An Allosteric Binding Site on Sortilin Regulates the Trafficking of VLDL, PCSK9, and LDLR in Hepatocytes

Abstract

ApoB lipoproteins (apo B-Lp) are produced in hepatocytes, and their secretion requires the cargo receptor sortilin. We examined the secretion of apo B-Lp-containing very low-density lipoprotein (VLDL), an LDL progenitor. Sortilin also regulates the trafficking of the subtilase PCSK9, which when secreted binds the LDL receptor (LDLR), resulting in its endocytosis and destruction at the lysosome. We show that the site 2 binding compound (cpd984) has multiple effects in hepatocytes, including (1) enhanced Apo-Lp secretion, (2) increased cellular PCSK9 retention, and (3) augmented levels of LDLR at the plasma membrane. We postulate that cpd984 enhances apo B-Lp secretion in part through binding the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3), which is present at higher levels on circulating VLDL form fed rats relative to after fasting. We attribute the enhanced VLDL secretion to its increased binding affinity for sortilin site 1 induced by cpd984 binding site 2. This hinders PCSK9 binding and secretion, which would subsequently prevent its binding to LDLR leading to its degradation. This suggests that site 2 is an allosteric regulator of site 1 binding. This effect is not limited to VLDL, as cpd984 augments binding of the neuropeptide neurotensin (NT) to sortilin site 1. Molecular dynamics simulations demonstrate that the C-terminus of NT (Ct-NT) stably binds site 1 through an electrostatic interaction. This was bolstered by the ability of Ct-NT to disrupt lower-affinity interactions between sortilin and the site 1 ligand PIP3. Together, these data show that binding cargo at sortilin site 1 is allosterically regulated through site 2 binding, with important ramifications for cellular lipid homeostasis involving proteins such as PCSK9 and LDLR.

Sorting through the extensive and confusing roles of sortilin in metabolic disease

“…Interestingly, binding of the propeptide to sortilin does not block the binding of all ligands, including proNGF and proBDNF (115), supporting the view that sortilin has multiple binding sites for ligands. Recently, a small molecule that specifically binds to "binding site 2" (the site where J o u r n a l P r e -p r o o f neurotensin does not bind), was shown to augment binding of neurotensin to sortilin binding site 1, suggesting that site 2 is an allosteric regulator of site 1 binding (169).…”

“…Sparks et al ( 115 , 155 , 168 ) developed another small molecule that binds to the C-terminal neurotensin binding site of sortilin, called cpd541, as well as the first small molecule that binds the N-terminal neurotensin binding site, cpd984. Similar to AF38469, cpd541 inhibits neurotensin binding ( 168 ). However, remarkably, cpd984 enhances neurotensin binding to sortilin ( 115 ).…”

Sortilin drives hypertension by modulating sphingolipid/ceramide homeostasis and by triggering oxidative stress

“…The novel findings summarized above will certainly open up broad research opportunities. Future endeavors could include identifying the molecular mechanisms leading to sortilin cleavage/shedding (most likely released by activated platelets) and determining whether changes in plasma levels of sortilin may have causative or consequential effects in arterial hypertension, studying the potential role of sortilin in endothelial autophagy ( 21 ), which might underlie the pathogenesis of hypertension and other cardiovascular diseases, and elucidating the involvement of endothelial sortilin in the pathophysiology of dyslipidemia, especially given the interaction between sortilin and PCSK9 ( 22 ).…”

Sortilin and hypertension

“…According to the latest studies, this protection could be attributable to a decrease in very-low-density lipoprotein (VLDL) production leading to reduced apoB containing lipoproteins in carriers of the SORT1 variants [61 ▪ ]. Furthermore, Sortilin augments secretion of PCSK9 (proprotein convertase subtilisin/kexin type 9 serine protease), which binds the LDL receptor resulting in its endocytosis [62], thereby increasing the risk of atherosclerosis [55,63,64].…”

“…&]. Furthermore, Sortilin augments secretion of PCSK9 (proprotein convertase subtilisin/kexin type 9 serine protease), which binds the LDL receptor resulting in its endocytosis[62], thereby increasing the risk of atherosclerosis[55,63,64].…”