A thumbwheel mechanism for APOA1 activation of LCAT activity in HDL[S]

Abstract

APOA1 is the most abundant protein in HDL. It modulates interactions that affect HDL's cardioprotective functions, in part via its activation of the enzyme, LCAT. On nascent discoidal HDL, APOA1 comprises 10 α-helical repeats arranged in an anti-parallel stacked-ring structure that encapsulates a lipid bilayer. Previous chemical cross-linking studies suggested that these APOA1 rings can adopt at least two different orientations, or registries, with respect to each other; however, the functional impact of these structural changes is unknown. Here, we placed cysteine residues at locations predicted to form disulfide bonds in each orientation and then measured APOA1's ability to adopt the two registries during HDL particle formation. We found that most APOA1 oriented with the fifth helix of one molecule across from fifth helix of the other (5/5 helical registry), but a fraction adopted a 5/2 registry. Engineered HDLs that were locked in 5/5 or 5/2 registries by disulfide bonds equally promoted cholesterol efflux from macrophages, indicating functional particles. However, unlike the 5/5 registry or the WT, the 5/2 registry impaired LCAT cholesteryl esterification activity ( < 0.001), despite LCAT binding equally to all particles. Chemical cross-linking studies suggest that full LCAT activity requires a hybrid epitope composed of helices 5-7 on one APOA1 molecule and helices 3-4 on the other. Thus, APOA1 may use a reciprocating thumbwheel-like mechanism to activate HDL-remodeling proteins.

The Mechanism of Bisphenol A Atherogenicity Involves Apolipoprotein A-I Downregulation through NF-κB Activation

“…Treatment of hepatocytes with BPA increased~2.4-fold the activity of the construct (NF-κB) 3 -luc (p < 0.01), as presented in Figure 4B. As a control, we overexpressed IKKB or MEKK1 together with the construct (NF-κB) 3 -luc in HepG2 cells and we obtained an increase in the luciferase activity ( Figure 4B).…”

“…In parallel, we assessed the capacity of BPA to activate a promoter containing NF-κB binding sites ( Figure 4B). For this purpose, we transiently transfected hepatocytes with the construct (NF-κB) 3 -luc containing three copies of the NF-κB binding site cloned upstream of the minimal SV40 promoter and the luciferase reporter gene in pGL2-Promoter Vector (Promega). Treatment of hepatocytes with BPA increased~2.4-fold the activity of the construct (NF-κB) 3 -luc (p < 0.01), as presented in Figure 4B.…”

“…For this purpose, we transiently transfected hepatocytes with the construct (NF-κB) 3 -luc containing three copies of the NF-κB binding site cloned upstream of the minimal SV40 promoter and the luciferase reporter gene in pGL2-Promoter Vector (Promega). Treatment of hepatocytes with BPA increased~2.4-fold the activity of the construct (NF-κB) 3 -luc (p < 0.01), as presented in Figure 4B. As a control, we overexpressed IKKB or MEKK1 together with the construct (NF-κB) 3 -luc in HepG2 cells and we obtained an increase in the luciferase activity ( Figure 4B).…”

“…Initially, it was proposed that the α-helices of apoA-I are arranged around the circumference of discoidal HDL particles in the so-called "double belt model", but later other structural models of recombinant discoidal HDL were proposed, such as the "looped belt", the "solar flares", and the "double superhelix" model, as reviewed in [2]. Recently, it was revealed that the apoA-I rings on nascent discoidal HDL particles can adopt at least two helical orientations, influencing the activation of lecithin cholesterol acyltransferase (LCAT) in HDL [3]. ApoA-I is more than a structural scaffold that maintains lipid packaging, as apoA-I plays an important role in the transport of cellular cholesterol from the artery wall to the liver for catabolism and acts as a cofactor for LCAT, an enzyme responsible for the formation of most plasma cholesteryl esters [4].…”

Dynamics of murine brain protein synthesisin vivoidentify the hippocampus, cortex and cerebellum as highly active metabolic sites

“…Another key member of the apolipoprotein family is ApoA1 which is the major protein constituent of plasma HDL. In addition to high expression in several peripheral tissues including the liver and intestine, ApoA1 is also one of the most abundant apolipoproteins in CSF(cerebrospinal fluid) (Roheim et al, 1979, Harr et al, 1996 and also serves as an important cofactor on LCAT activation (Sorci-Thomas et al, 2009, Cooke et al, 2018. Indeed, our data indicated that ApoA1 is far more abundant than ApoA4 in the murine brain (emPAI value 33.8±13.1) while still displaying a high level of heavy labelling by pSIVOM analysis (average heavy/light abundance ratio 16.9±1.5).…”