Cholesterol in LDL receptor recycling and degradation

Journal Pre-proofs Review Cholesterol in LDL receptor recycling and degradation Hui-xian Yang, Min Zhang, Shi-yin Long, Qin-hui Tuo, Ying Tian, Jianxiong Chen, Cai-ping Zhang, Duan-fang Liao PII: DOI: Reference:

S0009-8981(19)32053-4 https://doi.org/10.1016/j.cca.2019.09.022 CCA 15859

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Clinica Chimica Acta

Received Date: Revised Date: Accepted Date:

29 March 2019 18 September 2019 18 September 2019

Please cite this article as: H-x. Yang, M. Zhang, S-y. Long, Q-h. Tuo, Y. Tian, J-x. Chen, C-p. Zhang, D-f. Liao, Cholesterol in LDL receptor recycling and degradation, Clinica Chimica Acta (2019), doi: https://doi.org/ 10.1016/j.cca.2019.09.022

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Cholesterol in LDL receptor recycling and degradation Author names:　Hui-xian Yanga,c,1, Min Zhangc,1, Shi-yin Longc, Qin-hui Tuob, Ying Tianc, Jian-xiong Chenb,d, Cai-ping Zhangc*, Duan-fang Liaob*. Affiliation: a: Institute of Cardiovascular Disease, Medical College, University of South China, 28# W Changsheng Rd, Hengyang 421001, Hunan, China. b:

Division of Stem Cell Regulation and Application, State Key Laboratory of Chinese Medicine

Powder and Medicine Innovation in Hunan (incubation), Hunan University of Chinese Medicine, 300# Xueshi Rd, Hanpu Science & Education District, Changsha 410208, Hunan, China. c:

Department of Biochemistry and Molecular Biology, Medical College, University of South

China, 28# W Changsheng Rd, Hengyang, 421001, Hunan, China. d:

Department Pharmacology & Toxicology, University of Mississippi Medical Center, USA.

1:

These authors contributed equally to the work.

*Corresponding author: Cai-ping Zhang, PhD (C.–p, Zhang). Address: Department of Biochemistry and Molecular Biology, Medical College, University of South China, 28# W Changsheng Rd, Hengyang, 421001, Hunan, China. Tel: +8607348281372, Fax: +8607348281372, Email addresses: [email protected]. Duan-fang Liao, PhD (D.–f, Liao). Address: Hunan University of Chinese Medicine, 1# Xiangzui Rd, Hanpu Science & Education District Changsha 410208, Hunan, China. Tel: +86073188458002, Fax: +86073188458002, Email addresses: [email protected].

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Abstract The SREBP2/LDLR pathway is sensitive to cholesterol content in the endoplasmic reticulum (ER), while membrane low-density lipoprotein receptor (LDLR) is influenced by sterol response element binding protein 2 (SREBP2), pro-protein convertase subtilisin/kexin type 9 (PCSK9) and inducible degrader of LDLR (IDOL). LDL-C, one of the risk factors in cardiovascular disease, is cleared through endocytosis recycling of LDLR. Therefore, we propose that a balance between LDLR endocytosis recycling and PCSK9-mediated and IDOL-mediated lysosomal LDLR degradation is responsible for cholesterol homeostasis in the ER. For statins that decrease serum LDL-C levels via cholesterol synthesis inhibition, the mechanism by which the statins increase the membrane LDLR may be regulated by cholesterol homeostasis in the ER.

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Keywords: LDL-C; LDLR; SREBP2; PCSK9; IDOL; Dyslipidemia Nonstandard Abbreviations and Acronyms: CM

