Lipoprotein receptor interactions are not required for monocyte oxidation of LDL.
19
Citation
37
Reference
10
Related Paper
Abstract:
Upon activation, human peripheral blood monocytes and U937 cells oxidized low density lipoprotein (LDL), converting it to a cytotoxin.The oxidized LDL loses its ability to interact specifically with the native LDL (apoB/E) receptor and becomes a ligand for the scavenger receptors and two other receptors, FqRII (CD32) and CD36.We performed a series of studies to evaluate the potential contribution of each of these receptors to the process of monocyte-mediated LDL oxidation.To assess the participation of the apoB/E recep tor, we tested the ability of activated human monocytes to oxidize LDL after u p and down-regulation of apoB/E recep tors.Neither up-regulation nor down-regulation of the apoB/E receptor significantly modified the level of LDL lipid oxidation.Acetylated LDL, a ligand for scavenger receptors, was also oxidized by the activated monocytes.Methylated LDL, a chemically modified LDL that is not recognized by the apoB/E or scavenger receptors, was oxidized as we1i.mThus, LDL does not need to interact with either the apoB/E receptor or scavenger receptors in order to undergo lipid oxidation.Additionally, monoclonal antibodies to CD36 and CD32 were used to block these two receptors that recognize oxidized LDL.Although both antibodies interfered with oxidized LDL binding to these receptors, neither treatment interfered with LDL lipid oxidation mediated by activated human monocytes.Our results suggest that interaction with these receptors is not a requirement for LDL lipid oxidation by activated human monocytes.-Cathcart,M.Keywords:
Low-density lipoprotein
Monocyte
PCSK9
Lipoprotein(a)
Intervention with drugs to reduce Low Density Lipoprotein-cholesterol (LDL-C) has proven to decrease the risk of subsequent cardiovascular events, including mortality. Our goal is to develop novel, small molecule, LDL-C lowering drugs by targeting the Low Density Lipoprotein Receptor (LDLR) degradation pathway, which is modulated by the protease proprotein convertase subtilisin-like kexin type 9 (PCSK9). PCSK9 has been shown to bind to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR; it enhances the degradation of the LDLR by an unknown. We successfully integrated virtual screening methods and cell based assays into a simple, efficient procedure and identified compounds that interfere with the PCSK9-LDLR interaction. Using the atomic coordinates of the crystal structure of the PCSK9/LDLR-EGF-A complex, about a million drug-like compounds were docked in silico into the region of PCSK9 where LDLR-EGF-A binds. We selected 179 top scoring compounds that showed the best fit in the binding site as well as the greatest number of interactions with PCSK9. These compounds were tested in (a) our PCSK9/LDLR dependent HEK293 cell-based recombinant assay and (b) the endogenously expressed LDLR in HepG2/PCSK9 dependent cell based assay for their ability to upregulate the LDLR. From these screening campaigns, eleven compounds consistently exhibited concentration dependent increase in the LDLR as comparable to control, with IC 50 s in the low micromolar range. Three of the top picks were shown to exhibit a 5 to 10 fold up-regulation at 1.6 μM in two cell lines. In addition, these compounds also exhibited an increase in the fluorescently labeled DiI-LDL uptake in the nanomolar range in situ . Furthermore, in a two week animal study, subcutaneous injection of 4 mg/kg of one of these compounds resulted in a 32% reduction of the total cholesterol level in mice fed a high fat/high cholesterol diet as compared to 27% cholesterol reduction obtained with 40 mg/kg Atorvastatin. To our knowledge, no small molecule antagonist against PCSK9 has been reported, only mAbs. Thus, identifying such an oral small molecule PCSK9 inhibitor represents a tremendous advance and opportunity for drug development.
