Inducible Degrader Of Low-density Lipoprotein Receptor Research Articles

A Review of Progress on Targeting LDL Receptor-Dependent and -Independent Pathways for the Treatment of Hypercholesterolemia, a Major Risk Factor of ASCVD.

Since the discovery of the LDL receptor in 1973 by Brown and Goldstein as a causative protein in hypercholesterolemia, tremendous amounts of effort have gone into finding ways to manage high LDL cholesterol in familial hypercholesterolemic (HoFH and HeFH) individuals with loss-of-function mutations in the LDL receptor (LDLR) gene. Statins proved to be the first blockbuster drug, helping both HoFH and HeFH individuals by inhibiting the cholesterol synthesis pathway rate-limiting enzyme HMG-CoA reductase and inducing the LDL receptor. However, statins could not achieve the therapeutic goal of LDL. Other therapies targeting LDLR include PCSK9, which lowers LDLR by promoting LDLR degradation. Inducible degrader of LDLR (IDOL) also controls the LDLR protein, but an IDOL-based therapy is yet to be developed. Among the LDLR-independent pathways, such as angiopoietin-like 3 (ANGPTL3), apolipoprotein (apo) B, apoC-III and CETP, only ANGPTL3 offers the advantage of treating both HoFH and HeFH patients and showing relatively better preclinical and clinical efficacy in animal models and hypercholesterolemic individuals, respectively. While loss-of-LDLR-function mutations have been known for decades, gain-of-LDLR-function mutations have recently been identified in some individuals. The new information on gain of LDLR function, together with CRISPR-Cas9 genome/base editing technology to target LDLR and ANGPTL3, offers promise to HoFH and HeFH individuals who are at a higher risk of developing atherosclerotic cardiovascular disease (ASCVD).

Effect of type 2 diabetes on the inducible degrader of LDL receptor

The inducible degrader of LDL receptor (IDOL) acts as a post-transcriptional degrader of the LDL receptor (LDLR). IDOL is functionally active in the liver and in peripheral tissues. We have evaluated IDOL expression in circulating monocytes in subjects with and without type 2 diabetes and determined whether changes in IDOL expression could affect macrophage function like cytokine production in vitro. One hundred forty individuals with type 2 diabetes and 110 healthy control subjects were recruited. Cellular expression of IDOL and LDLR in peripheral blood CD14+ monocytes was measured by flow cytometry. The expression of intracellular IDOL was lower in individuals with diabetes than control (21.3 ± 4.6 mean fluorescence intensity × 1,000 vs. 23.8 ± 6.2, P < 0.01), and this was accompanied by an increase in cell surface LDLR (5.2 ± 3.0 mean fluorescence intensity × 1,000 vs. 4.3 ± 1.5, P < 0.01), LDL binding, and intracellular lipid (P < 0.01). IDOL expression correlated with HbA1c (r = −0.38, P < 0.01) and serum fibroblast growth factor-21 (FGF21) (r = −0.34, P < 0.01). Multivariable regression analysis, including age, sex, BMI, smoking, HbA1c, and log(FGF21), showed that HbA1c and FGF21 were significant independent determinants of IDOL expression. IDOL knockdown human monocyte-derived macrophages produced higher concentrations of interleukin 1 beta, interleukin 6, and TNFα than control macrophages upon stimulation with lipopolysaccharide (all P < 0.01). In conclusion, the expression of IDOL in CD14+ monocytes was decreased in type 2 diabetes and was associated with glycemia and serum FGF21 concentration.

Deficiency of Nardilysin in the Liver Reduces Serum Cholesterol Levels.

