Swapping the N- and C-terminal domains of human apolipoprotein E3 and AI reveals insights into their structure/activity relationship

Apolipoprotein (apo) E3 and apoAI are exchangeable apolipoproteins that play a dominant role in regulating plasma lipoprotein metabolism. ApoE3 (299 residues) is composed of an N-terminal (NT) domain bearing a 4-helix bundle and a C-terminal (CT) domain bearing a series of amphipathic α-helices. ApoAI (243 residues) also comprises a highly helical NT domain and a less structured CT tail. The objective of this study was to understand their structural and functional role by generating domain swapped chimeras: apoE3-NT/apoAI-CT and apoAI-NT/apoE-CT. The bacterially overexpressed chimeras were purified by affinity chromatography and their identity confirmed by immunoblotting and mass spectrometry. Their α-helical content was comparable to that of the parent proteins. ApoE3-NT/apoAI-CT retained the denaturation profile of apoE3 NT domain, with apoAI CT tail eliciting a relatively unstructured state; its lipid binding ability improved dramatically compared to apoE3 indicative of a significant role of apoAI CT tail in lipid binding interaction. The LDL receptor interaction and ability to promote ABCA1-mediated cholesterol efflux of apoE3-NT/apoAI-CT was comparable to that of apoE3. In contrast, apoAI-NT/apoE-CT elicited an unfolding pattern and lipid binding ability that were similar to that of apoAI. As expected, DMPC/apoAI-NT/apoE-CT discoidal particles did not elicit LDLr binding ability, and promoted SR-B1 mediated cellular uptake of lipids to a limited extent. However, apoAI-NT/apoE-CT displayed an enhanced ability to promote cholesterol efflux compared to apoAI, indicative of a significant role for apoE CT domain in mediating this function. Together, these results indicate that the functional attributes of apoAI and apoE3 can be conferred on each other and that NT-CT domain interactions significantly modulate their structure and function

The Interaction of ApoA-I and ABCA1 Triggers Signal Transduction Pathways to Mediate Efflux of Cellular Lipids

Reverse cholesterol transport (RCT) has been characterized as a crucial step for antiatherosclerosis, which is initiated by ATP-binding cassette A1 (ABCA1) to mediate the efflux of cellular phospholipids and cholesterol to lipid-free apolipoprotein A-I (apoA-I). However, the mechanisms underlying apoA-I/ABCA1 interaction to lead to the lipidation of apoA-I are poorly understood. There are several models proposed for the interaction of apoA-I with ABCA1 as well as the lipidation of apoA-I mediated by ABCA1. ApoA-I increases the levels of ABCA1 protein markedly. In turn, ABCA1 can stabilize apoA-I. The interaction of apoA-I with ABCA1 could activate signaling molecules that modulate posttranslational ABCA1 activity or lipid transport activity. The key signaling molecules in these processes include protein kinase A (PKA), protein kinase C (PKC), Janus kinase 2 (JAK2), Rho GTPases and Ca2+, and many factors also could influence the interaction of apoA-I with ABCA1. This review will summarize these mechanisms for the apoA-I interaction with ABCA1 as well as the signal transduction pathways involved in these processes

Genome duplication in a protein rich environment

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