Quercetin inhibits SARS-CoV-2 infection and prevents syncytium formation by cells co-expressing the viral spike protein and human ACE2.

BACKGROUND: Several in silico studies have determined that quercetin, a plant flavonol, could bind with strong affinity and low free energy to SARS-CoV-2 proteins involved in viral entry and replication, suggesting it could block infection of human cells by the virus. In the present study, we examined the ex vivo ability of quercetin to inhibit of SARS-CoV-2 replication and explored the mechanisms of this inhibition. METHODS: Green monkey kidney Vero E6 cells and in human colon carcinoma Caco-2 cells were infected with SARS-CoV-2 and incubated in presence of quercetin; the amount of replicated viral RNA was measured in spent media by RT-qPCR. Since the formation of syncytia is a mechanism of SARS-CoV-2 propagation, a syncytialization model was set up using human embryonic kidney HEK293 co-expressing SARS-CoV-2 Spike (S) protein and human angiotensin converting enzyme 2 (ACE2), [HEK293(S + ACE2) cells], to assess the effect of quercetin on this cytopathic event by microscopic imaging and protein immunoblotting. RESULTS: Quercetin inhibited SARS-CoV-2 replication in Vero E6 cells and Caco-2 cells in a concentration-dependent manner with a half inhibitory concentration (IC50) of 166.6 and 145.2 µM, respectively. It also inhibited syncytialization of HEK293(S + ACE2) cells with an IC50 of 156.7 µM. Spike and ACE2 co-expression was associated with decreased expression, increased proteolytic processing of the S protein, and diminished production of the fusogenic S2' fragment of S. Furin, a proposed protease for this processing, was inhibited by quercetin in vitro with an IC50 of 116 µM. CONCLUSION: These findings suggest that at low 3-digit micromolar concentrations of quercetin could impair SARS-CoV-2 infection of human cells partly by blocking the fusion process that promotes its propagation.

The biological relevance of PCSK9: when less is better .

Proprotein Convertase Subtilisin/Kexin-type 9 (PCSK9) is a circulating negative regulator of hepatic low-density lipoprotein receptor (LDLR), which clears cholesterol from blood. Gain-of-function genetic mutations that amplify PCSK9 activity have been found to cause potentially lethal familial hypercholesterolemia. Inversely, reduction of its activity through loss-of-function genetics or with pharmaceuticals was shown to increase hepatic LDLR, to lower blood cholesterol, and to protect against cardiovascular diseases. New epidemiological and experimental evidence suggests that this reduction could also attenuate inflammation, reinforce cancer immunity, provide resistance to infections, and protect against liver pathologies. In this review, we question the relevance of this protein under normal physiology. We propose that PCSK9 is an important, but nonessential, modulator of cholesterol metabolism and immunity, and that its pathogenicity results from its chronic overexpression.

Isoquercetin as an Anti-Covid-19 Medication: A Potential to Realize.

Isoquercetin and quercetin are secondary metabolites found in a variety of plants, including edible ones. Isoquercetin is a monoglycosylated derivative of quercetin. When ingested, isoquercetin accumulates more than quercetin in the intestinal mucosa where it is converted to quercetin; the latter is absorbed into enterocytes, transported to the liver, released in circulation, and distributed to tissues, mostly as metabolic conjugates. Physiologically, isoquercetin and quercetin exhibit antioxidant, anti-inflammatory, immuno-modulatory, and anticoagulant activities. Generally isoquercetin is less active than quercetin in vitro and ex vivo, whereas it is equally or more active in vivo, suggesting that it is primarily a more absorbable precursor to quercetin, providing more favorable pharmacokinetics to the latter. Isoquercetin, like quercetin, has shown broad-spectrum antiviral activities, significantly reducing cell infection by influenza, Zika, Ebola, dengue viruses among others. This ability, together with their other physiological properties and their safety profile, has led to the proposition that administration of these flavonols could prevent infection by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), or arrest the progression to severity and lethality of resulting coronavirus disease of 2019 (Covid-19). In silico screening of small molecules for binding affinity to proteins involved SARS-CoV-2 life cycle has repeatedly situated quercetin and isoquercetin near to top of the list of likely effectors. If experiments in cells and animals confirm these predictions, this will provide additional justifications for the conduct of clinical trials to evaluate the prophylactic and therapeutic efficacy of these flavonols in Covid-19.

The loss-of-function PCSK9Q152H variant increases ER chaperones GRP78 and GRP94 and protects against liver injury.

