MacKenzie, A.,Farahani, Reza.2009-03-192009-03-1919981998Source: Dissertation Abstracts International, Volume: 59-10, Section: B, page: 5307.9780612324442http://hdl.handle.net/10393/4469http://dx.doi.org/10.20381/ruor-13872IAPs (inhibitor of apoptosis proteins) are a new family of proteins capable of interacting with the components of the cell death machinery thus, inhibiting apoptosis. IAPs contain two or three 70 amino acid domains in their N-terminus called BIR (baculovirus IAP repeat) domains and a C-terminus $\rm C\sb3HC\sb4$ type RING zinc finger. Four human genes called naip, xiap (X-linked IAP), hiap-1 and hiap-2 (human IAP-1 and 2) have been cloned in the past several years. Although all of these genes exhibit some degree of apoptosis suppression capability, it is not clear which region of the protein is responsible for the anti-apoptotic function. miap-3, the mouse homologue of xiap was cloned by cDNA and genomic library screening and characterized. miap-3 has a coding sequence of 1491 bp translating into a protein with 496 amino acids and predicted size of 55 kDa. The mouse gene has 898 and 94% homology with human xiap at DNA and amino acid levels respectively and has been assigned to mouse chromosome X in the A3-A5 region. Northern blot analysis showed that miap-3 expresses as an over 8 kb message in all tissues examined indicating large 3' and/or 5' UTRs. The capacity of various miap-3 and xiap constructs to protect cells against apoptotic insult was assessed. Expression of full-length xiap or miap-3 cDNAs in CHO cells conferred only modest protection above the level observed for controls (i.e. CHO cells expressing plasmids with no insert which demonstrate 15-20% cell survival level). Expression of the RING zinc finger provides no protection to CHO cells. In contrast, expression of a construct encoding only the BIR domains (and not the RING zinc finger) resulted in four fold enhancement of survival after 72 hours of serum deprivation. It was of interest to investigate the cellular distribution of IAPs and any possible change in their distribution pattern under normal and apoptotic conditions. Immunofluorescent microscopy performed on CHO cells expressing xiap revealed that under normal conditions, Xiap concentrates in the cytoplasm. During apoptosis, however, Xiap appears in the nucleus as well. Change of localization pattern during apoptosis was further investigated by immunofluorescent studies. These revealed that full-length Xiap and Miap-3 as well as RING zinc-finger proteins concentrate in the cytoplasm. In contrast, BIR domains appeared to localize both in the cytoplasm and nucleus, suggesting that BIR domains may be cleaved and translocalized to the nucleus during apoptosis. The anti-apoptotic effect of IAPs during apoptosis may depend on the cleavage and/or translocation of BIR domains to the nucleus. (Abstract shortened by UMI.)135 p.Biology, Molecular.Thesis