Nardilysin (NRDC) has been shown to be involved in post-translational histone modifications, in addition to enhancement in ectodomain shedding of membrane-anchored protein, which play significant roles in various pathophysiology, including glucose homeostasis, inflammatory diseases and cancer. The present study sought to determine roles of NRDC in the liver on lipid and lipoprotein metabolism. We established liver-specific NRDC deficient mice by use of NRD1 floxed mice and albumin promoter-Cre recombinase (Cre) transgenic mice, and found that their serum low-density lipoprotein (LDL) cholesterol levels were significantly lower than those in control littermate mice. In the liver, LDL receptor (LDLR) mRNA expression was significantly upregulated, while inducible degrader of LDLR (IDOL) and microsomal triglyceride transfer protein (MTP) mRNA expression was significantly downregulated, in liver-specific NRDC deficient mice. Hepatic cell-surface LDLR expression levels were significantly elevated and serum pro-protein convertase subtilisin-kexin type 9 (PCSK9) levels were significantly reduced in mice with hepatic NRDC deficiency. In cultured hepatocytes, NRDC deficiency significantly reduced secreted PCSK9 and increased cell-surface LDLR expression. On the other hand, NRDC overexpression in cultured hepatocytes significantly increased secreted PCSK9 and lowered cell-surface LDLR expression. Thus, NRDC in murine hepatocytes appears to play key roles in cholesterol homeostasis, although the precise molecular mechanisms remain to be determined.

Regulation of intestinal LDLR by the LXR-IDOL axis

Background and aimsCholesterol metabolism is tightly regulated by transcriptional and post-transcriptional mechanisms. Accordingly, dysregulation of cholesterol metabolism is a major risk factor for the development of coronary artery disease and associated complications. In recent years, it has become apparent that next to the liver, the intestine plays a key role in systemic cholesterol metabolism by governing cholesterol absorption, secretion, and incorporation into lipoprotein particles. We have previously demonstrated that the Liver X receptor (LXR)-regulated E3 ubiquitin ligase inducible degrader of LDLR (IDOL) is a regulator of cholesterol uptake owing to its ability to promote the ubiquitylation of the low-density lipoprotein receptor (LDLR). However, whether the LXR-IDOL-LDLR axis regulates the LDLR in the intestine and whether this influences intestinal cholesterol homeostasis is not known. MethodsIn this study, we evaluated the role of the LXR-IDOL-LDLR axis in enterocyte cell models and in primary enterocytes isolated from Idol(−/−) and wild type mice. Furthermore, we studied the regulation of intestinal LDLR in Idol(−/−) and in wild type mice treated with the LXR agonist GW3965. Finally, we assessed ezetimibe-induced trans-intestinal cholesterol efflux in Idol(−/−) mice. ResultsWe show that in a wide range of intestinal cell lines LXR activation decreases LDLR protein abundance, cell surface occupancy, and LDL uptake in an IDOL-dependent manner. Similarly, we find that pharmacological dosing of C57BL6/N mice with the LXR agonist GW3965 increases Idol expression across the intestine with a concomitant reduction in Ldlr protein. Conversely, primary enterocytes isolated from Idol(−/−) mice have elevated Ldlr. To test whether these changes contribute to trans-intestinal cholesterol efflux, we measured fecal cholesterol in mice following ezetimibe dosing, but found no differences between Idol(−/−) and control mice in this setting. ConclusionsIn conclusion, our study establishes that the LXR-IDOL-LDLR axis is active in the intestine and is part of the molecular circuitry that maintains cholesterol homeostasis in enterocytes.

Hydrogen sulfide accumulates LDL receptor precursor via downregulating PCSK9 in HepG2 cells

Endogenous hydrogen sulfide (H2S) affects cholesterol homeostasis and liver X receptor α (LXRα) expression. However, whether low-density lipoprotein (LDL) receptor (LDLR), a key player in cholesterol homeostasis, is regulated by exogenous H2S through LXRα signaling has not been determined. We investigated the effects of sodium hydrosulfide (NaHS, H2S donor) on LDLR expression in the presence or absence of LXR agonists, T0901317 or GW3965 in HepG2 cells. We found that H2S strongly accumulated LDLR precursor in the presence of T0901317. Hence, LDLR transcription and the genes involved in LDLR precursor maturation and degradation were studied. T0901317 increased the LDLR mRNA level, whereas H2S did not affect LDLR transcription. H2S had no significant effect on the expression of LXRα and inducible degrader of LDLR (IDOL). H2S and T0901317 altered mRNA levels of several enzymes for N- and O-glycosylation and endoplasmic reticulum (ER) chaperones assisting LDLR maturation, but did not affect their protein levels. H2S decreased proprotein convertase subtilisin/kexin type 9 (PCSK9) protein levels and its mRNA level elevated by T0901317. T0901317 with PCSK9 siRNA also accumulated LDLR precursor as did T0901317 with H2S. High glucose increased PCSK9 protein levels and attenuated LDLR precursor accumulation induced by T0901317 with H2S. Taken together, H2S accumulates LDLR precursor by downregulating PCSK9 expression but not through the LXRα-IDOL pathway, LDLR transcriptional activation, or dysfunction of glycosylation enzymes and ER chaperones. These results also indicate that PCSK9 plays an important role in LDLR maturation in addition to its well-known effect on the degradation of LDLR mature form.

