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Dissecting the Function of IAP (Inhibitor of Apoptosis) Protein Domains in Inhibiting an Apoptotic Caspase
Author(s) -
Storm Mackenzie Bryan,
Junker Matthew
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.528.15
Subject(s) - apoptosis , caspase , inhibitor of apoptosis , microbiology and biotechnology , linker , caspase 3 , caspase 7 , xiap , caspase 8 , inhibitor of apoptosis domain , biology , chemistry , programmed cell death , biochemistry , computer science , operating system
Apoptosis (programmed cell death) is an essential process in all animals for normal development and tissue regeneration. Apoptosis also eliminates cells at risk for becoming cancerous or spreading a viral infection. Apoptosis requires the activity of intracellular caspase enzymes that are normally kept inhibited in living cells by inhibitor of apoptosis proteins (IAPs). Certain apoptosis‐promoting proteins bind IAPs to relieve caspase inhibition. IAPs contain 2–3 BIR (baculovirus IAP repeat) domains with intervening linkers. In many IAPs, residues in a linker bind and block the caspase active site but the BIR domains also influence caspase inhibition. To determine how the BIRs contribute to caspase inhibition, we compared the ability of different fragments of the Drosophila IAP DIAP1 to inhibit a Drosophila caspase using purified recombinant proteins and an in vitro caspase assay. 2 μM of a DIAP1 fragment containing the BIR1 and BIR2 domains with intervening linker inhibited the caspase by 72 ± 1%. Removing either BIR domain reduced the inhibition to 39–45 ± 10%. Neither BIR domain alone inhibited the caspase even at 6 μM. However, 6 μM added BIR1 domain interfered with the 2 μM BIR1‐linker, reducing its caspase inhibition from 56 ± 1% to 30 ± 2%. An affinity chromatography binding assay detected direct BIR1 domain binding to the caspase. This suggests that the BIR1 interfered with the BIR1‐linker by binding to and blocking a critical BIR1‐binding site on the caspase. Thus, the BIR1 likely functions within DIAP1 by helping anchor the linker to the caspase active site. The BIR2 domain did not interfere with the linker‐BIR2 nor show direct binding to the caspase, indicating that its mechanism for enhancing caspase inhibition by DIAP1 must differ from that of the BIR1 domain. The role of the BIR2 as well as of other linker residues is being further investigated. Support or Funding Information This research was funded by a Kutztown University Research Grant to M. Junker and a Kutztown University BEARS grant and a Kutztown University Undergraduate Research Committee Grant to M. Storm. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .