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How does prolyl hydroxylase-3 induce apoptosis in neuronal cells ?

Prolyl hydroxylase-3 (PHD3) is an important cellular oxygen-sensor that hydroxylates substrates in an oxygen-dependent manner. PHD3 activity is required during development for the induction of apoptosis in sympathoadrenal progenitor cells. Human PHD3 localizes to the nucleus and cytoplasm, while the highly conserved rat orthologue SM20 has been reported to localize to mitochondria. Given that protein localization is likely to determine the ability of PHD3 enzyme to hydroxylate specific substrate proteins, the aim of the project was to reevaluate the localization of both endogenous and transfected SM-20. Using Immunoblotting and immunofluorescence, it was found that despite presence of an N-terminal mitochondrial targeting sequence (MTS), transfected SM-20 localized to both nucleus and mitochondria. Human PHD3 lacks a MTS and localizes to nucleus and cytosol, but not to mitochondria. Localization analysis of endogenous SM20 in rat cells and tissue showed no clear evidence of mitochondrial localization. Interestingly, inclusion of the 5’untranslated region (5’UTR) in the SM-20 expression plasmid prevented the translation of the MTS, thus explaining the partial nuclear localization. It is hypothesized that the 5’UTR, via secondary structure in the mRNA, causes utilization of a second start codon downstream of the MTS, which corresponds to the start codon in the human PHD3. INTRODUCTION Pheochromocytoma is a cancer of the adrenal medulla, and these tumors comprise of chromaffin cells derived from the sympathetic neural progenitor cells (Lee et al., 2005). Appropriate removal of neuronal progenitor cells found in adrenal medulla, via apoptosis during development is important to prevent pheochromocytoma. In the absence of nerve growth factor, the cells undergo apoptosis that is dependent on c-Jun and its transcriptional target, PHD3 (Lee et al., 2005). PHD3, like its homologues PHD1 and PHD2, has the ability to hydroxylate HIF-1α in vitro. However, it has been shown that in vivo only the knockdown of PHD2, but not PHD3 or PHD1, causes an increase in HIF-1α (Berra et al., 2003). The physiological role of PHD3 is therefore not to regulate HIF-1α and may play an important role in regulating neural apoptosis. The human PHD3 orthologue was found in nucleus and cytoplasm (Metzen et al., 2003) whereas the rat orthologue was reported to be localized to mitochondria (Lipscomb et al., 2001). Interestingly, despite the differences in subcellular localization of human and rat PHD3, both have been reported to induce apoptosis in neural cells (Lipscomb et al., 1999). The subcellular localization of pro-apoptotic proteins is important for cell death. This is demonstrated in mitochondrial proteins such as cytochrome c, apoptosis inducing factor and endonuclease G. 1 Student 2 Supervisor Given that both human PHD3 and the rat homologue SM-20 can induce apoptosis in neural cells, but appear to be localized to different intracellular compartments, the aim of this project was to reevaluate the localization of both endogenous and transfected forms of PHD3 and SM20. A further goal was to characterize the mechanisms that govern the different localizations of human and rat orthologues of PHD3. MATERIALS AND METHODS Rat SM-20 cDNA was a gift from Dr. R. S. Freeman. Methods used in the experiments include cell culture and transfection techniques, immunoblotting, immunofluorence and plasmid construct of SM-20-V5, 5’UTR-SM-20, 5’UTR-SM-20 ATG codon mutants, PHD3-HA and untagged PHD3. RESULTS AND DISCUSSION Transfected SM-20 is localised to both mitochondria and nucleus To confirm that the rat SM-20 localizes to the mitochondria, SM-20 cDNA was PCR amplified and transfected into NIH-3T3 cells western blot analysis was carried out on fractionated nuclear and cytoplasmic fractions. Results showed that transfected SM-20 can translocate partially to the nucleus. Smaller V5-immunoreactive bands were also observed and may represent forms of SM20 after cleavage by the mitochondrial signal peptidase. To confirm the unexpected nuclear localization, NIH-3T3 and HEK293 cells were transfected with SM-20-V5 and analyzed by immunofluorescence. Apart from mitochondrial localization, nuclear localization was also detected (Fig. 1). We also determined the localization of transfected human PHD3-HA. Localization to both nucleus and cytosol, where it showed granular staining, is consistent with previous reports (Metzen et al., 2003). However, there was no mitochondrial staining, consistent with the absence of a MTS human PHD3. Figure 1: Immunofluorence of transfected SM20-V5 in NIH-3T3 and HEK293. NIH-3T3