PCSK9
Kexin
Proprotein Convertases
Subtilisin
Low-density lipoprotein
Cite
Citations (3)
PCSK9
Kexin
Proprotein Convertases
Low-density lipoprotein
Cite
Citations (74)
Elevated serum level of low density lipoprotein cholesterol (LDL-C) is the leading risk factor for cardiovascular disease. LDL receptor (LDLR)-mediated LDL clearance is the major factor determining the LDL-C level in the circulation. LDL binds to the LDLR on the cell surface and enters the cells through classical clathrin-coated vesicles. In the acidic endosome, LDLR is uncoupled from LDL and recycles back to the cell surface. The released LDL is transported to the lysosome for degradation. The proprotein convertase subtilisin kexin type 9 (PCSK9) gene encodes a hepatic secretory protein, and its mutations are strongly associated with levels of LDL-C. We and others have shown that PCSK9 directly interacts with LDLR on the cell surface and both are internalized through the clathrin-coated vesicles. However, in the acidic endosome, PCSK9 and LDLR form a tight complex and are targeted to lysosome for degradation, thereby reducing the level of LDLR on the surface of hepatocytes and decreasing hepatic clearance of LDL-C, which plays an important role in maintaining a relatively constant level of LDL in the plasma. Thus, blocking PCSK9 function has become a new strategy to treat hypercholesterolemia.In this review, we will summarize the latest progress in the functional and mechanistic studies of PCSK9 and also highlight the research progress of PCSK9 inhibitors. It aims to provide a reference for the study of PCSK9-LDLR pathway and the regulation of cholesterol metabolism.血清低密度脂蛋白胆固醇(low density lipoprotein cholesterol, LDL-C)水平的升高是导致心血管疾病发生的主要危险因素。低密度脂蛋白受体(LDL receptor, LDLR)介导的低密度脂蛋白(low density lipoprotein, LDL)清除是决定循环中LDL-C水平的主要因素。LDL与细胞表面的LDLR结合后通过经典的网格蛋白小窝(clathrin-coated vesicles)内化进入细胞。在酸性核内体中,LDLR与LDL解离并循环回到细胞表面,释放的LDL将被运送到溶酶体中降解。前蛋白转化酶枯草溶菌素9 (proprotein convertase subtilisin kexin type 9, PCSK9)编码一种肝脏分泌型蛋白,其突变与LDL-C水平密切相关。前期研究已经证明,PCSK9直接与细胞表面的LDLR相互作用,二者一起通过网格蛋白小窝内化进入细胞。然而,在酸性核内体中,PCSK9和LDLR形成紧密的复合物,并进入溶酶体中进行降解,从而减少肝细胞表面LDLR的水平,降低肝脏对LDL-C的清除,该过程对于维持血浆中LDL在相对恒定的水平具有重要作用。因此,阻断PCSK9功能已成为治疗高胆固醇血症的新策略。本文综述了PCSK9的功能和机制研究的最新进展,并着重介绍了PCSK9抑制剂的研究进展,旨在为PCSK9-LDLR通路的研究和胆固醇代谢的调控提供参考。.
PCSK9
Degradation
Low-density lipoprotein
Cite
Citations (2)
Proprotein convertase subtilisin kexin 9 (Pcsk9) is a subtilisin serine protease with a putative role in cholesterol metabolism. Pcsk9 expression is down-regulated by dietary cholesterol, and mutations in Pcsk9 have been associated with a form of autosomal dominant hypercholesterolemia. To study the function of Pcsk9 in mice, an adenovirus constitutively expressing murine Pcsk9 (Pcsk9-Ad) was used. Pcsk9 overexpression in wild-type mice caused a 2-fold increase in plasma total cholesterol and a 5-fold increase in non-high-density lipoprotein (HDL) cholesterol, with no increase in HDL cholesterol, as compared with mice infected with a control adenovirus. Fast protein liquid chromatography analysis showed that the increase in non-HDL cholesterol was due to an increase in low-density lipoprotein (LDL) cholesterol. This effect appeared to depend on the LDL receptor (LDLR) because LDLR knockout mice infected with Pcsk9-Ad had no change in plasma cholesterol levels as compared with knockout mice infected with a control adenovirus. Furthermore, whereas overexpression of Pcsk9 had no effect on LDLR mRNA levels, there was a near absence of LDLR protein in animals overexpressing Pcsk9. These results were confirmed in vitro by the demonstration that transfection of Pcsk9 in McA-RH7777 cells caused a reduction in LDLR protein and LDL binding. In summary, these results indicate that overexpression of Pcsk9 interferes with LDLR-mediated LDL cholesterol uptake. Because Pcsk9 and LDLR are coordinately regulated by cholesterol, Pcsk9 may be involved in a novel mechanism to modulate LDLR function by an alternative pathway than classic cholesterol inhibition of sterol regulatory element binding protein-mediated transcription.
PCSK9
Kexin
Proprotein Convertases
Knockout mouse
Cite
Citations (589)
Low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) play a pivotal role by regulating plasma low-density lipoprotein cholesterol (LDL-c) levels. Dihydromyricetin (DMY), the most abundant natural flavonoid in rattan tea, has proven anti-atherogenic effects, but the underlying molecular mechanisms remain poorly understood. Therefore, we studied the effects of DMY on LDLR and PCSK9. The results showed DMY promoted LDLR protein and mRNA expression and increased LDL uptake in HepG2 cells. DMY inhibited intracellular PCSK9 protein and mRNA expression. And it also significantly reduced PCSK9 levels in the cell culture medium. Furthermore, DMY inhibited the expression of PCSK9 through the liver nuclear transcription factor 1 A (HNF1A) and increased the protein expression of LDLR. Taken together, our results support the idea that DMY regulates LDL-c metabolism through PCSK9/LDLR signaling. This study reveals the potential mechanism of DMY’s anti-atherogenic effect and provides a theoretical basis for dietary DMY supplementation.
