Genomic organization and characterization of the human inhibitor of apoptosis genes hiap1 and hiap2.
|Title:||Genomic organization and characterization of the human inhibitor of apoptosis genes hiap1 and hiap2.|
|Authors:||Young, Sean Steven.|
|Abstract:||Evolution from single celled to multicellular and complex organisms brings with it several challenges. Chief among these is the necessity to control cellular proliferation, differentiation and cell death. These controls ensure the proper development of anatomical structures and the cellular homeostasis of the organism. Multicellularity also entails the active elimination of cells which have outlived their usefulness or have become malignant by virtue of being oncogenic, microbially infected or otherwise dangerous. Although initiating programmed cell death, or apoptotis, is catastrophic to the affected cell, failure to do so may be detrimental to the viability of the organism as a whole. Ultimately most, if not all, apoptotic death results from the activation of a series of proteases. These powerful proteases, termed caspases, orchestrate the regulated disassembly of the apoptotic cell. In order to ensure the capacity to inhibit or prevent the inappropriate induction of cell death, several negative regulatory mechanisms have evolved. Quite recently, it was found that a novel class of anti-apoptotic proteins are direct caspase inhibitors. The mammalian inhibitor of apoptotis (IAP) family is comprised of, minimally, four core members, all of which contain three protein-protein interaction domains termed BIR domains (Baculoviral IAP repeat). It is through these domains, originally described in the baculoviral counterpart of the mammalian IAPs, that their interaction with caspases is mediated. Two of the mammalian members, HIAP1 and HIAP2, are the subject of the following investigations. First the complete genomic organization of both hiap1 and hiap2 was elucidated. Results from this study indicated that both genes lie in tandem (head to tail) on the genome (at 11q23) separated by approximately 7 kbp. Furthermore, it was revealed that it is likely that these genes, as well as that of xiap, arose by gene duplication. By comparison to the murine organization, it was also determined that these duplication events occurred prior to the divergence of the species'. It was furthermore found that both genes possess unusually long 5'UTR's. Although the presence of several possible initiation codons within of their respective 5'UTR's would preclude translation initiation by traditional scanning, no evidence was found to support the presence of a standard internal ribosome entry site (IRES) upstream of either open reading frame. Studies into the transcriptional regulation of these genes found that although hiap2 transcription was unresponsive to a variety of stimuli, that of hiap1 was markedly enhanced by agents which activate NF-kappaB. Analysis of the 5' region of hiap1 has revealed the presence of two conserved putative NF-kappaB binding sites as well as an NF-IL-6 (CIEBPP) site and a putative NF-kappaB negative regulatory element. Gel shift analysis has shown that all are able to bind their respective factors although reporter gene analysis coupled with site directed mutagenesis has shown that only the NF-kappaB and NF-IL6 sites are functional under the conditions examined. Partial characterization of a naturally occurring hiap1/hiap2 double knockout cell line derived from the Burkitt's lymphoma line BJAB was also carried out. By comparing the effect of TNFalpha on both the derivative and parental line, it was found that the presence of these genes alleviated the negative impact of TNFalpha on cellular proliferation. Furthermore, transient re-introduction of hiap1 into the derivative line or anti-sense hiap1 into the parental line partially reversed their phenotype with respect to TNFalpha response.|
|Collection||Thèses, 1910 - 2010 // Theses, 1910 - 2010|