The N-terminus of apolipoprotein A-V adopts a helix bundle molecular architecture

Previous studies of recombinant full-length human apolipoprotein A-V (apoA-V) provided evidence of the presence of two independently folded structural domains. Computer-assisted sequence analysis and limited proteolysis studies identified an N-terminal fragment as a candidate for one of the domains. C-Terminal truncation variants in this size range, apoA-V(1-146) and apoA-V(1-169), were expressed in Escherichia coli and isolated. Unlike full-length apoA-V or apoA-V(1-169), apoA-V(1-146) was soluble in neutral-pH buffer in the absence of lipid. Sedimentation equilibrium analysis yielded a weight-average molecular weight of 18811, indicating apoA-V(1-146) exists as a monomer in solution. Guanidine HCl denaturation experiments at pH 3.0 yielded a one-step native to unfolded transition that corresponds directly with the more stable component of the two-stage denaturation profile exhibited by full-length apoA-V. On the other hand, denaturation experiments conducted at pH 7.0 revealed a less stable structure. In a manner similar to that of known helix bundle apolipoproteins, apoA-V(1-146) induced a relatively small enhancement in 8-anilino-1-naphthalenesulfonic acid fluorescence intensity. Quenching studies with single-Trp apoA-V(1-146) variants revealed that a unique site predicted to reside on the nonpolar face of an amphipathic alpha-helix was protected from quenching by KI. Taken together, the data suggest the 146 N-terminal residues of human apoA-V adopt a helix bundle molecular architecture in the absence of lipid and, thus, likely exist as an independently folded structural domain within the context of the intact protein.     

The C terminus of apolipoprotein A-V modulates lipid-binding activity

Human apolipoprotein A-V (apoA-V) is a potent modulator of plasma triacylglycerol (TG) levels. To probe different regions of this 343-amino-acid protein, four single Trp apoA-V variants were prepared. The variant with a Trp at position 325, distal to the tetraproline sequence at residues 293-296, displayed an 11-nm blue shift in wavelength of maximum fluorescence emission upon lipid association. To evaluate the structural and functional role of this C-terminal segment, a truncated apoA-V comprising amino acids 1-292 was generated. Far UV circular dichroism spectra of full-length apoA-V and apoA-V-(1-292) were similar, with approximately 50% alpha-helix content. In guanidine HCl denaturation experiments, both full-length and truncated apoA-V yielded biphasic profiles consistent with the presence of two structural domains. The denaturation profile of the lower stability component (but not the higher stability component) was affected by truncation. Truncated apoA-V displayed an attenuated ability to solubilize l-alpha-dimyristoylphosphatidylcholine phospholipid vesicles compared with full-length apoA-V, whereas a peptide corresponding to the deleted C-terminal segment displayed markedly enhanced kinetics. The data support the concept that the C-terminal region is not required for apoA-V to adopt a folded protein structure, yet functions to modulate apoA-V lipid-binding activity; therefore, this concept may be relevant to the mechanism whereby apoA-V influences plasma TG levels.     

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3’UTRs (3’LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3’UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3’LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3’LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3’UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3''LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.     

Apolipoprotein A-V N-terminal Domain Lipid Interaction Properties in Vitro Explain the Hypertriglyceridemic Phenotype Associated with Natural Truncation Mutants

The N-terminal 146 residues of apolipoprotein (apo) A-V adopt a helix bundle conformation in the absence of lipid. Because similarly sized truncation mutants in human subjects correlate with severe hypertriglyceridemia, the lipid binding properties of apoA-V(1–146) were studied. Upon incubation with phospholipid in vitro, apoA-V(1–146) forms reconstituted high density lipoproteins 15–17 nm in diameter. Far UV circular dichroism spectroscopy analyses of lipid-bound apoA-V(1–146) yielded an α-helix secondary structure content of 60%. Fourier transformed infrared spectroscopy analysis revealed that apoA-V(1–146) α-helix segments align perpendicular with respect to particle phospholipid fatty acyl chains. Fluorescence spectroscopy of single Trp variant apoA-V(1–146) indicates that lipid interaction is accompanied by a conformational change. The data are consistent with a model wherein apoA-V(1–146) α-helices circumscribe the perimeter of a disk-shaped bilayer. The ability of apoA-V(1–146) to solubilize dimyristoylphosphatidylcholine vesicles at a rate faster than full-length apoA-V suggests that N- and C-terminal interactions in the full-length protein modulate its lipid binding properties. Preferential association of apoA-V(1–146) with murine plasma HDL, but not with VLDL, suggests that particle size is a determinant of its lipoprotein binding specificity. It may be concluded that defective lipoprotein binding of truncated apoA-V contributes to the hypertriglyceridemia phenotype associated with truncation mutations in human subjects.     

Characterization of apolipoprotein A-V structure and mode of plasma triacylglycerol regulation

PURPOSE OF REVIEW: Cardiovascular disease is the leading cause of death in the USA, and hypertriglyceridemia represents an independent risk factor contributing to its premature onset. Apolipoprotein (apo)A-V has been shown to be a potent regulator of plasma triacylglycerol. We highlight structural aspects of apoA-V and discuss recent findings that provide mechanistic insight into its function as a regulator of plasma triacylglycerol metabolism. RECENT FINDINGS: Recent findings indicate that apoA-V is comprised of two independently folded domains. Fluorescence spectroscopy and truncation analysis revealed that the carboxyl-terminal region functions in apoA-V lipid binding, consistent with its known association with plasma lipoproteins. An indirect triacylglycerol-modulating effect of apoA-V has been attributed to heparan sulfate proteoglycan binding, as confirmed by structural studies. Furthermore, apoA-V has been shown to interact with cell surface receptors, potentially facilitating lipoprotein particle endocytosis. SUMMARY: Several features of apoA-V, including extremely low plasma concentration, lack of correlation with plasma cholesterol levels despite its association with HDL, and insolubility at neutral pH in the absence of lipid, are unlike those of other exchangeable apolipoproteins. Current and future studies of apoA-V will help to shed light on the molecular basis whereby this protein functions to modulate plasma lipid homeostasis.