ABCA transporters and associated genes in lipid metabolism

The ATP-binding cassette transporters A1 (ABCA1) and A7 (ABCA7) are inversely regulated under loading and deloading conditions and we were interested in protein/protein interactions of these transporters. The main topic was to identify, verify and characterize these interactions. In case of ABCA7, the yeast two-hybrid approach led to false positive results, and therefore we focused on the expression and regulation of the two published isoforms. Albeit having similar tissue distribution, the two isoforms show different regulation by stimulation, indicating the use of alternative promoters. This caused us to analyze the promoter of the long isoform in more detail and we were able to map the core promoter region. Analyzes of the putative alternative promoter might enable us to understand the regulatory differences between both isoforms. Due to our initial experiments and data from the literature, we would suggest different functions for the two isoforms of ABCA7. Isoform I localizes to the plasma membrane and is upregulated during monocyte to macrophage differentiation, indicating involvment in phagocytic processes. In contrast, isoform II localizes to intracellular membranes and is upregulated together with phosphatase and tensin homolog in HL-60 cells and during terminal keratinocyte differentiation, pointing to a function in the autophagic pathway. Further experiments are necessary to confirm this hypothesis. ABCA1 regulates plasma high-density lipoprotein levels and its cellular function is mediated through various protein interactions. One aim was to identify and confirm new ABCA1 C-terminus interacting PDZ proteins. By different methods seven new PDZ candidates were identified, and four of them, namely GAIP C-terminus-interacting protein1 (GIPC1), Tax1 (human T-cell leukemia virus type I) binding protein 3 (TAX1BP3), Scribble, and Membrane associated guanylate kinase, WW and PDZ domain containing 3 (MAGI3) were confirmed to bind to ABCA1 by different methods. Together with the known ABCA1 interacting PDZ proteins and their different tissue expression this may indicate that PDZ proteins are able to regulate ABCA1 trafficking and/or function in a tissue or even cell compartment specific manner. The cytosolic molybdo-flavoenzyme aldehyde oxidase 1 (AOX1), known as xenobiotic metabolizing enzyme, was previously suggested as an ABCA1 interacting protein. In this work it was shown that knock-down of AOX1 by siRNA significantly reduced ABCA1-dependent lipid-efflux and enhanced phagocytic uptake in HepG2 cells. ABCA1 and AOX1 were found coexpressed in certain human cell types, namely hepatocytes, kidney proximal tubular epithelial cells, Leydig cells and cells of the adrenal cortex. Deregulation of ABCA1 and AOX1 in hepatocellular- and renal cell-carcinomas was observed, suggesting that AOX1, perhaps in context with ABCA1, might be used as tumor marker. The involvement of AOX1 in metabolism of ethanol and xenobiotics is of particular interest, as it might link ABCA1 to detoxification, a process in which many ABC-transporters are involved. Finally, as ABCA1 through its interaction with Fas-associated via death domain (FADD) might influence apoptosis, we searched for apoptotic genes regulated by modified lipoproteins in a similar manner as ABCA1. Uptake of modified lipoproteins by macrophages causes foam cell formation and promotes the development of atherosclerotic lesions. Atherogenic lipoproteins exert cytotoxic effects and induce necrosis or apoptosis under certain conditions but may also enhance macrophage survival. GeneChip experiments were performed to identify genes that are regulated in macrophages treated with enzymatically modified low-density lipoprotein (E-LDL). Expression of TOSO, protecting cells against CD95- or tumor necrosis factor-mediated apoptosis, was found induced by E-LDL. Concomitantly, reduced apoptosis was detected in E-LDL loaded macrophages compared to oxidized LDL incubated cells or controls. Abundance of the caspase inhibitor FLICE-like inhibitory protein long form (FLIPL) was suggested to mediate the antiapoptotic properties of TOSO; however, FLIPL expression is induced neither in E-LDL laden macrophages nor in COS-7 cells overexpressing TOSO. E-LDL represents coreless liposome like particles and may be taken up by Fc- and complement-receptor dependent phagocytosis. Internalization of phagobeads by monocytes and macrophages upregulates TOSO but phagocytosis was not altered by TOSO in COS-7 cells. These data indicate that E-LDL-generated foam cells are protected from cell death most likely through the expression of TOSO by a mechanism independent of FLIPL. ABCA1 and ABCA7 may be related but exert distinct functions in the lipid-efflux/apoptosis/autophagy complex. While the function of ABCA1 is highly modulated by protein interactions, ABCA7 might be more affected by transcriptional regulation