A Nutrient-Dependent ANGPTL3–PCSK9-Containing Complex in HepG2 Cells and Human Plasma

ANGPTL3 and PCSK9 are key regulators of lipoprotein metabolism, with emerging evidence suggestive of a functional regulatory interrelationship. Indeed, patients lacking ANGPTL3 due to loss-of-function variants exhibit reduced circulating PCSK9 levels. This study aimed to determine whether ANGPTL3 and PCSK9 are part of the same complex. Co-immunoprecipitation (Co-IP) experiments in HepG2 cells and human plasma assessed whether ANGPTL3 and PCSK9 co-immunoprecipitated. A hypothetical interaction between the two proteins was also modeled in silico using two software packages: AlphaFold2 and ZDOCK. HepG2 cells overexpressing ANGPTL3, PCSK9, and ANGPTL3-PCSK9 were generated to investigate the relationship between ANGPTL3 and PCSK9 under different nutrient conditions. Reciprocal Co-IP experiments in HepG2 cells and human plasma showed that ANGPTL3 and PCSK9 could be recovered in the same co-immunoprecipitated sample, both intracellularly and in the culture medium. Structural modeling supported the experimental observations and identified a 2:1 ANGPTL3:PCSK9 arrangement as a plausible configuration. We observed higher association under fasting conditions and lower association under simulated feeding conditions. The latter was also confirmed in postprandial human plasma, where ANGPTL3 is known to bind ANGPTL8 in a 2:1 complex. Moreover, incubation of HepG2 cells with LDL appeared to reduce ANGPTL3-PCSK9 association. In summary, our study identifies a dynamic ANGPTL3–PCSK9 association that is regulated in a nutrient-dependent fashion. These findings may broaden current understanding of ANGPTL3 and PCSK9 biology and support further mechanistic studies, particularly in the context of therapeutic inhibition of these proteins.