Proprotein Convertase Subtilisin/kexin 9 Gene Research Articles

The Application of Peptide Nucleic Acids (PNA) in the Inhibition of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Gene Expression in a Cell-Free Transcription/Translation System.

Proprotein convertase subtilisin/kexin 9 (PCSK9) is a protein that plays a key role in the metabolism of low-density lipoprotein (LDL) cholesterol. The gain-of-function mutations of the PCSK9 gene lead to a reduced number of surface LDL receptors by binding to them, eventually leading to endosomal degradation. This, in turn, is the culprit of hypercholesterolemia, resulting in accelerated atherogenesis. The modern treatment for hypercholesterolemia encompasses the use of biological drugs against PCSK9, like monoclonal antibodies and gene expression modulators such as inclisiran-a short, interfering RNA (siRNA). Peptide nucleic acid (PNA) is a synthetic analog of nucleic acid that possesses a synthetic peptide skeleton instead of a phosphate-sugar one. This different structure determines the unique properties of PNA (e.g., neutral charge, enzymatic resistance, and an enormously high affinity with complementary DNA and RNA). Therefore, it might be possible to use PNA against PCSK9 in the treatment of hypercholesterolemia. We sought to explore the impact of three selected PNA oligomers on PCSK9 gene expression. Using a cell-free transcription/translation system, we showed that one of the tested PNA strands was able to reduce the PCSK9 gene expression down to 74%, 64%, and 68%, as measured by RT-real-time PCR, Western blot, and HPLC, respectively. This preliminary study shows the high applicability of a cell-free enzymatic environment as an efficient tool in the initial evaluation of biologically active PNA molecules in the field of hypercholesterolemia research. This cell-free approach allows for the omission of the hurdles associated with transmembrane PNA transportation at the early stage of PNA selection.

The Causal Relationship between PCSK9 Inhibitors and Malignant Tumors: A Mendelian Randomization Study Based on Drug Targeting.

Objective: This study explores the potential causal association between proprotein convertase subtilisin/kexin 9 (PCSK9) inhibitors and tumor development using Mendelian randomization (MR) based on drug targets. Methods: Instrumental variables within ±100 kb of the PCSK9 gene locus, impacting low-density lipoprotein cholesterol (LDL-C), were utilized for MR analysis. Coronary heart disease (CHD) served as a positive control to validate the causal relationship between PCSK9 inhibitors and various cancers. We employed reverse MR to address the reverse causation concerns. Data from positive controls and tumors were sourced from OpenGWAS. Results: MR analysis suggested a negative causal relationship between PCSK9 inhibitors and both breast and lung cancers (95%CIBreast cancer 0.81~0.99, p = 2.25 × 10-2; 95%CILung cancer 0.65~0.94, p = 2.55 × 10-3). In contrast, a positive causal link was observed with gastric, hepatic, and oral pharyngeal cancers and cervical intraepithelial neoplasia (95%CIGastric cancer 1.14~1.75, p = 1.88 × 10-2; 95%CIHepatic cancer 1.46~2.53, p = 1.16 × 10-2; 95%CIOral cavity and pharyngeal cancer 4.49~6.33, p = 3.36 × 10-4; 95%CICarcinoma in situ of cervix uteri 4.56~7.12, p = 6.91 × 10-3), without heterogeneity or pleiotropy (p > 0.05). Sensitivity analyses confirmed these findings. The results of MR of drug targets suggested no causal relationship between PCSK9 inhibitors and bladder cancer, thyroid cancer, pancreatic cancer, colorectal cancer, malignant neoplasms of the kidney (except for renal pelvis tumors), malignant neoplasms of the brain, and malignant neoplasms of the esophagus (p > 0.05). Reverse MR helped mitigate reverse causation effects. Conclusions: The study indicates a divergent causal relationship of PCSK9 inhibitors with certain cancers. While negatively associated with breast and lung cancers, a positive causal association was observed with gastric, hepatic, oral cavity, and pharyngeal cancers and cervical carcinoma in situ. No causal links were found with bladder, thyroid, pancreatic, colorectal, certain kidney, brain, and esophageal cancers.