chylomicron remnants

CRE

cholesterol regulation element

ER

endoplasmic reticulum

FERM

frame of ezrin/radixin/moesin homology

HMGCR

3-hydroxy-3-methylglutaryl-coenzyme A reductase

IDOL

inducible degrader of LDLR

LDL-C

low-density lipoprotein cholesterol

LDLR

low-density lipoprotein receptor

LXRs

liver X receptors

MVB

multivesicular body

MYLIP

myosin regulatory light chain interacting protein

Non-HDL-C

non-high density lipoprotein cholesterol

PCSK9

pro-protein convertase subtilisin/kexin type 9

PTB

phosphotyrosine-binding

RING

really interesting new gene

SCAP

SREBP cleavage-activating protein

SREBP2

sterol response element binding protein 2

3

1. Introduction Non-high-density lipoprotein cholesterol (non-HDL-C) and low-density lipoprotein (LDL) cholesterol (LDL-C) are still the coprimary targets for lipid treatment recommended by the National Lipid Association (NLA) in the Annual Summary of Clinical Lipidology [1, 2]. Non– HDL-C content is equal to that total cholesterol content minus HDL-C content in serum, and it includes LDL-C, intermediate density lipoproteins (IDL), very low-density lipoproteins (VLDL), VLDL remnants, chylomicron remnants (CM), and lipoprotein (a)[3]. Loading non-HDL-C proteins are mostly composed of Apo lipoprotein B100 (Apo B100) and Apo lipoprotein E (Apo E), which are recognized by LDLR (LDL receptor) for the clearance of serum cholesterol; and ~75% of the circulating cholesterol is removed by LDLR endocytic circulation [1, 4]. Furthermore, loss-of-function mutations of the ldlr gene lead to familial hypercholesterolemia (FH) [5]. Consequently, it is important to investigate the mechanisms by which LDLR levels are regulated in the clearance of serum LDL-C. LDLR is regulated in transcriptional and posttranscriptional phases. At the transcriptional level, LDLR mRNA expression is regulated by sterol response element-binding protein 2 (SREBP2), which is sensitive to cholesterol content in the endoplasmic reticulum (ER)[6]. At the posttranscriptional level, proprotein convertase subtilisin/kexin type 9 (PCSK9) reroutes the internalized LDLR degradation in lysosome[7], while inducible degrader of low-density lipoprotein receptor (IDOL), also called the myosin regulatory light chain interacting protein (MYLIP), acts as an E3 ligase to ubiquitylate LDLR, followed by degradation in lysosome[8]. In fact, the cholesterol content of the ER is regulated by cholesterol homeostasis in mammalian cells. The percentages of cholesterol are defined as moles of cholesterol divided by 4

moles of total lipid (sterols plus phospholipids) × 100[6]. When cholesterol content drops below 5% molar in the ER, cholesterol synthesis is activated by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), and cholesterol uptake (endocytosed by binding LDLR) is increased. However, with excess cholesterol accumulation in the ER, the level of both cholesterol synthesis and uptake is reduced, accompanied by an increase in LDLR degradation in lysosome[6]. Statins lower serum LDL-C levels via inhibiting the activity of HMGCR, the limiting enzyme in cholesterol synthesis; at the same time, they promote cytoplasmic membrane LDLR distribution due to activation of the SREBP2/LDLR pathway[9]. LDLR levels on cell surfaces reflect a balance between transcriptional and posttranscriptional processes. Consequently, the mechanism by which statins activate the SREBP2/LDLR pathway and the resulting effect on LDLR distribution on the cell surface are important topics that remain to be addressed. Therefore, we review the current research progress in studies of LDLR distribution on the cell surface regulated at the transcriptional and posttranscriptional levels. 2. SREBP2/LDLR transcriptional pathway is sensitive to cholesterol content in the ER SREBP2 was identified as a transcriptional regulator to maintain cholesterol homeostasis in mammalian cells[10]. The study of the role of SREBP2/LDLR pathway in cholesterol metabolism was carried out in the Chinese hamster ovary (CHO) K1 cells and the human fibroblasts[11]. In order to accurately measure cholesterol content in the ER, Radhakrishnan et al reported the method to obtain highly purified ER membranes in the CHO cell lines, which is also suitable to cultured human fibroblasts[6]. In addition we investigated the SREBP2 homology in five species, such as Homo sapiens, Mus musculus, Rattus norvegicus, Cricetulus griseus and Xenopus tropicalis, and found that their average identity is 92.18% in homology (the ratio of Mus 5

