GPIHBP1 is a membrane protein of endothelial cells that transports lipoprotein lipase (LPL), the key enzyme in plasma triglyceride metabolism, from the interstitial space to its site of action on the Show more
GPIHBP1 is a membrane protein of endothelial cells that transports lipoprotein lipase (LPL), the key enzyme in plasma triglyceride metabolism, from the interstitial space to its site of action on the capillary lumen. An intrinsically disordered highly negatively charged N-terminal domain of GPIHBP1 contributes to the interaction with LPL. In this work, we investigated whether the plethora of heparin-binding proteins with positively charged regions found in human plasma affect this interaction. We also wanted to know whether the role of the N-terminal domain is purely non-specific and supportive for the interaction between LPL and full-length GPIHBP1, or whether it participates in the specific recognition mechanism. Using surface plasmon resonance, affinity chromatography, and FRET, we were unable to identify any plasma component, besides LPL, that bound the N-terminus with detectable affinity or affected its interaction with LPL. By examining different synthetic peptides, we show that the high affinity of the LPL/N-terminal domain interaction is ensured by at least ten negatively charged residues, among which at least six must sequentially arranged. We conclude that the association of LPL with the N-terminal domain of GPIHBP1 is highly specific and human plasma does not contain components that significantly affect this complex. Show less
Pancreatic lipase (PNLIP) is a digestive enzyme that is a potential drug target for the treatment of obesity. A better understanding of its regulation mechanisms would facilitate the development of ne Show more
Pancreatic lipase (PNLIP) is a digestive enzyme that is a potential drug target for the treatment of obesity. A better understanding of its regulation mechanisms would facilitate the development of new therapeutics. Recent studies indicate that intestinal lipolysis by PNLIP is reduced by Angiopoietin-like protein 4 (ANGPTL4), whose N-terminal domain (nANGPTL4) is a known inactivator of lipoprotein lipase (LPL) in blood circulation and adipocytes. To elucidate the mechanism of PNLIP inhibition by ANGPTL4, we developed a novel approach, using isothermal titration calorimetry (ITC). The obtained results were compared with those of well-described inhibitors of PNLIP - ε-polylysine (EPL), (-)-epigallocatechin-3-gallate (EGCG) and tetrahydrolipstatin. We demonstrate that ITC allows to investigate PNLIP inhibition mechanisms in complex substrate emulsions and that the ITC-based assay is highly sensitive - the lowest concentration for quantification of PNLIP is 1.5 pM. Combining ITC with surface plasmon resonance and fluorescence measurements, we present evidence that ANGPTL4 is a lipid-binding protein that influences PNLIP activity through interactions with components of substrate emulsions (bile salts, phospholipids and triglycerides), and this promotes the aggregation of triglyceride emulsions similarly to the PNLIP inhibitors EPL and EGCG. In the absence of substrate emulsion, unlike in the case of LPL, ANGPTL4 did not induce the inactivation of PNLIP. Our data also prove that due to various interactions with components of substrate systems, the effect of a PNLIP inhibitor depends on whether its effect is measured in a complex substrate emulsion or in a simple substrate system. Show less