Schisandrin A in Schisandra chinensis Upregulates the LDL Receptor by Inhibiting PCSK9 Protein Stabilization in Steatotic Model.

Schisandra chinensis extract (SCE) protects against hypocholesterolemia by inhibiting proprotein convertase subtilisin/kexin 9 (PCSK9) protein stabilization. We hypothesized that the hypocholesterolemic activity of SCE can be attributable to upregulation of the PCSK9 inhibition-associated low-density lipoprotein receptor (LDLR). Male mice were fed a low-fat diet or a Western diet (WD) containing SCE at 1% for 12 weeks. WD increased final body weight and blood LDL cholesterol levels as well as alanine transaminase and aspartate aminotransferase expression. However, SCE supplementation significantly attenuated the increase in blood markers caused by WD. SCE also attenuated WD-mediated increases in hepatic LDLR protein expression in the obese mice. In addition, SCE increased LDLR protein expression and attenuated cellular PCSK9 levels in HepG2 cells supplemented with delipidated serum (DLPS). Non-toxic concentrations of schisandrin A (SA), one of the active components of SCE, significantly increased LDLR expression and tended to decrease PCSK9 protein levels in DLPS-treated HepG2 cells. High levels of SA-mediated PCSK9 attenuation was not attributable to reduced PCSK9 gene expression, but was associated with free PCSK9 protein degradation in this cell model. Our findings show that PCSK9 secretion can be significantly reduced by SA treatment, contributing to reductions in free cholesterol levels.

Role of PCSK9 in Homocysteine-Accelerated Lipid Accumulation in Macrophages and Atherosclerosis in ApoE-/- Mice.

Background: Homocysteine (Hcy) has been established as an independent risk factor for atherosclerosis, and the involvement of hyperhomocysteinemia (HHcy) in atherosclerotic lesions is complex. Proprotein convertase subtilisin kexin 9 (PCSK9) has vital importance in lipid metabolism, and its inhibitors have intense lipid-lowering and anti-atherosclerotic effects. However, the underlying effect of PCSK9 on HHcy-accelerated dyslipidemia of macrophages is still uncertain. The purpose of this study was to investigate the potential role of PCSK9 in Hcy-induced lipid accumulation and atherosclerotic lesions.Methods: In vitro, gene and protein expressions were assessed by real-time quantitative PCR and western blot in THP-1 macrophages with Hcy incubation. Lipid accumulation and cholesterol efflux were evaluated with Hcy treatment. SBC-115076 was used to examine the role of PCSK9 in ATP-binding cassette transporter A1 and G1 (ABCA1 and ABCG1)-dependent cholesterol efflux. In vivo, lesion area, lipid deposition and collagen contents were determined in aortas of ApoE−/− mice under a methionine diet. SBC-115076 was subcutaneously injected to explore the potential effects of PCSK9 inhibition on alleviating the severity of HHcy-related atherosclerotic lesions.Results: In THP-1 macrophages, Hcy dose- and time-dependently promoted PCSK9 gene and protein levels without regulating the translation of Low-density lipoprotein receptor (LDLR). SBC-115076 used to inhibit PCSK9 largely alleviated lipid accumulation and reversed the cholesterol efflux to apolipoprotein-I(apoA-I) and high-density lipoprotein (HDL) mediated by ABCA1 and ABCG1. In ApoE−/− mice, methionine diet induced HHcy caused larger lesion area and more lipid accumulation in aortic roots. SBC-115076 reduced atherosclerotic severity by reducing the lesion area and lipid accumulation and increasing expressions of ABCA1 and ABCG1 in macrophages from atherosclerotic plaque. In addition, SBC-115076 decreased plasma Hcy level and lipid profiles significantly.Conclusion: PCSK9 promoted lipid accumulation via inhibiting cholesterol efflux mediated by ABCA1 and ABCG1 from macrophages and accelerated atherosclerotic lesions under HHcy treatment. Inhibiting PCSK9 may have anti-atherogenic properties in HHcy-accelerated atherosclerosis.