musculus, Rattus norvegicus and Rattus norvegicus to Homo sapiens, respectively), whereas only 67.13% homology in Xenopus tropicalis (Fig. 2). LDLR mRNA expression is regulated by transcriptional factor SREBP2 in response to the ER cholesterol levels in mammalian cells. When cholesterol levels drop below 5% of total lipids in the ER, SREBP cleavage-activating protein (SCAP), a polytopic membrane protein, forms a complex with SREBP2, is coated by CopII proteins, as a vehicle, and transports the SCAP/SREBP2 complex from the ER to the Golgi. In the Golgi, SREBP2 is cleaved by two proteases, site-1 protease and site-2 protease, to release an activation transcriptional factor, nucleus SREBP2, with the NH2-terminal basic-helix-loop-helix-leucine zipper (bHLH-Zip) domain[6] that binds the cholesterol regulation element (CRE) of the LDLR gene promoter to activate LDLR mRNA transcription in the nucleus of mammalian cells[12]. However, when excess cholesterol accumulates in the ER, cholesterol binds SCAP, which induces a conformational change in SCAP that blocks the binding of COPII-coated proteins to a hexapeptide sequence, Met–Glu–Leu–Ala–Asp–Leu (MELADL) of SCAP. In addition, oxysterol, by binding INSIG, also blocks the binding of COPII-coated proteins[13] that remain the SREBP2/SCAP complex in the ER. Moreover, inactivated SREBP2 fails to transcript LDLR mRNA expression, reducing cholesterol uptake to maintain cholesterol homeostasis in mammalian cells[14]. In additional to LDLR, HMGCR is also regulated by SREBP2 [5]. It is well known that statins are competitive inhibitors of HMGCR. Statins were proven to lower serum LDL-C via diminishing cholesterol synthesis, which is their principal mechanism in lowering serum LDL-C to reduce the major vascular events in all age groups[15]; however, they also activate SREBP2, 6

promoting the expression of LDLR and improving cell cholesterol uptake in mammalian cells[16-18]. 3. The mechanism of statins promoting LDL-C uptake by elevated LDLR abundance on cell surfaces. From studies of the role of statins in lowering serum LDL-C and activating transcriptional LDLR mRNA expression, Besides, we also observed that Rosuvastatin lowers the serum LDL-C levels and hepatocyte lipid deposition of Wistar rats in vivo, whereas Rosuvastatin causes an increase both in total cholesterol content and lipid deposition of hepatocyte in vitro (data unpublished). In order to figure out this contrary phenomenon, we put forward a theory that statins ameliorate dyslipidemia by lowering serum LDL-C, making the cholesterol content less than 5% of total lipids in the ER, which promotes SREBP2 transformation into nucleus SREBP2, which is then transported from the Golgi into the nucleus to bind the sterol regulation element (SRE) of LDLR gene promoter, activating LDLR mRNA transcription. Furthermore, at the same time, statins also increase transcriptional HMGCR and PCSK9 mRNA expression via the activation of nucleus SREBP2, which helps balance cholesterol homeostasis in mammalian cells[14]. Free cholesterol is the precursor of steroid hormones, bile acids, and vitamin D3, and is an important component of the cell membrane, maintaining its fluidity in mammalian cells. Statins inhibit the activity of HMGCR, limiting cholesterol synthesis, but transcriptionally activate LDLR expression to improve cholesterol uptake in mammalian cells. We surmise that this is the reason why statins can dramatically lower plasma cholesterol levels but rarely cause side effects in human beings (Fig. 1). Thus, based on the studies described above, we propose a new mechanism by which statins 7

lower serum LDL-C levels: the active SREBP2/LDLR pathway achieved via diminishing cholesterol content (to <5% molar in total lipids) in the ER in mammalian cells. 4. Cholesterol homeostasis may result from a balance between LDLR endocytosis and PCSK9-mediated and IDOL-mediated lysosomal LDLR degradation in mammalian cells 4.1 Diminished

LDLR

levels

in

cytoplasmic

membrane

through

PCSK9

as

a

posttranscriptional regulator When two mutations, S127R and F216L, in the PCSK9 gene that are highly expressed in liver were first reported to result in autosomal dominant hypercholesterolemia[19], many investigations focused on the mechanism of PCSK9 affecting serum cholesterol levels by studying people of different geographical origins, such as Japan and Utah. It was demonstrated that the gain-of-function mutations in the PCSK9 gene elevated serum cholesterol levels and caused autosomal dominant hypercholesterolemia[20-22]. Maxwell et al revealed that PCSK9 posttranscriptionally modulates LDLR function and further affects serum cholesterol levels[23]. Park et al proved that changing LDLR protein levels was due to the influence of PCSK9 on the internalized cycles of LDLR via a posttranscriptional mechanism in 2004[7]. The four loss-of-function mutations, R46L, G106R, N157K and R237W[24], and the two gain-of-function mutations, S127R[18] and D374Y[25], in the PCSK9 gene were designed and then transiently transfected into HepG2 cells to observe the effect on LDLR levels[26]. The above studies demonstrated that the four loss-of-function mutations in the PCSK9 gene increase the amount of cell surface LDLR and internalization of LDLR, whereas by contrast the two gain-of-function mutations decrease the amount of cell surface LDLR. Thereafter, it was proven that PCSK9 is finely upregulated by the basic amino acid convertases furin, 8

