Structural Studies Of P53 Upregulated Modulator Of Apoptosis

p53 upregulated modulator of apoptosis(PUMA)is a member of the BH3-only group in the BCL-2 family,and plays a prominent role in promoting apoptosis.By either a p53 dependent or a p5 3 independent mechanism,PUMA can activate the homo-oligomerization of BAX and BAK,permeabilize the mitochondrial outer membrane,release cytochrome c and induce apoptosis.PUMA is essential for p53-dependent apoptosis and promotes apoptosis in cancer regardless the status of p53.It makes PUMA a potential target for anticancer therapy.Despite significant progress in its research since its discovery in 2001,the mechanism of PUMA promoting apoptosis is still under debate.It will be of great importance to elucidate the high resolution structure of PUMA for better understanding of the associated molecular mechanism in induction of apoptosis and for the development of therapeutics targeting cancer.In this study,we use E.coli expression system to obtain the protein amenable for structural studies.The expression condition was first optimized by varying factors such as expression strains,temperature and IPTG concentration.Mutants that mimic phosphorylation were generated for better hydrodynamic behavior.Stable and soluble proteins were eventually generated by making truncations at the C-and N-termini.The proteins were purified by affinity chromatography and gel chromatography.Despite intensive effort,no crystal for the native protein was obtained.However it is discovered that the deletion of resides 150-163 would transform PUMA from larger oligomers to monomer.In addition,residues 134-165 are important for the possible coiled coil structure.We have also been able to generate a complex of PUMA and BCL-xL for crystallization.