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![Cellular itinerary of the LDLR. (1) Receptors are first synthesized by ribosomes and folded in the endoplasmic reticulum. (2) Next, the receptors are glycosylated in the Golgi and transported to the cell surface. (3) At the plasma membrane, receptors bind lipoprotein ligands. (4) Internalization occurs via clathrin-coated pits, which ultimately deliver receptor-ligand complexes to endosomes. (5) After the bound lipoproteins are released, the receptors recycle back to the cell surface. Each of these 5 steps is also associated with a corresponding class of LDLR mutations found in FH. Initially adapted from Ref. [25], and modified, with permission, from Ref. [66] (www.els.net).](profile/Natalia-Beglova/publication/7791566/figure/fig1/AS:548671341031429@1507824849848/Cellular-itinerary-of-the-LDLR-1-Receptors-are-first-synthesized-by-ribosomes-and.png)
Cellular itinerary of the LDLR. (1) Receptors are first synthesized by ribosomes and folded in the endoplasmic reticulum. (2) Next, the receptors are glycosylated in the Golgi and transported to the cell surface. (3) At the plasma membrane, receptors bind lipoprotein ligands. (4) Internalization occurs via clathrin-coated pits, which ultimately deliver receptor-ligand complexes to endosomes. (5) After the bound lipoproteins are released, the receptors recycle back to the cell surface. Each of these 5 steps is also associated with a corresponding class of LDLR mutations found in FH. Initially adapted from Ref. [25], and modified, with permission, from Ref. [66] (www.els.net).
Source publication
The low-density lipoprotein receptor normally carries lipoprotein particles into cells, and releases them upon delivery to the low pH milieu of the endosome. Recent structural and functional studies of the receptor, combined with the plethora of prior knowledge about normal receptor function and the effects of disease-associated mutations that caus...
Contexts in source publication
Context 1
... pathway traversed by individual LDLR molecules in the cell is schematically illustrated in Figure 2. Immedi- ately after synthesis, the precursor of the mature receptor migrates with an apparent molecular weight of 120 kDa on SDS-PAGE. ...
Context 2
... patients with FH, more than 1000 mutations in the LDLR have been identified that interfere with one or more of these central events required for normal receptor function [27,28]. The mutations have been grouped into five classes, depending on what kind of receptor defect is observed ( Figure 2). Thus, class 1 mutants fail to produce detectable amounts of protein; class 2 mutants have a partial or complete transport defect; class 3 mutants are impaired in ligand binding; class 4 mutants fail to localize in clathrin- coated pits and are internalization-defective; and class 5 mutants exhibit a ligand release and recycling defect [1]. ...
Citations
... The role of the PCSK9 protein is to bind to the LDLR and promote its breakdown within the cell. Physiologically, LDLR recirculates 100-150 times between the cell plasma and the cell surface [24,25]; the recirculation process is shortened by the PCSK9 protein, resulting in a decreased number of LDLRs on the surface of the hepatocytes [26]. Gain-of-function mutations showed increased PCSK9 activity, resulting in significantly higher cholesterol levels in the bloodstream and enhanced atherosclerosis [24]. ...
Cardiovascular disease is the leading cause of mortality worldwide. Despite the availability of effective low-density lipoprotein cholesterol (LDL-C) lowering agents, an increased cardiovascular risk is still observed in individuals with therapeutic LDL-C levels. One of these cardiovascular risk factors is elevated plasma lipoprotein(a) (Lp(a)) concentration, which maintains chronic inflammation through the increased presence of oxidized phospholipids on its surface. In addition, due to its 90 percent homology with the fibrinolytic proenzyme plasminogen, Lp(a) exhibits atherothrombotic effects. These may also contribute to the increased cardiovascular risk in individuals with high Lp(a) levels that previous epidemiological studies have shown to exist independently of LDL-C and other lipid parameters. In this review, the authors overview the novel therapeutic options to achieve effective Lp(a) lowering treatment, which may help to define tailored personalized medicine and reduce the residual cardiovascular risk in high-risk patients. Agents that increase LDL receptor expression, including statins, proprotein convertase subtilisin kexin type 9 inhibitors, and LDL production inhibitors, are also discussed. Other treatment options, e.g., cholesterolester transfer protein inhibitors, nicotinic acid derivatives, thyroid hormone mimetics, lipoprotein apheresis, as well as apolipoprotein(a) reducing antisense oligonucleotides and small interfering RNAs, are also evaluated.