PC5/6A[25] and SREBP2[14, 27] and conversely downregulated by the farnesoid X receptor[28]. Because of the gain-of-function mutations in PCSK9 that result in autosomal dominant hypercholesterolemia and diminish the levels of LDLR on the cell plasma membrane, the details of how PCSK9 influences cell surface LDLR levels attracted more research interest. An important discovery was made by McNutt et al, who demonstrated that the ability of PCSK9 to degrade LDLRs is independent of catalytic activity, and the maturity of PCSK9 may be as a chaperone to prevent LDLR endocytosis recycling or target LDLR for degradation in lysosome[29]. Soon after, the idea of inhibiting PCSK9 as a novel strategy for the treatment of hypercholesterolemia was proposed by Lopez[30] and Seidah[31]. The success of this approach was demonstrated by using A H306 mutation in the EGF-A domain of LDLR, which enhanced the binding affinity with PCSK9-treated HepG2 cells and restored the LDLR levels to those of control cells[32]. Then, the neutralizing antibody against PCSK9 was injected in mice and nonhuman primates, which lowered serum cholesterol levels[33]. More and more evidence has identified PCSK9 as a new target to ameliorate dyslipidemia. AMG145 (Evolocumab) and Alirocumab, both monoclonal antibodies to PCSK9[34-36], have brought about significant decreases in LDL cholesterol levels in Phase II and III clinical trials[37, 38]. Almost simultaneously, Evolocumab and Alirocumab were approved as drugs to lower serum LDL cholesterol levels for treating dyslipidemia by the U.S. Food and Drug Administration in 2015[39, 40]. The 2017 NLA Expert Panel recommendations stated that PCSK9 inhibitor therapy was applicable to the following ASCVD associated disease categories: (1) stable ASCVD;

(2)

progressive

ASCVD;

(3)

LDL-C

≥190

mg/dL

(including

polygenic

hypercholesterolemia, heterozygous familial hypercholesterolemia [FH] and the homozygous FH 9

phenotype); and (4) very-high-risk patients with statin intolerance[41]. Further clinical study results showed that in the FOURIER (Further Cardiovascular Outcomes Research with PCSK9 Inhibition in Subjects with Elevated Risk), the primary outcome (a composite of fatal cardiovascular events, nonfatal myocardial infarction, and nonfatal stroke) was decreased 1.5% over 2.2 years by Evolocumab, and in ODYSSEY OUTCOMES (Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab), it is lowered 1.6% over 2.8 years by Alirocumab[42]. It may take some time to evaluate the long-term benefits of both Evolocumab and Alirocumab in lowering serum LDL-C levels. 4.2 Diminished LDLR endocytosis recycling due to LDLR ubiquitination catalyzed by IDOL The other posttranslational modulator of LDLR is IDOL, originally named MYLIP for its interaction with myosin regulatory light chain[43]. To identify the influence of LXR (liver X

receptors) on cholesterol uptake or/and cholesterol effluence in HepG2 cells and primary mouse macrophages, Zelcer et al[8] found that a synthetic LXR ligand reduced LDL uptake. Furthermore, they discovered that the LXR ligand did not change LDLR mRNA levels or the appearance of immature protein but decreased levels of the glycosylated form of LDLR protein and redistributed LDLR protein from the plasma membrane to intracellular compartments. In analyzing their array 9430057C20Rik to reveal which one plays a crucial role in this process, they identified a potential mediator that corresponds to MYLIP, renamed IDOL to match its biological function (Inducible Degrader of the LDLR) [8]. With an N-terminal frame of ezrin/radixin/moesin homology (FERM) domain and a C-terminal really interesting new gene (RING) domain[44, 45], IDOL actually acts as an 10