New Pharmacological Approaches to Target PCSK9.

Proprotein convertase subtilisin kexin 9 (PCSK9) plays a crucial role in regulating circulating levels of LDL-C as a consequence of its ability to inhibit LDL receptor recycling in the liver. Loss of function variants in the PCSK9 gene result in low LDL-C levels and associate with reduced cardiovascular risk, whereas gain of-function variants associate with hypercholesterolemia and increased risk of early cardiovascular events. Thus, PCSK9 inhibition has been established as an additional approach for the treatment of hypercholesterolemia. The aim of this review is to provide a brief overview of current strategies targeting PCSK9 and discuss clinical results of the emerging approaches. Two monoclonal antibodies targeting circulating PCSK9 (evolocumab and alirocumab) have been approved for the treatment of hypercholesterolemia and cardiovascular disease. Later, a gene silencing approach (inclisiran), which inhibits hepatic PCSK9 synthesis, was shown to be as effective as monoclonal antibodies but with a twice a year injection and is currently under evaluation for approval. Due to the elevated costs of such therapies, several other approaches have been explored, including peptide-based anti PCSK9 vaccination, and small oral PCSK9 inhibitors, which are still in preclinical phase. In the coming years, we will assist to a progressive introduction of novel anti-PCSK9 approaches in the clinical practice for the treatment of patients with hypercholesterolemia as well as patients at high cardiovascular risk.

1441LRP5 and PCSK9 in inflammatory cells immunomodulation

Abstract Background Atherosclerosis, the leading cause of cardiovascular diseases, is driven by high blood cholesterol levels and chronic inflammation. The disruption of the hepatic interaction between Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) and Low-Density Lipoprotein Receptor (LDLR) downregulates blood cholesterol levels and reduces cardiovascular events. Recent data suggest that other members of the LDLR superfamily may be targets of PCSK9. Purpose The aim of this work is to determine if LDLR-related protein 5 (LRP5) is a PCSK9 target, and to study the role of PCSK9 and LRP5 in foam cell formation and hence, in the mechanism of lipid accumulation and atherosclerotic plaque formation. Methods Intracellular protein and lipid localization, cholesteryl esters (CE) accumulation; quantification of structural and inflammatory proteins expression and immunoprecipitation analyses were performed in primary cultures of human inflammatory cells (monocytes and macrophages) silenced for LRP5 or PCSK9 and challenged with modified LDLs. Results We first show that LRP5 is needed for macrophage lipid uptake since LRP5-silenced macrophages have less intracellular CE accumulation. In LDL treated macrophages internalization of LRP5-bound LDL starts after 30 minutes of incubation and lasts up to 24hours. The SREBP-2 promoter is not involved in LRP5 regulation but it does regulate macrophage PCSK9 expression. Immunoprecipitation experiments show that LRP5 forms a complex with PCSK9 in lipid-loaded macrophages. Finally we studied the role of PCSK9 and LRP5 in the inflammatory response by TLR4/NFkB signaling pathway. We show decreased TLR4 protein expression levels and decreased nuclear translocation of NFκB in PCSK9 silenced-inflammatory cells after lipid loading indicating a downregulation of the proinflammatory pathway TLR4/NFκB. Increased gene expression is observed in TNF-α and IL1β after lipid-loading that is abolished in PCSK9-silenced macrophages. Furthermore release of the proinflammatory cytokines TNF-α and IL1β is decreased in PCSK9-silenced macrophages. LRP5 protein expression is increased in lipid-loaded macrophages independent of the presence or absence of PCSK9. Conclusions These results demonstrate that, in human macrophages, LRP5 is internalized with lipids. Furthermore, PCSK9 and LRP5 can form a complex in the cytoplasm of lipid-loaded macrophages opening the possibility that PCSK9 induces lysosomal LRP5 degradation in a similar manner than it does with LDLR. Finally we also show that PCSK9 gene interference decreases inflammation and supports a role for PCSK9 as an inflammatory mediator in atherosclerosis. Acknowledgement/Funding CN16/11/00411-LB; TERCEL RD16/0011/018-LB; FIS2016-02014 -MBP; SEC2015 to MBP