Individuals harboring the loss-of-function (LOF) proprotein convertase subtilisin/kexin type 9 Gln152His variation (PCSK9Q152H) have low circulating low-density lipoprotein cholesterol levels and are therefore protected against cardiovascular disease (CVD). This uncleavable form of proPCSK9, however, is retained in the endoplasmic reticulum (ER) of liver hepatocytes, where it would be expected to contribute to ER storage disease (ERSD), a heritable condition known to cause systemic ER stress and liver injury. Here, we examined liver function in members of several French-Canadian families known to carry the PCSK9Q152H variation. We report that PCSK9Q152H carriers exhibited marked hypocholesterolemia and normal liver function despite their lifelong state of ER PCSK9 retention. Mechanistically, hepatic overexpression of PCSK9Q152H using adeno-associated viruses in male mice greatly increased the stability of key ER stress-response chaperones in liver hepatocytes and unexpectedly protected against ER stress and liver injury rather than inducing them. Our findings show that ER retention of PCSK9 not only reduced CVD risk in patients but may also protect against ERSD and other ER stress-driven conditions of the liver. In summary, we have uncovered a cochaperone function for PCSK9Q152H that explains its hepatoprotective effects and generated a translational mouse model for further mechanistic insights into this clinically relevant LOF PCSK9 variant.

Association of the rs562556 PCSK9 Gene Polymorphism with Reduced Mortality in Severe Malaria among Malian Children.

Recent evidence suggests that proprotein convertase subtilisin/kexin type 9 (PCSK9), a downmodulator of cellular uptake of blood cholesterol, also negatively impacts host immune response to microbial infection. In this study, we investigated whether carrying the loss-of-function (LOF) rs562556 (c.1420 A > G; p.I474 V) PCSK9 single nucleotide polymorphism (SNP) affected the outcome of severe malaria in children. Archival DNA of a cohort of 207 Malian children suffering from severe malaria was genotyped for the rs562556 SNP. Sixty-four children were either heterozygous or homozygous for the minor G allele (carriers); 143 children were homozygous for the common A allele (noncarriers). Among carriers, there was one mortality case (1.6%), compared to 15 cases (10.5%) among noncarriers (p=0.0251), suggesting that the G allele is associated with better survival in severe malaria. Intriguingly, this allele did not negatively segregate with any of the clinical symptoms linked to mortality in this cohort. Studies are needed to determine whether PCSK9 inactivation promotes a protective immune response to malaria infection.

The enigma of soluble LDLR: could inflammation be the key?

Soluble low-density lipoprotein receptor (sLDLR) is the circulating ectodomain of transmembrane LDLR. Its blood level strongly correlates with that of triglycerides (TG). This correlation has eluded satisfactory explanation. Hypertriglyceridemia and shedding of the ectodomain of many transmembrane receptors often accompany inflammatory states. The shedding mostly occurs through cleavage by a disintegrin-and-metalloproteinase-17 (ADAM-17), an enzyme activated by inflammation. It reduces the cellular uptake of TG-loaded lipoproteins, causing their accumulation in circulation; hence the correlation between plasma sLDLR and TG. Soluble LDLR could become a new surrogate marker of inflammation.

Associations Between Soluble LDLR and Lipoproteins in a White Cohort and the Effect of PCSK9 Loss-of-Function.

Context: Elevated circulating cholesterol-rich low-density lipoprotein (LDL) particles increase coronary artery disease risk. Cell-surface hepatic LDL receptors (LDLRs) clear 70% of these particles from circulation. The ectodomain of LDLR is shed into circulation, preventing it from removing LDL particles. The role that LDLR ectodomain shedding plays as a regulatory mechanism is unknown. Objective: We describe LDLR shedding via the relationships between circulating soluble LDLRs (sLDLRs) and serum lipoproteins, serum proprotein convertase subtilin/kexin type 9 (PCSK9; a negative regulator of LDLR), and clinical parameters in a white Canadian population. Design: Population-based, cross-sectional study. Settings: Clinical Research Center, The Ottawa Hospital, and Faculty of Medicine, University of Ottawa. Participants: Two hundred seventy-three white Canadians. Intervention: None. Main Outcome Measures: sLDLR measured by ELISA; serum lipids and PCSK9, PCSK9 genotypes, and clinical parameters from previous analyses. Results: sLDLRs correlated strongly with triglycerides (TG; r = 0.624, P < 0.0001) and moderately with LDL cholesterol (r = 0.384, P < 0.0001), and high-density lipoprotein cholesterol (r = -0.307, P = 0.0003). Only TG correlations were unaffected by PCSK9 variations. sLDLR levels were significantly elevated in those with TG >50th or LDL cholesterol >75th percentiles. Conclusions: Serum sLDLR levels correlate with several lipoprotein parameters, especially TG, and the presence of PCSK9 loss-of-function variants alters sLDLR levels and correlations, except for TG. Ectodomain LDLR shedding has a role in LDL metabolism, distinct from PCSK9, with interplay between these two pathways that regulate cell-surface LDLRs. Findings suggest alteration of LDLR shedding could emerge as a target to treat dyslipidemia.

