IAP: Antagonizing the Antagonist

For this article, Laura DeFrancesco interviewed David Vaux, principal research fellow, Walter and Eliza Hall Institute, Melbourne, Australia. Data from the Web of Science show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. A.M. Verhagen et al., "Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins," Cell, 102: 43-53, July 7, 2000. (Cited in 176 papers) Apoptosis, or programmed cell death, provides organisms a way to remove unwanted cells, such as during morphogenesis, or to defend against viral infection. Of course, certain molecules exist to prohibit apoptosis. One of these proteins, aptly named Inhibitor of Apoptosis Protein (IAP), was first found in viruses, which use them to keep host cells alive while the virus replicates and propagates. These IAPs interfere with key effectors of apoptosis, the family of proteases known as caspases, which, when activated, literally digest the cell from the inside out. But the story doesn't end there: antagonists exist to antagonize the IAPs; these molecules send the cell back down the apoptosis pathway. For example, three IAP antagonists, Reaper, Grim and HID, have been identified in Drosophila. These proteins promote cell death by binding to the IAPs, keeping them from suppressing caspase activity. However, no similar IAP antagonists had been identified in mammals until the labs of David Vaux, Xiaodong Wang, and the editors of Cell crossed paths. Vaux's lab was one of the first to identify mammalian homologs of the baculoviral IAPs (XIAP, cIAP1 and cIAP2). Vaux, principal research fellow at the Walter and Eliza Hall Institute, Melbourne, Australia, hypothesized that, like their Drosophila counterparts, these IAPs could be controlled by pro-apoptotic proteins. When homologs for Reaper, Grim or HID couldn't be found by searching genomic databases, researchers took a different approach. Anne Verhagen, a postdoc in Vaux's lab, expressed XIAP in mammalian cells and immunoprecipitated it from cell lysates along with associated proteins. Several novel proteins were found by running the coimmunoprecipitated proteins on two-dimensional gels. Using nanoelectrospray tandem mass spectrometry, Lisa Connolly, a research assistant in the lab of Vaux's collaborator, Richard Simpson, obtained amino acid sequences on four IAP binding proteins, one of which was named DIABLO (for Direct IAP binding protein of low Pi) and another HtrA2 (also known as Omi). ©2002 Cell PressLocalizing DIABLO: The N-terminal 53 amino acids of DIABLO were fused to GFP, and the fusion protein was expressed in 293T cells (left) or NT2cells (right). Yellow indicates co-localization of mitotracker red and GFP fluorescence. Working independently on apoptosis in HeLa cell lysates, Wang, a Howard Hughes Medical Institute investigator at the University of Texas Southwestern Medical Center at Dallas, identified several components of the cell death mechanism (including Apaf-1, caspase 9, cytochrome c, and DFF) using a classical biochemical approach. He found an additional activity in cell lysates that promoted efficient caspase activation. Purifying this activity led his team to identify a protein they termed Smac (for second mitochondrial activator of cell death), so-called because the protein had a mitochondrial targeting sequence, and antibodies showed it localized to the mitochondria in healthy cells. The two groups independently submitted papers to Cell. The reviewers requested revisions, and then asked for more work because they still weren't satisfied. However, in an unusual move, the editors suggested that the two labs exchange their papers to see, with each other's help, if they could reach solutions. In doing so, the two groups realized that they had purified the same protein. Wang hadn't determined how Smac promotes apoptosis, but after learning that Vaux had found that it was an IAP binding protein, he looked for and confirmed IAP binding activity by Smac. And while Vaux's group knew that DIABLO was processed, they didn't know this occurred in the mitochondria. Using antibody provided by Wang, they confirmed its presence in the mitochondria of healthy cells, but released into the cytosol by UV irradiation, an apoptotic inducer. Vaux remembers, "Wang was surprised and delighted to learn from our paper that DIABLO/Smac functioned by binding to IAPs. From reading his paper we were blown away to learn that the reason DIABLO/Smac is processed is that it is targeted to the mitochondria, where its amino-terminus is removed." Laura DeFrancesco (defrancesco1@earthlink.net) is a freelance writer in Pasadena, Calif. 1. C. Du et al., "Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition," Cell, 102:33-42, July 7, 2000. (Cited in 196 papers)

IAP: Antagonizing the Antagonist

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