Spectrum of mutations of familial hypercholesterolemia in the 22 Arab countries

Background and aimsFamilial hypercholesterolemia (FH) is an inherited genetic disorder of lipid metabolism characterized by a high serum LDL-cholesterol profile and xanthoma formation, and FH increases the risk of premature atherosclerosis and cardiovascular disease (CVD). Mutations in the low-density lipoprotein (LDLR), apolipoprotein B (APOB), proprotein convertase subtilisin/kexin 9 (PCSK9), and LDLRAP1 genes have been associated with FH. Although FH is a major risk for CVD, the disease prevalence and its underlying molecular basis in the 22 Arab countries are still understudied. This study aimed to analyze all peer-reviewed studies related to the prevalence of FH and its causative mutations in the 22 Arab countries. MethodsWe searched five literature databases (Scopus, Science Direct, Web of Science, PubMed, and Google Scholar) from inception until June 2018, using all possible search terms to capture all of the genetic and prevalence data related to Arab patients with FH. ResultsA total of 5,484 titles and abstracts were identified; 51 studies met our inclusion criteria for the final systematic review. Fifty-one mutations in Arab patients with FH were identified in only eight Arab countries; 47 were identified in the LDLR gene, two in the PCSK9 gene, and two in LDLRAP1 gene. Twenty mutations in the LDLR gene were distinctive to Arab patients. A few studies reported prevalence estimates, ranging from 0.4% to 6.8%. ConclusionsThis is the first systematic review to dissect the up-to-date status of the genetic epidemiology of Arab patients with FH. It seems that FH is underdiagnosed and that its prevalence is understudied due to the dearth of published information about Arab patients with FH. Therefore, there is a need for well-controlled genetic epidemiological studies on Arab patients with FH.

Targeting pro-protein convertase subtilisin kexin-9 as a novel therapy of hypercholesterolemia

Reducing low density lipoprotein (LDL) cholesterol level is an established primary and secondary prevention strategy for coronary heart disease. However, not all patients are able to achieve their LDL targets as recommended by the guidelines. Over the last 10 years, high plasma LDL level is known to be associated with a higher level of pro-protein convertase subtilisin kexin-9 (PCSK-9). Loss-of-function mutations in the PCSK-9 gene is associated with lower plasma LDL level and cardiovascular risk. Since its discovery in 2003, PCSK-9 has triggered many researchers to design a PCSK-9 inhibitor to reduce LDL cholesterol through competitive inhibition of this molecule. Some phase III clinical trials have showed promising results of PCSK-9 inhibitor efficacy in lowering LDL level and improving clinical outcome. This article aims to discuss the role of PCSK-9 in LDL metabolism and the efficacy of PCSK-9 inhibitor in reducing plasma LDL level.

Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Inhibition in Patients With or at High Risk of Coronary Heart Disease

Proprotein convertase subtilisin/kexin 9 (PCSK9) is an enzyme that binds and inactivates the low-density lipoprotein (LDL) receptor onhepatocytes, leading to higher levels of serum LDL cholesterol (LDL-C). Individuals with ‘loss of function’ mutations in the PCSK9 gene havelower LDL-C levels and are at decreased risk of coronary heart disease. In light of this, inhibition of PCSK9 activity has become a target forreducing LDL-C levels, with approaches including monoclonal antibodies, antisense oligonucleotides, and RNA interference technology. Twomonoclonal antibodies to PCSK9, alirocumab and evolocumab, have recently been FDA approved. Although large-scale clinical trials to assesscardiovascular outcomes are awaited, reductions in LDL-C levels have been demonstrated, with no increase in muscle-related adverse effects.In addition to these monoclonal antibody approaches, inclisiran – a molecule that inhibits PCSK9 synthesis by RNA interference – is currentlyunder development. Phase 1 and 2 studies indicate an LDL-C-lowering effect comparable to that of monoclonal antibodies, with effectspersisting to as long as 180 days. Phase 3 studies are planned investigating dose administration two to three times per year. The aim of this report is to summarize the role of PCSK9 inhibition in the lowering of LDL-C. Approaches to PCSK9 inhibition are discussed, and an up-todate insight into current developments in the field is provided.

