microscopy(cryo-EM)are the three main tools employed in structural investigation.Each of them has its unique advantages as well as limitations.As the first developed research method of structural biology,X-ray crystallography is widely used and 85%of the structures deposited in Protein Data Bank are determined by the technique.In this chapter,basic principlesof X-ray crystallography will be introduced,which include diffraction theory,crystallization,diffraction data collection,the phase problem and molecular replacement,model building and refinement.2 Structural studies of Anthropogenic PelotaPelota is an evolutionarily conservative RNA binding protein.Pelota is widely distributed and can be found in Archaea,yeast,fruit flies,mice,and humans.Human and mouse Pelota share a high homology of95%.Pelota is composed of three domains,the N-terminal,Center,and C-terminal domains.Pelota contains a conservative nuclear localization signal(PRKRK)in its N-ter minal and its Center and C-terminal domains are analogous to the Center and C-terminal structural of eRFl.Pelota involves in the regulation of cell cycle and the formation of reproductive cells.Its knockout will result in infertility.Pelota proteins participate in the regulation of protein translation processby strictly controlling the mRNA-decay to ensure the correct protein translation and normal cell activity.Pelota also plays an important role in the monitoring mechanism of mRNA and the ribosome recycling.Due to the effect in mRNA-decay and the ribosome recycling,Pelota may be employed as drug targets for the development of therapeutics.This chapter describes the structural studies of Pelota.We solved the crystal structure of Pelota C terminal domain at 2.6A,which resembles the eRF1 C-domain.3 Structural Studies of p53 Upregulated Modulator ofApoptosisPUMA is a member of the BH3-only group in the BCL-2 family and is the essential factor in the p53-associated apoptosis pathway.PUMA can activate the homo-oligomerization of BAX and BAK through p53 dependent and independent pathways,which result in permeabilizethe mitochondrial outer membrane and releasing cyto-chrome c to induce apoptosis.PUMA's activity is independent to the status of p53,PUMA plays more direct role in cell apotosis and may be employed as drug targets for the development of the rapeutics for cancer.Hsc70 is a constitutive expressed heat shock protein in mammalian cells,and plays an important role in the regulation of protein metabolism and processing.Hsc70 interacts with PUMA and may be involved in PUMA,s folding or degradation.The structure of PUMA or PUMA-Hsc70 complex can help us to understand the molecular mechanism in PUMA apoptosis.In this study,we tried a variety of techniques,but failed to obtain the crystal of PUMA.We found that the 134-165 amino acids of PUMA form a coiled-coil structure,which can promotes PUMA polymerization.And wo also demonstrated that PUMA interacts with the Hsc70.Finally,we got two ATPase structures in different binding states.One has a ATP molecule and the other do not,the active site of the structure bound ATP becomes smaller.We tried to get the complex crystals of a peptide derived from PUMA and ATPase domain,but ultimately there is no peptide in the complex structure.