Bioinformatic predictions and image analysis of localization and interactions of endonuclease G, AIF, and AMID in human cells
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| Year of publication | 2007 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | During apoptosis several mitochondrial proteins are released. They participate in caspase-independent nuclear DNA degradation, namely apoptosis-inducing factor (AIF, also PDCD8 or AIFM1) and endonuclease G. Another interesting protein, which is not located inside mitochondria, was expected to act similarly as AIF due to high sequence homology with AIF. This protein is AIF-homologous mitochondrion-associated inducer of death (AMID, also PRG3 or AIFM2). We studied the cellular localization and colocalization of proteins AIF, endonuclease G and AMID experimentally using designed mammalian expression vectors, which carry genes encoding the proteins of interest fused to the fluorescent proteins and using bioinformatic predictions, that analyze the amino acid sequence of the proteins with various algorithms. We also designed and applied the novel method of single-cell image analysis of the translocation of the fluorescent proteins into the nucleus. We confirmed the colocalization of AIF and endonuclease G in the mitochondria of human cells. We also analyzed their translocation from mitochondria to the nucleus during apoptosis. AMID was found to be cytoplasmic protein, bound to various cellular surfaces from their cytoplasmic side. Overexpression of fusion protein AMID-HcRed-tandem was not lethal to the cells or mitochondria. We did not observe its translocation into the nucleus during staurosporine-induced apoptosis. The possible role of AMID in apoptosis was not observed. Bioinformatic predictions and time-lapse FRET experiments were conducted to analyze the interactions of the studied proteins in living and fixed human cells. Our results contribute to the comprehension of localization, interactions and functions of AMID, AIF, and endonuclease G in human cells. |
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