The hepatic WASH complex is required for efficient plasma LDL and HDL cholesterol clearance

The evolutionary conserved Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex is one of the crucial multiprotein complexes that facilitates endosomal recycling of transmembrane proteins. Defects in WASH components have been associated with inherited developmental and neurological disorders in humans. Here, we show that hepatic ablation of the WASH component Washc1 in chow-fed mice increases plasma concentrations of cholesterol in both LDLs and HDLs, without affecting hepatic cholesterol content, hepatic cholesterol synthesis, biliary cholesterol excretion, or hepatic bile acid metabolism. Elevated plasma LDL cholesterol was related to reduced hepatocytic surface levels of the LDL receptor (LDLR) and the LDLR-related protein LRP1. Hepatic WASH ablation also reduced the surface levels of scavenger receptor class B type I and, concomitantly, selective uptake of HDL cholesterol into the liver. Furthermore, we found that WASHC1 deficiency increases LDLR proteolysis by the inducible degrader of LDLR, but does not affect proprotein convertase subtilisin/kexin type 9-mediated LDLR degradation. Remarkably, however, loss of hepatic WASHC1 may sensitize LDLR for proprotein convertase subtilisin/kexin type 9-induced degradation. Altogether, these findings identify the WASH complex as a regulator of LDL as well as HDL metabolism and provide in vivo evidence for endosomal trafficking of scavenger receptor class B type I in hepatocytes.

IDOL, inducible degrader of low-density lipoprotein receptor, serves as a potential therapeutic target for dyslipidemia

Low-density lipoprotein cholesterol (LDL-C) is the hall marker for the atherosclerotic cardiovascular disease (ASCVD). It has been shown that over 70% of circulating LDL-C is metabolized through binding and activation of hepatic LDL receptor (LDLR). Genetic LDLR mutations cause hypercholesterolemia in the patients. Therefore, elevation of LDLR levels is beneficial for the treatment of dyslipidemia. LDLR expression is regulated by the SREBP2/PCSK9 pathways. Targeting SREBP2/PCSK9 pathways by statins and human monoclonal PCSK9 antibody has been shown to reduce the progression of ASVCD. Recent studies identified that inducible degrader of LDLR (IDOL) is a novel regulator of LDLR. IDOL is an E3-ubiquitin ligase regulated via liver X receptors (LXRs) binding to the upstream of translation start site of IDOL. IDOL modulates LDLR distribution through ubiquitination and degradation of LDLR in lysosomes. Genome-wide association studies (GWAS) have revealed that the nonsynonymous substitution rs9370867 of IDOL probably contributes to the variability of circulating LDL levels. Recently studies also demonstrated that IDOL influences PCSK9 expression in a LDLR/SREBP2-dependent manner. Based upon these novel findings, we hypothesize that IDOL and PCSK9 would have a synergistic effect on LDLR distribution. Specifically, loss of IDOL increases LDLR distribution in the hepatic cell, and subsequently reduces serum LDL-C levels in dyslipidemic patients. IDOL might be a potential therapeutic target for the treatment of ASCVD.