E3-ubiquitin ligase via its F3b subdomain recognizing the sequence WxxKNxxSI/MxF of the N-terminal to the NPxY motif, which is unique to LDLR, VLDLR, and ApoER2[45], and it triggers the ubiquitination of LDLR, VLDLR, and ApoER2 followed by their degradation in lysosome. IDOL is proven to be transcriptionally regulated by LXR, corresponding to intracellular cholesterol levels[8, 46]. Therefore, Zelcer et al[8] put forward a theory that the LXR-IDOL-LDLR axis is a complementary pathway to SCAP-SREBP for regulation of the uptake and effluence of cellular cholesterol. IDOL as a negative regulator of LDLR began to be noticed and investigated in various fields. Dong et al[47] demonstrated that suppression of Idol gene expression is an additional underlying mechanism by which statins lower serum cholesterol levels in cell models of HepG2 and primary hepatocytes from hamster liver. Moreover, Scotti et al [48] confirmed again that the LXR-IDOL pathway also plays a feedback regulation role in intracellular cholesterol homeostasis in a null-Idol mutation from mouse embryonic stem cell. Moreover, compared to wild-type mice, diminished Idol expression resulted in lower cholesterol and even protected against lipid accumulation in brown adipose tissue[49]. When investigating 122 Mexican individuals (≤10th age 56 subjects

with low LDL-C; and ≥90th age 66 subjects with high LDL-C), Weissglas-Volkov et al[50] revealed that the nonsynonymous SNP rs9370867 (N342S in IDOL) was associated with high total cholesterol, which resulted in more LDLR degradation and decreased LDL uptake. However, in a study of 1384 Italian individuals, Dhyani et al[51] elucidated that the N342S variant neither influenced the plasma lipid profile nor was associated with arteriosclerotic cardiovascular disease; furthermore, they suggested that other single nucleotide polymorphisms in the Idol gene might be associated with cholesterol metabolism. Hong et al[52] demonstrated that treated with GW3965 (a 11

synthetic LXR agonist) in primates, LXR activation induces hepatic IDOL protein expression and lessens LDLR levels, which elevates plasma LDL-C levels, while diminishing IDOL with an antisense oligonucleotide lowers plasma LDL-C levels. The idea of inhibiting IDOL as a novel strategy for improving dyslipidemia has taken hold. First, Lindholm et al[53] proposed that studies on LDLR ubiquitinated by IDOL and then degraded in lysosome opened new possibilities for influencing cholesterol metabolism in various diseases. Furthermore, inhibiting the activity of IDOL was considered a novel approach for reducing cholesterol and ameliorating cardiovascular disease[54-57]. The molecular mechanism by which IDOL influences ubiquitinated LDLR degradation in lysosome attracted numerous researchers. Traditionally, LDLR in the plasma membrane recognizes and binds serum LDL, which is then enclosed within a clathrin-coated pit, followed by separation from the plasma membrane to form a clathrin-coated vesicle in mammalian cells. At the late endosome stage, LDLR releases LDL at low pH and then is recycled into plasma membrane of the mammalian cell while LDL is degraded in the late endosomal fusion lysosomes[58-60]. However, LDLR accumulation on lipid rafts of mammalian plasma membrane triggers ubiquitin enzymes E1, E2, and E3 (IDOL) to cascade-catalyze, resulting in LDLR ubiquitination within epsins-coated vesicles that are then transported by the endosomal sorting complex to multivesicular bodies (MVB) and finally degrade in lysosome[61, 62]. However deubiquitinated IDOL by USP2-69 and USP2-45, the two proteins of USP2 (ubiquitin-specific protease 2), maintains the stability of IDOL and then decrease LDL uptake of cells[63]. 4.3 Presume that a balance that maintains cholesterol homeostasis exists between the SREBP2/LDLR pathway and PCSK9-mediated and IDOL-mediated LDLR lysosomal 12

degradation in mammalian cells In view of the excellent work discussed above, we think that a balance that maintains cholesterol homeostasis exists between LDLR endocytosis recycling (clathrin-coated) and epsins-coated vesicle mediated LDLR degradation in lysosome in mammalian cells. Therefore, we propose that ER cholesterol levels control this balance. When cholesterol exceeds 5% of total lipids in the ER, the SCAP/SREBPs pathway is inhibited, and the LXR/IDOL/LDLR axis would be activated, decreasing cholesterol uptake and promoting cholesterol effluence in mammalian cells. In contrast, when cholesterol content is less than 5% of total lipids in the ER, the SCAP/SREBPs pathway is activated to enhance transcriptional LDLR mRNA levels, simultaneously accompanied by PCSK9 mRNA expression, while the LXR/IDOL/LDLR axis would be hindered, thereby elevating cholesterol uptake and lowering cholesterol effluence in mammalian cells. Of course, this hypothesis needs to be proven in in vitro and in vivo experiments (Fig. 3). 5. Conclusion and prospects Above all, lowering plasma LDL-C levels is a major therapeutic strategy for reducing the incidence of atherosclerotic cardiovascular disease. The majority of plasma LDL-C is recognized and eliminated by LDLR endocytosis recycling in hepatocytes. The goal of increasing LDLR abundance on the cell membrane surface remains a sound strategy for ameliorating dyslipidemia. LDLR is regulated by transcriptional SREBP2 and by posttranslational PCSK9 and IDOL. At the transcriptional level, statins inhibit the role of HMGCR in cholesterol biosynthesis and increase LDLR mRNA expression via activating SREBP2, which enhances cholesterol uptake to maintain cholesterol homeostasis. PCSK9, one of the nucleus SREBP2 transcriptional activation 13