... The domain structure of the LDLR, as well as sites of interaction and cleavage by other proteins (details of which are presented later), are shown in Figure 2. The LDLR binds LDL via an extracellular ligand binding domain composed of 7 cysteine-rich LDLR type A (LA) repeats, each separated by a short linker of a few amino acids [16] (Figure 2). This ligand binding domain recognises the protein component of LDL particles, apolipoprotein B100, and, to a lower affinity, apolipoprotein E which is present in very low-density lipoprotein particles and chylomicrons [17,18]. Glycosylation (both N-linked and O-linked) within the ligand binding domain has been shown to enhance the ligand binding affinity of the LDLR [19][20][21]. ...
The amount of the low-density lipoprotein receptor (LDLR) on the surface of hepatocytes is the primary determinant of plasma low-density lipoprotein (LDL)-cholesterol level. Although the synthesis and cellular trafficking of the LDLR have been well-documented, there is growing evidence of additional post-translational mechanisms that regulate or fine tune the surface availability of the LDLR, thus modulating its ability to bind and internalise LDL-cholesterol. Proprotein convertase subtilisin/kexin type 9 and the asialoglycoprotein receptor 1 both independently interact with the LDLR and direct it towards the lysosome for degradation. While ubiquitination by the E3 ligase inducible degrader of the LDLR also targets the receptor for lysosomal degradation, ubiquitination of the LDLR by a different E3 ligase, RNF130, redistributes the receptor away from the plasma membrane. The activity of the LDLR is also regulated by proteolysis. Proteolytic cleavage of the transmembrane region of the LDLR by γ-secretase destabilises the receptor, directing it to the lysosome for degradation. Shedding of the extracellular domain of the receptor by membrane-type 1 matrix metalloprotease and cleavage of the receptor in its LDL-binding domain by bone morphogenetic protein-1 reduces the ability of the LDLR to bind and internalise LDL-cholesterol at the cell surface. A better understanding of how the activity of the LDLR is regulated will not only unravel the complex biological mechanisms controlling LDL-cholesterol metabolism but also could help inform the development of alternative pharmacological intervention strategies for the treatment of hypercholesterolaemia.
... Note, while we did not observe an impact of Ca 2+ on CUB7,8 and albumin binding, this may be due to the small cubilin domain used for monitoring binding and a pH & Ca 2+ conformation change. In the large native protein, a change comparable to what has been observed for other recycling receptors such as the low-density lipoprotein may result in altered ligand binding (42,45). Future innovative intravital approaches may reveal the specific pH and Ca 2+ conditions for the dynamic handoff of albumin from cubilin to FcRn. ...
... Blocking glycosylation at N1285 did not result in albumin binding suggesting other glycosylation sites may also influence the albumin interaction possibly by preventing the binding conformation. To investigate a possible pH or Ca 2+ -induced conformation change, similar to what has been shown to occur for low-density lipoprotein receptors (42,45,94), TRP fluorescence was evaluated at different pH's plus or minus Ca 2+ . The magnitude of the change was largest in CUB6-8 and calcium had minimal impact compared to pH. ...