Mice Fed a High-Cholesterol Diet Supplemented with Quercetin-3-Glucoside Show Attenuated Hyperlipidemia and Hyperinsulinemia Associated with Differential Regulation of PCSK9 and LDLR in their Liver and Pancreas.

SCOPE: Hepatic LDL receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) regulate the clearance of plasma LDL-cholesterol (LDL-C): LDLR promotes it, and PCSK9 opposes it. These proteins also express in pancreatic ß cells. Using cultured hepatocytes, we previously showed that the plant flavonoid quercetin-3-glucoside (Q3G) inhibits PCSK9 secretion, stimulated LDLR expression, and enhanced LDL-C uptake. Here, we examine whether Q3G supplementation could reverse the hyperlipidemia and hyperinsulinemia of mice fed a high-cholesterol diet, and how it affects hepatic and pancreatic LDLR and PCSK9 expression. METHODS AND RESULTS: For 12 weeks, mice are fed a low- (0%) or high- (1%) cholesterol diet (LCD or HCD), supplemented or not with Q3G at 0.05 or 0.1% (w/w). Tissue LDLR and PCSK9 is analyzed by immunoblotting, plasma PCSK9 and insulin by ELISA, and plasma cholesterol and glucose by colorimetry. In LCD-fed mice, Q3G has no effect. In HCD-fed mice, it attenuates the increase in plasma cholesterol and insulin, accentuates the decrease in plasma PCSK9, and increases hepatic and pancreatic LDLR and PCSK9. In cultured pancreatic ß cells, however, it stimulates PCSK9 secretion. CONCLUSION: In mice, dietary Q3G could counter HCD-induced hyperlipidemia and hyperinsulinemia, in part by oppositely modulating hepatic and pancreatic PCSK9 secretion.

Malaria severity: Possible influence of the E670G PCSK9 polymorphism: A preliminary case-control study in Malian children.

AIM: Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) is a hepatic secretory protein which promotes the degradation of low-density lipoprotein receptors leading to reduced hepatic uptake of plasma cholesterol. Non-synonymous single-nucleotide polymorphisms in its gene have been linked to hypo- or hyper- cholesterolemia, depending on whether they decrease or increase PCSK9 activity, respectively. Since the proliferation and the infectivity of Plasmodium spp. partially depend on cholesterol from the host, we hypothesize that these PCSK9 genetic polymorphisms could influence the course of malaria infection in individuals who carry them. Here we examined the frequency distribution of one dominant (C679X) and two recessive (A443T, I474V) hypocholesterolemic polymorphisms as well as that of one recessive hypercholesterolemic polymorphism (E670G) among healthy and malaria-infected Malian children. METHODS: Dried blood spots were collected in Bandiagara, Mali, from 752 age, residence and ethnicity-matched children: 253 healthy controls, 246 uncomplicated malaria patients and 253 severe malaria patients. Their genomic DNA was extracted and genotyped for the above PCSK9 polymorphisms using Taqman assays. Associations of genotype distributions and allele frequencies with malaria were evaluated. RESULTS: The minor allele frequency of the A443T, I474V, E670G, and C679X polymorphisms in the study population sample was 0.12, 0.20, 0.26, and 0.02, respectively. For each polymorphism, the genotype distribution among the three health conditions was statistically insignificant, but for the hypercholesterolemic E670G polymorphism, a trend towards association of the minor allele with malaria severity was observed (P = 0.035). The association proved to be stronger when allele frequencies between healthy controls and severe malaria cases were compared (Odd Ratio: 1.34; 95% Confidence Intervals: 1.04-1.83); P = 0.031). CONCLUSIONS: Carriers of the minor allele of the E670G PCSK9 polymorphism might be more susceptible to severe malaria. Further investigation of the cholesterol regulating function of PCSK9 in the pathophysiology of malaria is needed.

The ever-expanding saga of the proprotein convertases and their roles in body homeostasis: emphasis on novel proprotein convertase subtilisin kexin number 9 functions and regulation.