Biology of proprotein convertase subtilisin kexin 9: beyond low-density lipoprotein cholesterol lowering.

Proprotein convertase subtilisin kexin 9 (PCSK9) is a key regulator of low-density lipoprotein receptor levels and LDL-cholesterol levels. Loss-of-function mutations in PCSK9 gene are associated with hypocholesterolaemia and protection against cardiovascular disease, identifying PCSK9 inhibition as a valid therapeutic approach to manage hypercholesterolaemia and related diseases. Although PCSK9 is expressed mainly in the liver, it is present also in other tissues and organs with specific functions, raising the question of whether a pharmacological inhibition of PCSK9 to treat hypercholesterolaemia and associated cardiovascular diseases might be helpful or deleterious in non-hepatic tissues. For example, PCSK9 is expressed in the vascular wall, in the kidneys, and in the brain, where it was proposed to play a role in development, neurocognitive process, and neuronal apoptosis. A link between PCSK9 and immunity was also proposed as both sepsis and viral infections are differentially affected in the presence or absence of PCSK9. Despite the increasing number of observations, the debate on the exact roles of PCSK9 in extrahepatic tissues is still ongoing, and as very effective drugs that inhibit PCSK9 have become available to the clinician, a better understanding of the biological roles of PCSK9 is warranted.

Proprotein convertase subtilisin/kexin 9 V4I variant with LDLR mutations modifies the phenotype of familial hypercholesterolemia

Familial hypercholesterolemia (FH) is caused by mutations in the genes encoding low-density lipoprotein receptor (LDLR), apolipoprotein B, or proprotein convertase subtilisin/kexin 9 (PCSK9). However, FH shows variability of the clinical phenotype modified by other genetic variants or environmental factors. Our objective was to determine the distribution of PCSK9 variants in Japanese FH heterozygotes and to clarify whether those variants and the combination of those variants and LDLR mutations modify the clinical phenotypes. A direct sequence analysis was performed for all 18 exons of LDLR gene and 12 exons of PCSK9 gene in 269 clinically diagnosed FH heterozygotes. The serum lipid levels of the carriers of each variant were compared to those of noncarriers. We also assessed Achilles tendon xanthoma and the prevalence of coronary artery disease (CAD) in the patients aged ≥30years. Eleven PCSK9 variants were detected. There were 4 frequent PCSK9 variants: L21_22insL, A53V, V4I, and E32K. The PCSK9 L21_22insL and A53V were in linkage disequilibrium with each other. There were no significant differences in serum lipids levels and the prevalence of CAD at the age of ≥ 30years between PCSK9 V4I, L21_22insL/A53V, or E32K variant carriers and noncarriers without LDLR mutations. In the patients carrying LDLR mutations and aged ≥ 30years, the additional PCSK9 V4I variant was linked to a significantly increased prevalence of CAD in accord with the elevation of the LDL-cholesterol level. The addition of the PCSK9 V4I was suggested to modify the phenotype of patients carrying LDLR mutations by affecting their LDLR metabolism.

One-shot, one cure with genome editing for dyslipidemia.

One of the most inspiring examples of rapid bench-to-bedside translation has been the development of proprotein convertase subtilisin/kexin 9 (PCSK9) inhibitory antibodies for the reduction of serum low-density lipoprotein and prevention of coronary artery disease. Astute genetic analysis of gain-of-function mutations in patients with elevated low-density lipoprotein (LDL) levels1 built the case for the role of PCSK9 in LDL receptor binding and lysosomal destruction.2 Now the prospect of lipid-lowering therapy delivered monthly via injection is on the horizon with successful clinical trials for the 2 leading monoclonal antibodies.3 The logical next expectation would be for a permanent disruption of the PCSK9 gene, rendering continued therapy unnecessary because the naturally occurring and protective loss-of-function mutation could be given as a 1-time treatment. Ding et al4 present evidence that the technology for this now exists with genome editing and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) …

Correlation of PCSK9 gene polymorphism with cerebral ischemic stroke in Xinjiang Han and Uygur populations.

