Structural And Functional Analysis Of Tn-IAP1v

Widely expressed in the species from insects to mammals, IAP (inhibitor of apoptosis protein) family is an extremely significant and evolutionarily conserved protein family, inhibiting apoptosis in the molecular apoptotic mechanism. Some mammalian IAP, such as XIAP, cIAP1, cIAP2 and Livin, can block mitochondria and death receptor induced apoptosis, as direct inhibitors of mammalian initiator caspase-9, and effector caspase-3, -7 in the caspase activation pathways. Although Tn-IAP1 derived from TN-368 cells, a cell strain of Trichoplusia ni, can rescue AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) lacking p35, actinomycin D, Hid and Grim induced Spodoptera frugiperda cells (Sf-21) from apoptosis, its potential molecular mechanism and biochemical pathway have not been deciphered as yet. By using RT-PCR, we cloned a variant gene of Tn-IAP1 from TN-5B1-4 cells, another cell strain of Trichoplusia ni. Six single nucleotide polymorphisms (SNPs) were found between the two variant genes, resulting in three single amino acid polymorphisms (SAPs) that are all located in the linker regions of characteristic IAP domians, BIRs (baculoviral IAP repeat) and RING (really interesting gene). Sequence homology analysis indicated that, among three BIRs of mammalian XIAP, Tn-IAP1v-BIR2 had the largest sequence similarity and the closest evolutionary relationship with XIAP-BIR3 that is responsible for the inhibition of caspase-9. Moreover, Tn-IAP1v lacked a similar fragment to the linker peptide between XIAP-BIR1 and XIAP-BIR2 that is responsible for the inhibition of caspase-3, -7. These data suggested a potentially distinct caspase inhibition mechanism of Tn-IAP1v from that of XIAP. With bacterial expression system, Tn-IAP1v and other functionally related proteins, caspase-8, caspase-9, XIAP, Sf-IAP,were expressed in the form of soluble protein in Escherichia coli, and purified with affinity chromatography at high purity. In the mammalian in vitro cell-free apoptotic system, Tn-IAP1v was experimentally proved that it could block cytochrome c but not initiator caspase-8 of death receptor pathway induced caspase activation. This result was confirmed by the observation that Tn-IAP1v could directly inhibit caspase-9 of cytochrome c pathway, but not effector caspase-3, -7, and initiator caspase-8 in the in vitro caspase inhibition assay. 3 Tn-IAP1v的结构与功能分析 Moreover, Tn-IAP1v could specifically bind to caspase-9 in the protein pull-down assay. Obviously, these experimental results totally match the functional prediction from sequence homology analysis. Meanwhile, we also proved that recombinant soluble Tn-IAP1v from its native TN-5B1-4 cells had the almost identical inhibitory effects on caspases with that fromEscherichia coli, although...