PCSK9
Kexin
Proprotein Convertases
Low-density lipoprotein
Cite
Citations (0)
Hypercholesterolemia characterized by excessively elevated levels of plasma low-density lipoprotein-cholesterol (LDLc) increases the risk of atherosclerosis, causing cardiovascular disease and cerebrovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9), which is secreted from liver, interacts with LDL-receptor (LDLR) that uptakes LDLc from plasma into hepatocytes. The PCSK9-LDLR interaction leads degradation of the LDLR on hepatocytes, resulting in elevation of plasma LDLc levels. Although anti-PCSK9 antibodies that inhibit PCSK9-LDLR interaction have been used for the treatment of hypercholesterolemia, the antibody agents are very expensive because they are produced using mammalian cells. To overcome this problem, it is necessary to develop PCSK9/LDLR interaction inhibitors that are produced by chemical synthesis to be low-cost in hypercholesterolemia treatment.
PCSK9
Kexin
Proprotein Convertases
Low-density lipoprotein
Proprotein Convertases
Cite
Citations (0)
Elevated LDL-cholesterol (LDLc) levels are a major risk factor for cardiovascular disease and atherosclerosis. LDLc is cleared from circulation by the LDL receptor (LDLR). Proprotein convertase subtilisin/kexin 9 (PCSK9) enhances the degradation of the LDLR in endosomes/lysosomes, resulting in increased circulating LDLc. PCSK9 can also mediate the degradation of LDLR lacking its cytosolic tail, suggesting the presence of as yet undefined lysosomal-targeting factor(s). Herein, we confirm this, and also eliminate a role for the transmembrane-domain of the LDLR in mediating its PCSK9-induced internalization and degradation. Recent findings from our laboratory also suggest a role for PCSK9 in enhancing tumor metastasis. We show herein that while the LDLR is insensitive to PCSK9 in murine B16F1 melanoma cells, PCSK9 is able to induce degradation of the low density lipoprotein receptor-related protein 1 (LRP-1), suggesting distinct targeting mechanisms for these receptors. Furthermore, PCSK9 is still capable of acting upon the LDLR in CHO 13-5-1 cells lacking LRP-1. Conversely, PCSK9 also acts on LRP-1 in the absence of the LDLR in CHO-A7 cells, where re-introduction of the LDLR leads to reduced PCSK9-mediated degradation of LRP-1. Thus, while PCSK9 is capable of inducing degradation of LRP-1, the latter is not an essential factor for LDLR regulation, but the LDLR effectively competes with LRP-1 for PCSK9 activity. Identification of PCSK9 targets should allow a better understanding of the consequences of PCSK9 inhibition for lowering LDLc and tumor metastasis.
PCSK9
Kexin
Proprotein Convertases
Internalization
Cite
Citations (211)
The interaction between proprotein convertase subtilisin/kexin type 9 (PCSK9) and the low-density lipoprotein receptor (LDLR) is a promising target for the treatment of hyperc-holesterolemia. In this study, a new method based on competitive affinity and tag detection was developed, which aimed to evaluate potent natural inhibitors preventing the interaction of PCSK9/LDLR directly. Herein, natural compounds with efficacy in the treatment of hypercholesterolemia were chosen to investigate their inhibitory activities on the PCSK9/LDLR interaction. Two of them, polydatin (1) and tetrahydroxydiphenylethylene-2-O-glucoside (2), were identified as potential inhibitors for the PCSK9/LDLR interaction and were proven to prevent PCSK9-mediated LDLR degradation in HepG2 cells. The results suggested that this strategy could be applied for evaluating potential bioactive compounds inhibiting the interaction of PCSK9/LDLR and this strategy could accelerate the discovery of new drug candidates for the treatment of PCSK9-mediated hypercholesterolemia.
PCSK9
Kexin
Proprotein Convertases
Subtilisin
Cite
Citations (13)
PCSK9
Kexin
Cite
Citations (43)
Proprotein convertase subtilisin/kexin type 9 (PCSK9) affects cholesterol homeostasis by targeting hepatic LDL receptor (LDLR) for lysosomal degradation. Clinically, PCSK9 inhibitors effectively reduce LDL-cholesterol (LDL-C) levels and the incidence of cardiovascular events. Because microRNAs (miRs) are integral regulators of cholesterol homeostasis, we investigated the involvement of miR-483 in regulating LDL-C metabolism. Using in silico analysis, we predicted that miR-483-5p targets the 3'-UTR of PCSK9 mRNA. In HepG2 cells, miR-483-5p targeted the PCSK9 3'-UTR, leading to decreased PCSK9 protein and mRNA expression, increased LDLR expression, and enhanced LDL-C uptake. In hyperlipidemic mice and humans, serum levels of total cholesterol and LDL-C were inversely correlated with miR-483-5p levels. In mice, hepatic miR-483 overexpression increased LDLR levels by targeting Pcsk9, with a significant reduction in plasma total cholesterol and LDL-C levels. Mechanistically, the cholesterol-lowering effect of miR-483-5p was significant in mice receiving AAV8 PCSK9-3'-UTR but not Ldlr-knockout mice or mice receiving AAV8 PCSK9-3'-UTR (ΔBS) with the miR-483-5p targeting site deleted. Thus, exogenously administered miR-483 or similarly optimized compounds have potential to ameliorate hypercholesterolemia.
PCSK9
Cite
Citations (42)