Kidney disease often manifests with an increase in proteinuria, which can result from both glomerular and/or proximal tubule injury. The proximal tubules are the major site of protein and peptide endocytosis of the glomerular filtrate, and cubilin is the proximal tubule brushborder membrane glycoprotein receptor that binds filtered albumin and initiates its processing in proximal tubules. Albumin also undergoes multiple modifications depending upon the physiologic state. We previously documented that carbamylated albumin had reduced cubilin binding, but the effects of cubilin modifications on binding albumin remain unclear. Here, we investigate the cubilin-albumin binding interaction to define the impact of cubilin glycosylation and map the key glycosylation sites while also targeting specific changes in a rat model of proteinuria. We identified a key Asn residues, N1285, was identified that when glycosylated reduced albumin binding. In addition, we found a pH-induced conformation change may contribute to ligand release. To further define the albumin-cubilin binding site, we determined the solution structure of cubilin’s albumin binding domain, CUB7,8, using small-angle X-ray scattering (SAXS) and molecular modeling. We combined this information with mass spectrometry crosslinking experiments of CUB7,8 and albumin that provides a model of the key amino acids required for cubilin-albumin binding. Together, our data supports an important role for glycosylation in regulating the cubilin interaction with albumin, which is altered in proteinuria and provides new insight into the binding interface necessary for the cubilin-albumin interaction.
... In contrast, in the lower pH environment of the early endosome, LDLR undergoes a conformational change to a 'closed' conformation, in which the R4 (residues Cys127 to Cys163) and R5 (Cys176 to Cys210) LBD repeats move inward to interact with the YWTD/β-propeller domain (Ile377 to Gly642) by hydrophobic and charged interactions, thus modulating LDLR conformation as a function of pH (Beglova et al. 2004;Rudenko et al. 2002). The closed LDLR conformation enables LDL-C to detach from the LDLR-LDL-C complex and LDLR to exit the endosome and recycle back to the cell surface (Beglova and Blacklow 2005;Jeon and Blacklow 2005;Martínez-Oliván et al. 2015). ...
LDL-receptor (LDLR)-mediated uptake of LDL-C into hepatocytes is impaired by lysosomal degradation of LDLR, which is promoted by proprotein convertase subtilisin/kexin type 9 (PCSK9). Cell surface binding of PCSK9 to LDLR produces a complex that translocates to an endosome, where the acidic pH strengthens the binding affinity of PCSK9 to LDLR, preventing LDLR recycling to the cell membrane. We present a new approach to inhibit PCSK9-mediated LDLR degradation, namely, targeting the PCSK9/LDLR interface with a PCSK9-antagonist, designated Flag-PCSK9PH, which prevents access of WT PCSK9 to LDLR. In HepG2 cells, Flag-PCSK9PH, a truncated version (residues 53-451) of human WT PCSK9, strongly bound LDLR at the neutral pH of the cell surface but dissociated from it in the endosome (acidic pH), allowing LDLR to exit the lysosomes intact and recycle to the cell membrane. Flag-PCSK9PH thus significantly enhanced cell-surface LDLR levels and the ability of LDLR to take up extracellular LDL-C.
... Thus, the binding of PCSK9 near the EGF-A N-terminal area may affect the known interdomain interactions, either via EGF-A or via the steric effects of PCSK9 itself, or influence the EGF-A conformation and the calcium ion coordination. The intramolecular interactions in EGF-A include the interdomain packing of EGF-A with EGF-B, which is essential for LDLR stability [33][34][35], as well as the interaction of EGF-A with the ligandbinding module R7, responsible for the stiff conformation of this LDL part in a broad pH range [36][37][38][39][40][41][42]. This rigidity seems to favor the pH-dependent closed conformation which enables ligand release and LDLR recuperation from the endosomal compartment [43]. ...
PCSK9 has now become an important target to create new classes of lipid-lowering drugs. The prevention of its interaction with LDL receptors allows an increase in the number of these receptors on the surface of the cell membrane of hepatocytes, which leads to an increase in the uptake of cholesterol-rich atherogenic LDL from the bloodstream. The PCSK9 antagonists described in this review belong to different classes of compounds, may have a low molecular weight or belong to macromolecular structures, and also demonstrate different mechanisms of action. The mechanisms of action include preventing the effective binding of PCSK9 to LDLR, stimulating the degradation of PCSK9, and even blocking its transcription or transport to the plasma membrane/cell surface. Although several types of antihyperlipidemic drugs have been introduced on the market and are actively used in clinical practice, they are not without disadvantages, such as well-known side effects (statins) or high costs (monoclonal antibodies). Thus, there is still a need for effective cholesterol-lowering drugs with minimal side effects, preferably orally bioavailable. Low-molecular-weight PCSK9 inhibitors could be a worthy alternative for this purpose.