PURPOSE OF REVIEW: The nine members of the proprotein convertase family play major physiological roles during development and in the adult, and their dysregulation leads to various diseases. The primary objective of this article is to review recent findings on the clinical importance of some of these convertases concentrating mostly on PCSK9, the ninth member of the convertase family. This includes the transcriptional and translational regulation of PCSK9, its ability to enhance the degradation of LDL receptor (LDLR), and the implication of PCSK9 in inflammation and sepsis. RECENT FINDINGS: PCSK9 levels are upregulated by E2F1 and reduced by specific miRNAs and by Annexin A2 that bind the 3' end of its mRNA. The implication of the LDLR in the clearance of pathogenic bacterial debris in mice and human puts in perspective a new role for PCSK9 in the regulation of sepsis. The specific implication of the LDLR in the clearance of Lp(a) is now confirmed by multiple studies of PCSK9 inhibition in human cohorts. SUMMARY: Emerging data suggest that PCSK9 can be regulated at the transcriptional and translational levels by specific factors and miRNAs. The identification of a novel pocket in the catalytic domain of PCSK9 represents a harbinger for a new class of small inhibitor drugs. The implication of the LDLR in reducing the effects of bacterially induced sepsis has been supported by both human and mouse data. Outcome studies confirmed the clinical importance of reducing PCSK9 levels. The present review puts in perspective new developments in the PCSK9 biology and its regulation of the LDLR. VIDEO ABSTRACT: http://links.lww.com/COL/A17.

Antiviral activity of quercetin-3-ß-O-D-glucoside against Zika virus infection.

Q3G is a natural derivative of quercetin and is already widely used in various foods and drinks. Our results clearly demonstrated that Q3G exerts antiviral activity against ZIKV in both tissue culture and knockout mice, and that post-exposure in vivo treatment with Q3G could have a beneficial effect. In the future, Q3G should be tested in human cell lines (such as Huh-7, HeLa, or K048, a fetal brain neural stem cell line) to provide further data supporting its potential efficacy in humans; in addition, live viral loads or viremia should be tested in treated animals to supplement the survival results observed in this study. Although the treatment regimens will need to be further optimized (i.e., dosage, frequency of treatment, and administration routes), our results support the results of Q3G efficacy studies in nonhuman primates against ZIKV infection. Further studies will also be needed to investigate the mechanism of Q3G antiviral action, in order to obtain valuable insights into the design of novel targets for antiviral therapeutics in the future.

The Effect of PCSK9 Loss-of-Function Variants on the Postprandial Lipid and ApoB-Lipoprotein Response.

Context: Proprotein convertase subtilisin kexin 9 (PCSK9) mediates degradation of the low-density lipoprotein receptor (LDLR), thereby increasing plasma low-density lipoprotein cholesterol (LDL-C). Variations in the PCSK9 gene associated with loss of function (LOF) of PCSK9 result in greater expression of hepatic LDLR, lower concentrations of LDL-C, and protection from cardiovascular disease (CVD). Apolipoprotein-B (apoB) remnants also contribute to CVD risk and are similarly cleared by the LDLR. We hypothesized that PCSK9-LOF carriers would have lower fasting and postprandial remnant lipoproteins on top of lower LDL-C. Objective: To compare fasting and postprandial concentrations of triglycerides (TGs), total apoB, and apoB48 as indicators of remnant lipoprotein metabolism in PCSK9-LOF carriers with those with no PCSK9 variants. Design: Case-control, metabolic study. Setting: Clinical Research Center of The Ottawa Hospital. Participants: Persons with one or more copies of the L10ins/A53V and/or I474V and/or R46L PCSK9 variant and persons with no PCSK9 variants. Intervention: Oral fat tolerance test. Main Outcomes Measures: Fasting and postprandial plasma TG, apoB48, total apoB, total cholesterol, and PCSK9 were measured at 0, 2, 4, and 6 hours after an oral fat load. Results: Participants with PCSK9-LOF variants (n = 22) had reduced fasting LDL-C (-14%) as well as lower fasting TG (-21%) compared with noncarrier controls (n = 23). LOF variants also had reduced postprandial total apoB (-17%), apoB48 (-23%), and TG (-18%). Postprandial PCSK9 declined in both groups (-24% vs -16%, respectively). Conclusions: Participants carrying PCSK9-LOF variants had attenuated levels of fasting and postprandial TG, apoB48, and total apoB. This may confer protection from CVD and further validate the use of PCSK9 inhibitors to lower CVD risk.