Ubiquitin ligase Idol Inhibitors for Treating Hypercholesterolemia

Despite widespread use of statins and other therapeutics, hypercholesterolemia remains a significant medical issue for a many patients unable to effectively regulate cholesterol levels. Cholesterol uptake is primarily mediated by the hepatic low density lipoprotein receptor (LDLR), which binds and internalizes plasma LDL. Elevated LDLR is associated with reduced LDL levels. Recently, the E3 ligase Idol (Inducible Degrader of LDLR) was shown to be a key regulator of LDLR levels. IDOL is a unique E3 ligase that utilizes its FERM domain to specifically target LDLR for polyubiquitylation and subsequent lysosomal degradation. Genetic ablation of IDOL raises levels of LDLR. Thus, inhibition of IDOL may be beneficial for the treatment of hypercholesterolemia. The ubiquitin‐proteasome system is a rich landscape for drug discovery. E3 ligases in particular are attractive therapeutic targets in various disorders, including metabolic disorders. Nevertheless, it has been challenging to discover and develop E3 ligase inhibitors as first in class clinical candidates. Here, we report the identification of novel IDOL inhibitors that modulate cellular cholesterol homeostasis. These compounds increased LDLR levels and increased LDL association in various cellular models. Biophysical characterization revealed direct binding of the compounds to IDOL and perturbation of IDOL:LDLR interactions. The most promising compounds were used as starting points to develop novel drug like molecules and the lead compounds are being evaluated in translational models of hypercholesterolemia. Data will be presented summarizing our progress to date targeting IDOL for the treatment of hypercholesterolemia. Work supported in part by NIH grant HL127893.

AAV Vectors Expressing LDLR Gain-of-Function Variants Demonstrate Increased Efficacy in Mouse Models of Familial Hypercholesterolemia

Familial hypercholesterolemia is a genetic disorder that arises because of loss-of-function mutations in the low-density lipoprotein receptor (LDLR) and homozygous familial hypercholesterolemia is a candidate for gene therapy using adeno-associated viral vectors. Proprotein convertase subtilisin/kexin type 9 (PCSK9) and inducible degrader of LDLR (IDOL) negatively regulate LDLR protein and could dampen adeno-associated viral vector encoded LDLR expression. We sought to create vectors expressing gain-of-function human LDLR variants that are resistant to degradation by human PCSK9 (hPCSK9) and IDOL and thereby enhance hepatic LDLR protein abundance and plasma LDL cholesterol reduction. Amino acid substitutions were introduced into the coding sequence of human LDLR cDNA to reduce interaction with hPCSK9 and human IDOL. A panel of mutant human LDLRs was initially screened in vitro for escape from PCSK9. The variant human LDLR-L318D was further evaluated using a mouse model of homozygous familial hypercholesterolemia lacking endogenous LDLR and apolipoprotein B mRNA editing enzyme catalytic, APOBEC-1 (double knockout). Administration of wild-type human LDLR to double knockout mice, expressing hPCSK9, led to diminished LDLR activity. However, LDLR-L318D was resistant to hPCSK9-mediated degradation and effectively reduced cholesterol levels. Similarly, the LDLR-K809R\C818A construct avoided human IDOL regulation and achieved stable reductions in serum cholesterol. An adeno-associated viral vector serotype 8.LDLR-L318D\K809R\C818A vector that carried all 3 amino acid substitutions conferred partial resistance to both hPCSK9- and human IDOL-mediated degradation. Amino acid substitutions in the human LDLR confer partial resistance to PCSK9 and IDOL regulatory pathways with improved reduction in cholesterol levels and improve on a potential gene therapeutic approach to treat homozygous familial hypercholesterolemia subjects.

Hepatic Overexpression of Idol Increases Circulating Protein Convertase Subtilisin/Kexin Type 9 in Mice and Hamsters via Dual Mechanisms

Low-density lipoprotein receptor (LDLR) is degraded by inducible degrader of LDLR (Idol) and protein convertase subtilisin/kexin type 9 (PCSK9), thereby regulating circulating LDL levels. However, it remains unclear whether, and if so how, these LDLR degraders affect each other. We therefore investigated effects of liver-specific expression of Idol on LDL/PCSK9 metabolism in mice and hamsters. Injection of adenoviral vector expressing Idol (Ad-Idol) induced a liver-specific reduction in LDLR expression which, in turn, increased very-low-density lipoprotein/LDL cholesterol levels in wild-type mice because of delayed LDL catabolism. Interestingly, hepatic Idol overexpression markedly increased plasma PCSK9 levels. In LDLR-deficient mice, plasma PCSK9 levels were already elevated at baseline and unchanged by Idol overexpression, which was comparable with the observation for Ad-Idol-injected wild-type mice, indicating that Idol-induced PCSK9 elevation depended on LDLR. In wild-type mice, but not in LDLR-deficient mice, Ad-Idol enhanced hepatic PCSK9 expression, with activation of sterol regulatory element-binding protein 2 and subsequently increased expression of its target genes. Supporting in vivo findings, Idol transactivated PCSK9/LDLR in sterol regulatory element-binding protein 2/LDLR-dependent manners in vitro. Furthermore, an in vivo kinetic study using (125)I-labeled PCSK9 revealed delayed clearance of circulating PCSK9, which could be another mechanism. Finally, to extend these findings into cholesteryl ester transfer protein-expressing animals, we repeated the above in vivo experiments in hamsters and obtained similar results. A vicious cycle in LDLR degradation might be generated by PCSK9 induced by hepatic Idol overexpression via dual mechanisms: sterol regulatory element-binding protein 2/LDLR. Furthermore, these effects would be independent of cholesteryl ester transfer protein expression.