... Multiple sources of evidence demonstrate that LDLR recycling requires organized cellular actions including luminal endosomal acidification and calcium (Ca 2+ ) signaling [45][46][47][48][49][50][51]. In particular, endosomal acidification plays a key role in the control of LDLR recycling [40,52,53]. ...
Apolipoprotein E (ApoE) is a protein that plays an important role in the transport of fatty acids and cholesterol and in cellular signaling. On the surface of the cells, ApoE lipoparticles bind to low density lipoprotein receptors (LDLR) that mediate the uptake of the lipids and downstream signaling events. There are three alleles of the human ApoE gene. Presence of ApoE4 allele is a major risk factor for developing Alzheimer’s disease (AD) and other disorders late in life, but the mechanisms responsible for biological differences between different ApoE isoforms are not well understood. We here propose that the differences between ApoE isoforms can be explained by differences in the pH-dependence of the association between ApoE3 and ApoE4 isoforms and LDL-A repeats of LDLR. As a result, the following endocytosis ApoE3-associated LDLRs are recycled back to the plasma membrane but ApoE4-containing LDLR complexes are trapped in late endosomes and targeted for degradation. The proposed mechanism is predicted to lead to a reduction in steady-state surface levels of LDLRs and impaired cellular signaling in ApoE4-expressing cells. We hope that this proposal will stimulate experimental research in this direction that allows the testing of our hypothesis.
... Plasma LDL enters cells via endocytosis mediated by cell-surface LDLRs that remove cholesterol-rich lipoprotein particles from the circulation, to be metabolized principally by the liver [30,31]. Accordingly, the activity of the LDLR in the liver is a major determinant of plasma LDL-C levels [32]. We found that animals fed the HCD had a significant decrease in hepatic LDLR mRNA and protein, along with an increase in serum LDL-C. ...
Background
Emodin has been widely used in traditional Chinese medicine, but few studies have tried to understand the mechanism of its anti-hypercholesterolemic effect.
Material/Methods
To delineate the underlying pathways, high-cholesterol diet (HCD)-fed Sprague-Dawley rats were orally administrated emodin or the lipid-lowering medicine simvastatin. Emodin was administered at 10, 30, or 100 mg/kg, while simvastatin was administered at 10 mg/kg. Parameters measured included lipid profiles (serum total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol, aorta endothelium-dependent vasorelaxation in response to acetylcholine, and nitric oxide (NO) production. RT-qPCR and western blotting were performed to evaluate aortic endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS), and hepatic LDL receptor (LDLR). Indices of liver and serum oxidation were also measured.
Results
The atherogenic index was increased by the HCD but significantly reduced in all treatment groups. The HCD-fed experimental group treated with emodin at 10 mg/kg had significantly lower serum total-C and LDL-C and improved aorta vasorelaxation and enhanced NO production. Also, emodin significantly attenuated the lipid profiles and restored endothelial function, as reflected by upregulated expression of hepatic LDLR and p-eNOS, respectively. Furthermore, emodin at 10 mg/kg significantly enhanced superoxide dismutase activity, lowered the malondialdehyde level in both liver and serum, and enhanced catalase activity in serum.
Conclusions
The ability of emodin to inhibit hypercholesterolemia in HCD-fed rats was associated with lower serum total-C and LDL-C, restoration of aortic endothelial function, and improved antioxidant capacity. Low-dose emodin showed better protection of aortic endothelium and better antioxidant activity than did higher doses.
... 71,78,79 However, the selectivity and affinity of CD320 and LDLR are very different: CD320 has the Cbl/TCN2 complex as its only natural ligand, whereas LDLR binds LDL but is not known to bind Cbl/TCN2. While LDLR-A1 and LDLR-A2 are present in both receptors, their spatial orientation in these receptors differs, 62,[80][81][82] which is critical to ligand binding affinity and specificity. It is likely that, as with natural ligands, CD320 and LDLR display differences in binding to TCPP. ...