BackgroundCerebral ischemic stroke (CIS) is a major cause of morbidity and mortality. Its main pathological basis is atherosclerosis (AS); in turn, the main risk factor in AS is dyslipidemia. Human proprotein convertase subtilisin/kexin9 (PCSK9) plays a key role in regulating plasma low-density lipoprotein (LDL) cholesterol levels. We sought to assess the association between PCSK9 and CIS in Chinese Han and Uygur populations.Material/MethodsWe selected 408 CIS patients and 348 control subjects and used a single-base terminal extension (SNaPshot) method to detect the genotypes of the 20 single-nucleotide polymorphisms (SNPs) in PCSK9.ResultsDistribution of SNP8 (rs529787) genotypes showed a significant difference between CIS and control participants (P=0.049). However, when analyzing Han and Uygur populations separately, we found that only Han subjects showed distribution of SNP1 (rs1711503), SNP2 (rs2479408), and SNP8 (rs529787) alleles that was significantly different between CIS and control participants (P=0.028, P=0.013, P=0.006, respectively), and distribution of SNP2 (rs2479408) in the dominant model (CC vs. CG + GG) was significantly different between CIS and control participants (P=0.013), even after adjustment for covariates (OR: 75.262, 95% confidence interval [CI]: 7.232–783.278, P<0.001). Distribution of the 2 haplotypes (A-C and G-C) (rs1711503 and rs2479408) was significantly different between CIS and control participants (both, P=0.011).ConclusionsBoth rs1711503 and rs2479408 of PCSK9 genes were associated with CIS in the Han population of China. A-C haplotype may be a genetic marker of CIS risk in this population.

Identification and characterization of new gain-of-function mutations in the PCSK9 gene responsible for autosomal dominant hypercholesterolemia

BackgroundThe identification of mutations in PCSK9 (proprotein convertase subtilisin kexin9) in autosomal dominant hypercholesterolemia (ADH), has revealed the existence of a new player in cholesterol homeostasis. PCSK9 has been shown to enhance the degradation of the LDL receptor (LDLR) at the cell surface. Gain-of-function mutations of PCSK9 induce ADH and are very rare, but their identification is crucial in studying PCSK9's role in hypercholesterolemia, its detailed trafficking pathway and its impact on the LDLR. MethodsIn order to identify new mutations and understand the exact mechanisms of action of mutated PCSK9, PCSK9 was sequenced in 75 ADH patients with no mutations in the LDLR or APOB genes. Functional analyses in cell culture were conducted and the impact of novel PCSK9 mutations on the quantitative and qualitative features of lipoprotein particles and on the HDL-mediated cellular cholesterol efflux was studied. ResultsAmong these 75 ADH probands with no mutations in the LDLR or APOB genes, four gain-of-function mutations of PCSK9 were identified, of which two were novel: the p.Leu108Arg and the p.Asp35Tyr substitutions. In vitro studies of their consequences on the activity of PCSK9 on cell surface levels of LDLR showed that the p.Leu108Arg mutation clearly results in a gain-of-function, while the p.Asp35Tyr mutation created a novel Tyr-sulfation site, which may enhance the intracellular activity of PCSK9. ConclusionThese data further contribute to the characterization of PCSK9 mutations and to better understanding of the impact on cholesterol metabolism of this new therapeutic target.

