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Apolipoprotein AV (APOA5) lowers plasma triglyceride (TG) levels by binding to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppressing its capacity to inhibit lipoprotein lipase (LPL) catalytic activity and its ability to detach LPL from binding sites within capillaries. However, the sequences in APOA5 that are required for suppressing ANGPTL3/8 activity have never been defined. A clue to the identity of those sequences was the presence of severe hypertriglyceridemia in two patients harboring an APOA5 mutation that truncates APOA5 by 35 residues ("APOA5Δ35"). We found that wild-type (WT) human APOA5, but not APOA5Δ35, suppressed ANGPTL3/8's ability to inhibit LPL catalytic activity. To pursue that finding, we prepared a mutant mouse APOA5 protein lacking 40 C-terminal amino acids ("APOA5Δ40"). Mouse WT-APOA5, but not APOA5Δ40, suppressed ANGPTL3/8's capacity to inhibit LPL catalytic activity and sharply reduced plasma TG levels in mice. WT-APOA5, but not APOA5Δ40, increased intracapillary LPL levels and reduced plasma TG levels in Apoa5 -/- mice (where TG levels are high and intravascular LPL levels are low). Also, WT-APOA5, but not APOA5Δ40, blocked the ability of ANGPTL3/8 to detach LPL from cultured cells. Finally, an antibody against a synthetic peptide corresponding to the last 26 amino acids of mouse APOA5 reduced intracapillary LPL levels and increased plasma TG levels in WT mice. We conclude that C-terminal sequences in APOA5 are crucial for suppressing ANGPTL3/8 activity in vitro and for regulating intracapillary LPL levels and plasma TG levels in vivo., Competing Interests: Competing interests statement:S.G.Y. is on the scientific advisory board of Kyttaro; he has received consulting fees from Kyttaro and holds stock in that company. All authors from Eli Lilly & Co. own stock in the company.

The lipolytic processing of triglyceride-rich lipoproteins (TRLs) by lipoprotein lipase (LPL) is crucial for the delivery of dietary lipids to the heart, skeletal muscle, and adipose tissue. The processing of TRLs by LPL is regulated in a tissue-specific manner by a complex interplay between activators and inhibitors. Angiopoietin-like protein 4 (ANGPTL4) inhibits LPL by reducing its thermal stability and catalyzing the irreversible unfolding of LPL's α/β-hydrolase domain. We previously mapped the ANGPTL4 binding site on LPL and defined the downstream unfolding events resulting in LPL inactivation. The binding of LPL to glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 protects against LPL unfolding. The binding site on LPL for an activating cofactor, apolipoprotein C2 (APOC2), and the mechanisms by which APOC2 activates LPL have been unclear and controversial. Using hydrogen-deuterium exchange/mass spectrometry, we now show that APOC2's C-terminal α-helix binds to regions of LPL surrounding the catalytic pocket. Remarkably, APOC2's binding site on LPL overlaps with that for ANGPTL4, but their effects on LPL conformation are distinct. In contrast to ANGPTL4, APOC2 increases the thermal stability of LPL and protects it from unfolding. Also, the regions of LPL that anchor the lid are stabilized by APOC2 but destabilized by ANGPTL4, providing a plausible explanation for why APOC2 is an activator of LPL, while ANGPTL4 is an inhibitor. Our studies provide fresh insights into the molecular mechanisms by which APOC2 binds and stabilizes LPL-and properties that we suspect are relevant to the conformational gating of LPL's active site.

The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL's α/β-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL's catalytic site, including β2, β3-α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on β3-α3 and progress to β5 and β4-α4, ultimately leading to the irreversible unfolding of regions that form LPL's catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL's α/β-hydrolase domain ( T m of 34.8 °C). GPIHBP1 binding dramatically increases LPL stability ( T m of 57.6 °C), while ANGPTL4 lowers the onset of LPL unfolding by ∼20 °C, both for LPL and LPL•GPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 °C but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism., Competing Interests: The authors declare no competing interest.

LPL is essential for intravascular lipid metabolism and is of high medical relevance. Since LPL is notoriously unstable, there is an unmet need for a robust expression system producing high quantities of active and pure recombinant human LPL (hLPL). We showed previously that bovine LPL purified from milk is unstable at body temperature (T m is 34.8°C), but in the presence of the endothelial transporter glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), LPL is stabile (T m increases to 57.6°C). Building on this information, we now designed an expression system for hLPL using Drosophila Schneider 2 cells grown in suspension at high cell density and at an advantageous temperature of 25°C. We cotransfected Schneider 2 cells with hLPL, lipase maturation factor 1, and soluble GPIHBP1 to provide an efficient chaperoning and stabilization of LPL in all compartments during synthesis and after secretion into the conditioned medium. For LPL purification, we used heparin-Sepharose affinity chromatography, which disrupted LPL-GPIHBP1 complexes causing GPIHBP1 to elute with the flow-through of the conditioned media. This one-step purification procedure yielded high quantities of pure and active LPL (4-28 mg/l). Purification of several hLPL variants (furin cleavage-resistant mutant R297A, active-site mutant S132A, and lipid-binding-deficient mutant W390A-W393A-W394A) as well as murine LPL underscores the versatility and robustness of this protocol. Notably, we were able to produce and purify LPL containing the cognate furin cleavage site. This method provides an efficient and cost-effective approach to produce large quantities of LPL for biophysical and large-scale drug discovery studies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Lipoprotein lipase (LPL) plays a major role in the lipid homeostasis mainly by mediating the intravascular lipolysis of triglyceride rich lipoproteins. Impaired LPL activity leads to the accumulation of chylomicrons and very low-density lipoproteins (VLDL) in plasma, resulting in hypertriglyceridemia. While low-density lipoprotein cholesterol (LDL-C) is recognized as a primary risk factor for atherosclerosis, hypertriglyceridemia has been shown to be an independent risk factor for cardiovascular disease (CVD) and a residual risk factor in atherosclerosis development. In this review, we focus on the lipolysis machinery and discuss the potential role of triglycerides, remnant particles, and lipolysis mediators in the onset and progression of atherosclerotic cardiovascular disease (ASCVD). This review details a number of important factors involved in the maturation and transportation of LPL to the capillaries, where the triglycerides are hydrolyzed, generating remnant lipoproteins. Moreover, LPL and other factors involved in intravascular lipolysis are also reported to impact the clearance of remnant lipoproteins from plasma and promote lipoprotein retention in capillaries. Apolipoproteins (Apo) and angiopoietin-like proteins (ANGPTLs) play a crucial role in regulating LPL activity and recent insights into LPL regulation may elucidate new pharmacological means to address the challenge of hypertriglyceridemia in atherosclerosis development. [ABSTRACT FROM AUTHOR]