On the molecular mechanism for regulating lipoprotein lipase activity

Intravascular lipolysis is a highly regulated process that controls lipid homeostasis by an intricate interplay between a potent lipase and its endogenous activators and inhibitors. Lipoprotein lipase (LPL) is the workhorse enzyme central to intravascular lipolysis. It catalyzes the breakdown of triglycerides in the capillary lumen of endothelial cells in heart, adipose tissue and skeletal muscles. A number of endogenous regulatory proteins ensures that the spatiotemporal activity of LPL matches the given need of lipid nutrients required for energy metabolism. At present, these positive regulators are GPIHBP1, APOC2, and APOA5, while the negative regulators are APOC3, ANGPTL3, ANGPTL4, and ANGPTL8. This thesis outlines the results from work carried out on the interactions between LPL, GPIHBP1, APOC2 and ANGPTL4. ANGPTL4 is an important inhibitor of LPL, which regulates TG levels primarily in the adipose compartment. By employing HDX-MS, we mapped the interaction site of ANGPTL4 on LPL [51–62 (region between β2 and α2), 84–101 (region between β3 and α3), and 220–226 (lid region)]. Building on our HDX-MS data, we furthermore defined the unfolding trajectory of LPL that leads to its permanent inhibition and this unfolding pathway is intriguingly catalyzed by ANGPTL4 binding. More specifically, we find that binding of ANGPTL4 changes the allosteric communication in the α/β-hydrolase domain (NTD) of LPL leading to an increased dynamics in 180–195/219 (β6–β7) and 239–249 (α5), which ultimately renders LPL prone to global unfolding. This propensity of LPL to unfolding is marked by emergence of bimodal isotope envelopes in peptides spanning catalytic triad i.e., 131–165 (β4–α4–β5). The unfolding of LPL by ANGPTL4 is irreversible, while the unfolding of LPL bound to GPIHBP1 is reversible inferring that GPIHBP1 introduces a kinetic block on the last step (point-of-no-return) in the unfolding trajectory. With nanoDSF and temperature-dependent pulse labelling HDX-MS, we emphasize the importance of an inherent LPL metastability in this regulatory process. We show that the α/β- hydrolase domain of LPL undergoes thermal unfolding with an apparent Tm of 34.8 °C. GPIHBP1 binding leads to a dramatic increase of >20°C in the apparent Tm (now 57.6 °C). In contrast, LPL binding of the inhibitor ANGPTL4 reduces the apparent Tm by more than 20°C to