Studies On The Specific Antagonists Of Tumor Anti-apoptotic Factor C-FLIP

TRAIL can selectively induce apoptosis of transformed cells,tumor cells and virus-transformed cells without apparent toxic effects on normal cells.Since it was discovered,TRAIL has been a focus of intensive research.However,from recent studies,we have found that many tumor cells are resistant to TRAIL-induced apoptosis due to the high levels expression of anti-apoptotic proteins such as IAPs,Bcl-2 and c-FLIP,and low level expression of apoptotic protein such as Procaspase-8.If we understand this apoptosis signaling process clearly,the cancer treatment research based on this apoptosis pathway can be progressing very smoothly.Through amino acid sequence and protein 3D structures/crystalline analysis,it has been proposed that c-FLIP and Procaspase-8 interact with FADD very differently.Our studies have confirmed and proved the hypothesis.We have designed affinity chromatogphy screening systems in cell free system with six ?-helix-peptides of DED domain of FADD.C-FLIPs(180aa)and Procaspase-8-DED proteins were expressed and purified from their corresponding E-Coli vectors of pET-28a(+).These proteins were then applied to the six ?-helix-peptides affinity columns separately.Helix-4 peptide affinity column binds c-FLIPs(180aa)protein strongly.Helix-5 peptide affinity column binds Procaspase-8-DED protein strongly.The smaller peptides "Ser-Met-Leu-Leu-Glu-Gln" and "Met-Leu-Leu-Glu" in the Helix-4 bind to c-FLIPs(180aa)and prevent its binding to helix-4 column,suggesting that c-FLIPs(180aa)binds to Met-Leu-Leu-Glu of DED of FADD.The peptide "SLRR" in the Helix-5 and "LRRHD" in the Helix-6 of FADD'DED bind to Procasapse-8-DED and prevent their binding to helix-5 affinity column,suggesting that Procaspase-8-DED binds to LRR sequence of DED domain of FADD.Furthermore,base on the peptides Ser-Met-Leu-Leu-Glu-Gln and Met-Leu-Leu-Glu,12 more peptides were synthesized and screened for their abilities to prevent the binding of c-FLIP0180aa)to the helix-4 affinity column.D-Met-Leu-Leu-Glu-D-Ala was the most active peptide and D-Leu-Leu-Glu is the smallest peptide of high activity.Finally the peptide Ac-D-Met-Leu-Leu-Glu-D-Ala-FMK was synthesized and will be tested for its antagonism to c-FLIP in cancer cells.In addition,pET28a(+)expression vectors of Procaspase-8,c-FLIPs(F114A),Procaspase-8-DED(?64L)and FADD were constructed successfully for further mechanism studies.