proteins, has been shown through antibody injections (such as Evolocumab and Alirocumab) to neutralize the role of mature PCSK9 in plasma and improve dyslipidemia. Given the observed role that IDOL in cynomolgus monkeys played in lowering serum LDL-C levels, it seems clear that, sooner or later, IDOL will become a new target to clear serum LDL-C and reduce the incidence of atherosclerotic cardiovascular disease. It is critical to delineate the balance that exists between SREBP2, PCSK9 and IDOL in maintaining cholesterol homeostasis.

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Conflict of interest The authors declare no conflict of interest.

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Acknowledgements This work was supported by the National Nature Science Fund of China (No. 81600291, No.81673722, No. 81670268 and No. 31871169), the Nature Science Fundation of Hunan province (No. 2018JJ2348 and No. 2018JJ2346), Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study (No. 0223-0002-0002000-54), and the Construct Program of the Pharmaceutical Science Key Discipline in Hunan Province.

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Fig. 1 The possible mechanism of statins activating the SREBP2/LDLR pathway via decreasing cholesterol content to less than 5% of total lipids in the endoplasmic reticulum Statins not only competitively inhibit the activity of HMGCR to diminish cholesterol synthesis but also activate the SREBP2/LDLR pathway to enhance transcriptional expression of LDLR. Thus, statins elevate levels of LDLR on cell surfaces to promote LDL-C uptake, the mechanism of which possibly involves cholesterol content dropping below 5% of total lipids in the endoplasmic reticulum.

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Fig. 2 Homology tree of SREBP2 in five species SREBP2 homology was analyzed in Homo sapiens, Mus musculus, Rattus norvegicus, Cricetulus griseus and Xenopus tropicalis. The protein sequence of each species is alignment with Homo sapiens, and the results showed that the percentages of Mus musculus, Rattus norvegicus, Cricetulus griseus and Xenopus tropicalis to Homo sapiens are 91.76%, 92.38%, 92.39%, 67.13%, respectively.

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Fig. 3 Hypothesis that LDLR distribution on cell surface may be regulated by cholesterol content in the ER The SREBP2 pathway (in green lines) is sensitive to cholesterol content in the ER. When cholesterol content drops below 5% of total lipids in the ER, which can be caused by statins that competitively inhibit the activity of HMGCR, the exposed hexapeptide MELADL sequence of SCAP binds CopII coat proteins, then transports the SCAP/SREBP2 complex from the ER to the Golgi where SREBP2 is split by two proteases to form the active nucleus SREBP2 (nSREBP2) with the N-terminal bHLH-Zip domain. Then, nSREBP2 enters the nucleus, binds to the sterol regulation element (SRE), and activates LDLR mRNA transcription. LDLR removes serum LDL-C by means of LDLR endocytosis recycling (in black lines). At the same time, IDOL protein expression (in red lines) is inhibited, therefore reducing IDOL-mediated LDLR ubiquitination, which is followed by degradation in lysosome. However, when there is excess cholesterol accumulation in the ER, the hexapeptide MELADL sequence of SCAP that binds with cholesterol to change its conformation fails to be covered with CopII coat proteins, and therefore, the SCAP/SREBP2 complex remains in the ER and inhibits LDLR mRNA transcription. When the active nSREBP2 is increased, both LDLR and PCSK9 mRNA transcriptional levels are elevated simultaneously. Because PCSK9 acts as a chaperone to mediate LDLR degradation in lysosome (in orange lines), we hypothesize that PCSK9 may be involved in fine-tuning cholesterol homeostasis in mammalian cells.

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