Porphyrins are used for cancer diagnostic and therapeutic applications, but the mechanism of how porphyrins accumulate in cancer cells remains elusive. Knowledge of how porphyrins enter cancer cells can aid the development of more accurate cancer diagnostics and therapeutics. To gain insight into porphyrin uptake mechanisms in cancer cells, we developed a flow cytometry assay to quantify cellular uptake of meso‐tetra (4‐carboxyphenyl) porphyrin (TCPP), a porphyrin that is currently being developed for cancer diagnostics. We found that TCPP enters cancer cells through clathrin‐mediated endocytosis. The LDL receptor, previously implicated in the cellular uptake of other porphyrins, only contributes modestly to uptake. We report that TCPP instead binds strongly (KD=42nM) to CD320, the cellular receptor for cobalamin/transcobalamin II (Cbl/TCN2). Additionally, TCPP competes with Cbl/TCN2 for CD320 binding, suggesting that CD320 is a novel receptor for TCPP. Knockdown of CD320 inhibits TCPP uptake by up to 40% in multiple cancer cell lines, including lung, breast, and prostate cell lines, which supports our hypothesis that CD320 both binds to and transports TCPP into cancer cells. Our findings provide some novel insights into why porphyrins concentrate in cancer cells. Additionally, our study describes a novel function for the CD320 receptor which has been reported to transport only Cbl/TCN2 complexes.
... PCSK9 interferes with the binding between LDL cholesterol and LDLR at the cell surface, and subsequently promotes the lysosomal degradation of LDLR [5]. Owing to this negative feedback response of PCSK9 on the LDL cholesterol-lowering effect of LDLR, PCSK9 inhibition has been suggested as an alternative strategy for the treatment of hypercholesterolemia and related-chronic diseases since its discovery in 2003 [6,7]. ...
The objective of the present study was to investigate the mechanism by which capsella bursa-pastoris ethanol extract (CBE), containing 17.5 milligrams of icaritin per kilogram of the extract, and icaritin, mediate hypocholesterolemic activity via the low-density lipoprotein receptor (LDLR) and pro-protein convertase subtilisin/kexin type 9 (PCSK9) in obese mice and HepG2 cells. CBE significantly attenuated serum total and LDL cholesterol levels in obese mice, which was associated with significantly decreased PCSK9 gene expression. HepG2 cells were cultured using delipidated serum (DLPS), and CBE significantly reduced PCSK9 and maintained the LDLR level. CBE co-treatment with rosuvastatin attenuated statin-mediated PCSK9 expression, and further increased LDLR. The icaritin contained in CBE decreased intracellular PCSK9 and LDLR levels by suppressing transcription factors SREBP2 and HNF-1α. Icaritin also significantly suppressed the extracellular PCSK9 level, which likely contributed to post-translational stabilization of LDLR in the HepG2 cells. PCSK9 inhibition by CBE is actively attributed to icaritin, and the use of CBE and icaritin could be an alternative therapeutic approach in the treatment of hypercholesterolemia.
... The YWTD region folds into a six-bladed μ-propeller, which is involved in the release of lipoprotein particles from the receptor within the endosome [16,17]. Next is the O-linked glycosylation domain (encoded by exon 15), which is rich in serine and threonine residues that get glycosylated in the Golgi leading to an increase in the molecular weight of the newly synthesized receptor from 120 kDa up to 160 kDa [15,18]. The following region is the transmembrane domain (encoded by exons 16 & 17) that anchors the receptor to the plasma membrane [15]. ...
The majority of the low-density lipoprotein (LDL) receptors present in the body are expressed in the liver. Therefore, plasma LDL levels significantly correlate with changes in the activity of the hepatic LDL receptor. Based on this, there is a need to understand the regulatory mechanisms that control the hepatic expression of the low-density lipoprotein (LDL) receptor. Herein, we have prepared a functional rat LDL receptor minigene construct that can produce mRNA after splicing. Sequence analysis suggests that this construct has the potential to code for a truncated version of LDL receptor protein. This minigene could be used as a research tool to identify small molecules, natural products, and regulators of the LDL receptor gene that could be developed into LDL receptor-specific activators for therapeutic use.