A new method for measurement of total plasma PCSK9: clinical applications

The proprotein convertase subtilisin kexin-9 (PCSK9) circulates in plasma as mature and furin-cleaved forms. A polyclonal antibody against human PCSK9 was used to develop an ELISA that measures total plasma PCSK9 rather than only the mature form. A cross-sectional study evaluated plasma levels in normal (n = 254) and hypercholesterolemic (n = 200) subjects treated or untreated with statins or statin plus ezetimibe. In controls, mean plasma PCSK9 (89.5 +/- 31.9 ng/ml) correlated positively with age, total cholesterol, LDL-cholesterol (LDL-C), triglycerides, and fasting glucose. Sequencing PCSK9 from individuals at the extremes of the normal PCSK9 distribution identified a new loss-of-function R434W variant associated with lower levels of circulating PCSK9 and LDL-C. In hypercholesterolemic subjects, PCSK9 levels were higher than in controls (99.3 +/- 31.7 ng/ml, P < 0.04) and increased in proportion to the statin dose, combined or not with ezetimibe. In treated patients (n = 139), those with familial hypercholesterolemia (FH; due to LDL receptor gene mutations) had higher PCSK9 values than non-FH (147.01 +/- 42.5 vs. 127.2 +/- 40.8 ng/ml, P < 0.005), but LDL-C reduction correlated positively with achieved plasma PCSK9 levels to a similar extent in both subsets (r = 0.316, P < 0.02 in FH and r = 0.275, P < 0.009 in non-FH). The detection of circulating PCSK9 in both FH and non-FH subjects means that this assay could be used to monitor response to therapy in a wide range of patients.

The unique role of proprotein convertase subtilisin/kexin 9 in cholesterol homeostasis

The LDL receptor (LDLR) plays an essential role in the regulation of plasma (LDL) cholesterol concentrations by virtue of its ability to clear plasma LDL. Down-regulation of the LDLR by proprotein convertase subtilisin/kexin 9 (PCSK9) has recently emerged as a regulatory mechanism that controls plasma LDL cholesterol concentrations. Studies in which PCSK9 is over-expressed in mice, have demonstrated that PCSK9, by enhancing hepatic LDLR degradation, decreases the availability of the LDLR for LDL uptake, resulting in increased plasma LDL cholesterol levels. However, PCSK9 has also recently been shown to mediate down-regulation of surface receptors other than the LDLR, suggesting that it may have much broader roles than initially thought.

Identification of mutations in the apolipoprotein B-100 gene and in the PCSK9 gene as the cause of hypocholesterolemia

Background Characterization of the normally occurring mutations as the cause of hypocholesterolemia may increase our understanding of the normal lipid metabolism. Methods DNA from 93 unrelated hypocholesterolemic subjects with a mean (±SD) value for total serum cholesterol of 3.3 (± 0.5) mmol/l) were subjected to DNA sequencing of the individual exons of the apolipoprotein B-100 (apoB-100) gene and of the proprotein convertase subtilisin/kexin 9 (PCSK9) gene. The same analyses were also performed in 23 unrelated subjects with autosomal dominant hypercholesterolemia who had unusually low levels of total serum cholesterol. Results Of the 93 hypocholesterolemic subjects, 9 subjects (9.7%) were heterozygous for a truncating mutation in the apoB-100 gene and six subjects (6.5%) were heterozygous for a loss-of-function mutation in the PCSK9 gene. Of the 23 subjects with autosomal dominant hypercholesterolemia, four subjects (17.4%) were heterozygous for mutations in the apoB-100 gene. Conclusion Truncating mutations in the apoB-100 gene are slightly more common as the cause of hypocholesterolemia compared to loss-of-function mutations in the PCSK9 gene. It appears that mutations in the apoB-100 gene may completely normalize the lipid profile in subjects with autosomal dominant hypercholesterolemia, whereas loss-of-function mutations in the PCSK9 gene do not have a sufficient cholesterol-lowering capacity.

Errata. PCSK9 function and physiology

PCSK9 has exploded onto center stage of plasma cholesterol metabolism, raising hopes for a new strategy to treat hypercholesterolemia. PCSK9 in a plasma protein that triggers increased degradation of the LDL receptor. Gain-of-function mutations in PCSK9 reduce LDL receptor levels in the liver, resulting in high levels of LDL cholesterol in the plasma and increased susceptibility to coronary heart disease. Loss-of-function mutations lead to higher levels of the LDL receptor, lower LDL cholesterol levels, and protection from coronary heart disease. Two papers in this issue of the Journal of Lipid Research exemplify the rapid pace of progress in understanding PCSK9 molecular interactions and physiology. Dr. Shilpa Pandit and coworkers from Merck Research Laboratories describe the functional basis for the hypercholesterolemia associated with gain-of-function missense mutations in PCSK9. Dr. Jay Horton's group at UT Southwestern describe the kinetics and metabolism of PCSK9 and the impact of PCSK9 on LDL receptors in the liver and adrenal gland.

