Identification and characterization of ABCA1-interactive proteins and their relevance to atherosclerosis

The ATP-binding cassette transporter 1 (ABCA1) has recently been identified as a major regulator of systemic HDL. The main focus of this work was to identify and further characterize ABCA1-interactive proteins. A Yeast-Two-Hybrid screening with the last 144 amino acids of the ABCA1 C-terminus as bait was performed. Our results indicate that ABCA1 forms a heteromeric complex with each of: Fas-associated death domain (FADD), beta2-syntrophin, syntaxin 13 and flotillin-1, indicating a role for ABCA1 beyond mere reverse cholesterol transport. The interactions of the identified putative ABCA1-associated proteins were confirmed by independent approaches. In addition, functional studies were performed to elucidate the physiological consequences of these protein complexes. The finding that FADD directly interacts with ABCA1 is surprising and links cellular HDL metabolism with a protein mainly described in the context of death receptor-induced apoptosis, since FADD plays a well-established role in transduction of apoptotic signals and other cellular processes. The FADD/ABCA1 interaction may indicate an anti-apoptotic ABCA1 function independent from a suggested phosphatidylserine translocase activity. The C-terminal sequence of ABCA1 has been described previously as a binding motif for the PDZ domains of syntrophin. We showed that ABCA1 binds beta2-syntrophin, a PDZ protein that acts as a general adaptor of membrane proteins to the actin cytoskeleton via utrophin, a protein highly homologous to dystrophin. The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC transporter family, has been reported to interact with syntaxin 1A. Thereupon, we investigated the interaction of ABCA1 with syntaxins and identified syntaxin 13 as a direct ABCA1-interactor in this family. Syntaxin 13, through its interacting protein pallidin (syntaxin 13 interacting protein), is linked to Hermansky-Pudlak syndrome (HPS) and Chediak-Higachi syndrome (CHS). These lysosomal storage diseases are associated with pulmonary fibrosis and prolonged bleeding caused by defects in the Adapter protein-3 complex (AP-3). Recent publications show that ABCA1 mutations in Tangier patients are related to impaired platelet activation due to the presence of ABCA1, syntaxin 13 and syntaxin 13-interacting protein in the AP-3 pathway. Moreover, ABCA1 deficiency leads to enhanced phagocytosis in Tangier macrophages and fibroblasts, related to disturbances of the AP-3 pathway. The enhanced uptake of apoA-I by endocytic and phagocytic ingestion may also explain the increased catabolism of apoA-I in Tangier patients. Syntaxin 13 deficiency causes ABCA1 protein degradation and therefore syntaxin 13 may be important in ABCA1 maturation and vesicular transport. This finding links ABCA1 to a potential regulator of phagocytosis indicating that both the phagosomal and lysosomal compartment may be involved in ABCA1-dependent choline-phospholipid efflux. The integral membrane protein flotillin-1 was also found to interact with ABCA1. Overexpression of flotillin-1 enhances filipodia formation, linking ABCA1 to migration and cytoskeletal changes. In the second part of the work human monocyte-derived macrophages were loaded with differently modified atherogenic LDLs, and subsequently deloaded by HDL3 in order to ascertain differential gene regulation within lipid-related pathways. This characterization was performed by Affymetrix U133a gene chips. Our results indicate that Ox-LDL induces less cytoplasmic cholesteryl ester accumulation than E-LDL, a result of the chemical alterations during oxidation. We further showed that cholesterol efflux influences the gene expression of a substantial portion of structural elements of the large and small ribosomal subunits. Deloading of E-LDL, but not Ox-LDL, loaded macrophages led to a distinguished upregulation of genes in the polymerase-II complex, indicating a close relationship between E-LDL, taken up outside clathrin coated pits by phagocytosis, and the control mechanisms at the transcriptional level. This E-LDL specific effect continues more downstream in the translation machinery represented by the 40S and 60S ribosomal genes as well as in the proteasomal degradation machinery where unneeded proteins are promptly degraded. In addition, ATP synthases were also upregulated during HDL3-induced deloading of E-LDL, but not Ox-LDL, loaded macrophages. In addition, LDL-receptor mediated cholesterol uptake downregulates intracellular cholesterol synthesis by inhibition of HMGCoA reductase and inhibition of LDL-receptor by feedback mechanism. This cholesterol sensitive regulatory system leads to subsequent deloading of E-LDL whereas Ox-LDL is trapped inside the cell. Thus, certain modification types of lipoproteins may indirectly control cellular translation machinery