Blockade of cholesterol absorption by ezetimibe reveals a complex homeostatic network in enterocytes

Enterocyte cholesterol homeostasis reflects aggregated rates of sterol synthesis, efflux, and uptake from plasma and gut lumen. Cholesterol synthesis and LDL uptake are coordinately regulated by sterol regulatory element-binding proteins (SREBP), whereas sterol efflux is regulated by liver X receptors (LXR). How these processes are coordinately regulated in enterocytes, the site of cholesterol absorption, is not well understood. Here, we treat mice with ezetimibe to investigate the effect of blocking cholesterol absorption on intestinal SREBPs, LXRs, and their effectors. Ezetimibe increased nuclear SREBP-2 8-fold. HMG-CoA reductase (HMGR) and LDL receptor (LDLR) mRNA levels increased less than 3-fold, whereas their protein levels increased 30- and 10-fold, respectively. Expression of inducible degrader of LDLR (IDOL), an LXR-regulated gene that degrades LDLRs, was reduced 50% by ezetimibe. Coadministration of ezetimibe with the LXR agonist T0901317 abolished the reduction in IDOL and prevented the increase in LDLR protein. Ezetimibe-stimulated LDLR expression was independent of proprotein convertase subtilisin/kexin type 9 (PSCK9), a protein that degrades LDLRs. To maintain cholesterol homeostasis in the face of ezetimibe, enterocytes boost LDL uptake by increasing LDLR number, and they boost sterol synthesis by increasing HMGR and other cholesterologenic genes. These studies reveal a hitherto undescribed homeostatic network in enterocytes triggered by blockade of cholesterol absorption.

An LXR Agonist Promotes Glioblastoma Cell Death through Inhibition of an EGFR/AKT/SREBP-1/LDLR–Dependent Pathway

Glioblastoma (GBM) is the most common malignant primary brain tumor of adults and one of the most lethal of all cancers. Epidermal growth factor receptor (EGFR) mutations (EGFRvIII) and phosphoinositide 3-kinase (PI3K) hyperactivation are common in GBM, promoting tumor growth and survival, including through sterol regulatory element-binding protein 1 (SREBP-1)-dependent lipogenesis. The role of cholesterol metabolism in GBM pathogenesis, its association with EGFR/PI3K signaling, and its potential therapeutic targetability are unknown. In our investigation, studies of GBM cell lines, xenograft models, and GBM clinical samples, including those from patients treated with the EGFR tyrosine kinase inhibitor lapatinib, uncovered an EGFRvIII-activated, PI3K/SREBP-1-dependent tumor survival pathway through the low-density lipoprotein receptor (LDLR). Targeting LDLR with the liver X receptor (LXR) agonist GW3965 caused inducible degrader of LDLR (IDOL)-mediated LDLR degradation and increased expression of the ABCA1 cholesterol efflux transporter, potently promoting tumor cell death in an in vivo GBM model. These results show that EGFRvIII can promote tumor survival through PI3K/SREBP-1-dependent upregulation of LDLR and suggest a role for LXR agonists in the treatment of GBM patients.

Nuclear Receptor LXR as a Novel Therapeutic Antitumoral Target in Glioblastoma

Both primary and transformed cells need cholesterol for their growth. Guo and colleagues unraveled the connection between epidermal growth factor receptor mutations in glioblastoma and increased cholesterol influx via sterol regulatory element-binding protein 1 and low-density lipoprotein receptor (LDLR) increase. They propose the activation of the liver X receptor-inducible degrader of LDLR-LDLR axis as a therapeutic approach to reduce intracellular cholesterol, block tumor growth, and induce cell death.