Abstract
During apoptosis several mitochondrial proteins are released. Some of them participate in caspase-independent nuclear DNA degradation, especially apoptosis-inducing factor (AIF) and endonuclease G (endoG). Another interesting protein, which was expected to act similarly as AIF due to the high sequence homology with AIF is AIF-homologous mitochondrion-associated inducer of death (AMID). We studied the structure, cellular localization, and interactions of several proteins in silico and also in cells using fluorescent microscopy. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the lipid membranes. Bioinformatic predictions were conducted to analyze the interactions of the studied proteins with each other and with other possible partners. We conducted molecular modeling of proteins with unknown 3D structures. These models were then refined by MolProbity server and employed in molecular docking simulations of interactions. Our results show data acquired using a combination of modern in silico methods and image analysis to understand the localization, interactions and functions of proteins AMID, AIF, endonuclease G, and other apoptosis-related proteins.
Highlights
During some forms of apoptosis the mitochondrial outer membrane becomes depolarized and partially permeable to proteins
Most of the analyzed proteins have known cellular location, we employed these server tools to validate the accuracy of a final prediction summary based on the combined results of these tools (Table 1). These results clearly indicate that the predicted cellular location of endonuclease G (endoG) and apoptosisinducing factor (AIF) is in the mitochondrion, cyclophilin A and HSP70-1 in the cytoplasm, and DNA topoisomerase II α in the nucleus
We clearly show that sequence homology of AIF and AMID is restricted to Ndh conserved domain, that corresponds to oxidoreductase function and not to apoptotic function of AIF, which was shown to reside in large C-terminal part of AIF sequence, which is totally missing in AMID sequence (Fig. 1A) [54]
Summary
During some forms of apoptosis the mitochondrial outer membrane becomes depolarized and partially permeable to proteins. This results in a massive nonspecific release of hydrophilic proteins from the intermembrane space into the cytoplasm [1]. Among these proteins are apoptosisinducing factor (AIF) and endonuclease G (endoG). The release of these proteins results in activation of the apoptotic caspases, degradation of nuclear DNA, and cell death [2,3]. EndoG, AIF and AMID have all been found to influence chromatin changes during apoptosis [6]
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