Elevated plasma triglycerides are a risk factor for coronary artery disease, which is the leading cause of death worldwide. Lipoprotein lipase (LPL) reduces triglycerides in the blood by hydrolyzing t Show more
Elevated plasma triglycerides are a risk factor for coronary artery disease, which is the leading cause of death worldwide. Lipoprotein lipase (LPL) reduces triglycerides in the blood by hydrolyzing them from triglyceride-rich lipoproteins to release free fatty acids. LPL activity is regulated in a nutritionally responsive manner by macromolecular inhibitors including angiopoietin-like proteins 3 and 4 (ANGPTL3 and ANGPTL4). However, the mechanism by which ANGPTL3 inhibits LPL is unclear, in part due to challenges in obtaining pure protein for study. We used a new purification protocol for the N-terminal domain of ANGPTL3, removing a DNA contaminant, and found DNA-free ANGPTL3 showed enhanced inhibition of LPL. Structural analysis showed that ANGPTL3 formed elongated, flexible trimers and hexamers that did not interconvert. ANGPTL4 formed only elongated flexible trimers. We compared the inhibition of ANGPTL3 and ANGPTL4 using human very-low-density lipoproteins as a substrate and found both were noncompetitive inhibitors. The inhibition constants for the trimeric ANGPTL3 (7.5 ± 0.7 nM) and ANGPTL4 (3.6 ± 1.0 nM) were only 2-fold different. Heparin has previously been reported to interfere with ANGPTL3 binding to LPL, so we questioned if the negatively charged heparin was acting in a similar fashion to the DNA contaminant. We found that ANGPTL3 inhibition is abolished by binding to low-molecular-weight heparin, whereas ANGPTL4 inhibition is not. Our data show new similarities and differences in how ANGPTL3 and ANGPTL4 regulate LPL and opens new avenues of investigating the effect of heparin on LPL inhibition by ANGPTL3. Show less
Cardiovascular disease has been the leading cause of death throughout the world for nearly 2 decades. Hypertriglyceridemia affects more than one-third of the population in the United States and is an Show more
Cardiovascular disease has been the leading cause of death throughout the world for nearly 2 decades. Hypertriglyceridemia affects more than one-third of the population in the United States and is an independent risk factor for cardiovascular disease. Despite the frequency of hypertriglyceridemia, treatment options are primarily limited to diet and exercise. Lipoprotein lipase (LPL) is an enzyme responsible for clearing triglycerides from circulation, and its activity alone can directly control plasma triglyceride concentrations. Therefore, LPL is a good target for triglyceride-lowering therapeutics. One approach for treating hypertriglyceridemia may be to increase the amount of enzymatically active LPL by preventing its inhibition by angiopoietin-like protein 4 (ANGPTL4). However, little is known about how these two proteins interact. Therefore, we used hydrogen-deuterium exchange MS to identify potential binding sites between LPL and ANGPTL4. We validated sites predicted to be located at the protein-protein interface by using chimeric variants of LPL and an LPL peptide mimetic. We found that ANGPTL4 binds LPL near the active site at the lid domain and a nearby α-helix. Lipase lid domains cover the active site to control both enzyme activation and substrate specificity. Our findings suggest that ANGPTL4 specifically inhibits LPL by binding the lid domain, which could prevent substrate catalysis at the active site. The structural details of the LPL-ANGPTL4 interaction uncovered here may inform the development of therapeutics targeted to disrupt this interaction for the management of hypertriglyceridemia. Show less