The Cellular Trafficking of the Secretory Proprotein Convertase PCSK9 and Its Dependence on the LDLR

Mutations in the proprotein convertase PCSK9 gene are associated with autosomal dominant familial hyper- or hypocholesterolemia. These phenotypes are caused by a gain or loss of function of proprotein convertase subtilisin kexin 9 (PCSK9) to elicit the degradation of the low-density lipoprotein receptor (LDLR) protein. Herein, we asked whether the subcellular localization of wild-type PCSK9 or mutants of PCSK9 and the LDLR would provide insight into the mechanism of PCSK9-dependent LDLR degradation. We show that the LDLR is the dominant partner in regulating the cellular trafficking of PCSK9. In cells lacking the LDLR, PCSK9 localized in the endoplasmic reticulum (ER). In cells expressing the LDLR, PCSK9 sorted to post-ER compartments (i.e. endosomes in cell lines and Golgi apparatus in primary hepatocytes), where it colocalized with the LDLR. In cell lines, PCSK9 also colocalized with the LDLR at the cell surface, requiring the presence of the C-terminal Cys/His-rich domain of PCSK9. We provide evidence that PCSK9 promotes the degradation of the LDLR by an endocytic mechanism, as small interfering RNA-mediated knockdown of the clathrin heavy chain reduced the functional activity of PCSK9. We also compared the subcellular localization of natural mutants of PCSK9 with that of the wild-type enzyme in human hepatic (HuH7) cells. Whereas the mutants associated with hypercholesterolemia (S127R, F216L and R218S) localized to endosomes/lysosomes, those associated with hypocholesterolemia did not reach this compartment. We conclude that the sorting of PCSK9 to the cell surface and endosomes is required for PCSK9 to fully promote LDLR degradation and that retention in the ER prevents this activity. Mutations that affect this transport can lead to hyper- or hypocholesterolemia.

Hepatic PCSK9 Expression Is Regulated by Nutritional Status via Insulin and Sterol Regulatory Element-binding Protein 1c

Familial autosomal dominant hypercholesterolemia is associated with high risk for cardiovascular accidents and is related to mutations in the low density lipoprotein receptor or its ligand apolipoprotein B (apoB). Mutations in a third gene, proprotein convertase subtilisin kexin 9 (PCSK9), were recently associated to this disease. PCSK9 acts as a natural inhibitor of the low density lipoprotein receptor pathway, and both genes are regulated by depletion of cholesterol cell content and statins, via sterol regulatory element-binding protein (SREBP). Here we investigated the regulation of PCSK9 gene expression during nutritional changes. We showed that PCSK9 mRNA quantity is decreased by 73% in mice after 24 h of fasting, leading to a 2-fold decrease in protein level. In contrast PCSK9 expression was restored upon high carbohydrate refeeding. PCSK9 mRNA increased by 4-5-fold in presence of insulin in rodent primary hepatocytes, whereas glucose had no effect. Moreover, insulin up-regulated hepatic PCSK9 expression in vivo during a hyperinsulinemic-euglycemic clamp in mice. Adenoviral mediated overexpression of a dominant or negative form of SREBP-1c confirmed the implication of this transcription factor in insulin-mediated stimulation of PCSK9 expression. Liver X receptor agonist T0901317 also regulated PCSK9 expression via this same pathway (a 2-fold increase in PCSK9 mRNA of primary hepatocytes cultured for 24 h in presence of 1 microm T0901317). As our last investigation, we isolated PCSK9 proximal promoter and verified the functionality of a SREBP-1c responsive element located from 335 bp to 355 bp upstream of the ATG. Together, these results show that PCSK9 expression is regulated by nutritional status and insulinemia.