Comparing expression and activity of PCSK9 in SPRET/EiJ and C57BL/6J mouse strains shows lack of correlation with plasma cholesterol.

OBJECTIVE: Low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) are opposing regulators of plasma LDL-cholesterol levels. The PCSK9 gene exhibits many single or compound polymorphisms within or among mammalian species. This is case between the SPRET/EiJ (SPRET) and C57BL/6J (B6) mouse strains. We examined whether these polymorphisms could be associated with differential expression and activity of their respective PCSK9 molecules. METHODS: Liver expression of LDLR and PCSK9 transcripts were assessed by RT-PCR, and that of their corresponding proteins by immunoblotting. Purified recombinant PCSK9 proteins were assayed for their ability to degrade LDLR. Pcsk9 gene proximal promoters were tested for activation of a luciferase reporter gene. RESULTS: SPRET and B6 mice carried comparable levels of plasma cholesterol in spite of the fact that SPRET mice expressed less PCSK9 and more LDLR in liver. There were indels and single-base differences between their Pcsk9 cDNA and promoter sequences. Ex vivo, SPRET PCSK9 protein was less secreted but was more active at degrading LDLR. Its gene promoter was more active at driving expression of the luciferase reporter. CONCLUSIONS: Collectively, these results suggest that, compared to the B6 mouse, the SPRET mouse may represent an example of absence of direct correlation between PCSK9 and cholesterol levels in plasma, due to genetic variations leading to reduced secretion of PCSK9 associated with greater LDLR-degrading activity.

Prophylactic Efficacy of Quercetin 3-ß-O-d-Glucoside against Ebola Virus Infection.

Ebola outbreaks occur on a frequent basis, with the 2014-2015 outbreak in West Africa being the largest one ever recorded. This outbreak has resulted in over 11,000 deaths in four African countries and has received international attention and intervention. Although there are currently no approved therapies or vaccines, many promising candidates are undergoing clinical trials, and several have had success in promoting recovery from Ebola. However, these prophylactics and therapeutics have been designed and tested only against the same species of Ebola virus as the one causing the current outbreak. Future outbreaks involving other species would require reformulation and possibly redevelopment. Therefore, a broad-spectrum alternative is highly desirable. We have found that a flavonoid derivative called quercetin 3-ß-O-d-glucoside (Q3G) has the ability to protect mice from Ebola even when given as little as 30 min prior to infection. Furthermore, we have demonstrated that this compound targets the early steps of viral entry. Most promisingly, antiviral activity against two distinct species of Ebola virus was seen. This study serves as a proof of principle that Q3G has potential as a prophylactic against Ebola virus infection.

60 YEARS OF POMC: From the prohormone theory to pro-opiomelanocortin and to proprotein convertases (PCSK1 to PCSK9).

Pro-opiomelanocortin (POMC), is a polyprotein expressed in the pituitary and the brain where it is proteolytically processed into peptide hormones and neuropeptides with distinct biological activities. It is the prototype of multipotent prohormones. The prohormone theory was first suggested in 1967 when Chrétien and Li discovered γ-lipotropin and observed that (i) it was part of ß-lipotropin (ß-LPH), a larger polypeptide characterized 2 years earlier and (ii) its C-terminus was ß-melanocyte-stimulating hormone (ß-MSH). This discovery led them to propose that the lipotropins might be related biosynthetically to the biologically active ß-MSH in a precursor to end product relationship. The theory was widely confirmed in subsequent years. As we celebrate the 50th anniversary of the sequencing of ß-LPH, we reflect over the lessons learned from the sequencing of those proteins; we explain their extension to the larger POMC precursor; we examine how the theory of precursor endoproteolysis they inspired became relevant for vast fields in biology; and how it led, after a long and arduous search, to the novel proteolytic enzymes called proprotein convertases. This family of nine enzymes plays multifaceted functions in growth, development, metabolism, endocrine, and brain functions. Their genetics has provided many insights into health and disease. Their therapeutic targeting is foreseeable in the near future. Thus, what started five decades ago as a theory based on POMC fragments, has opened up novel and productive avenues of biological and medical research, including, for our own current interest, a highly intriguing hypocholesterolemic Gln152His PCSK9 mutation in French-Canadian families.

Variable effects of gender and Western diet on lipid and glucose homeostasis in aged PCSK9-deficient C57BL/6 mice CSK9PC57BL/6.

BACKGROUND: Proprotein convertase subtilisin/kexin-type 9 (PCSK9) downregulates clearance of plasma cholesterol by liver. Its inactivation increases this clearance, reducing cardiovascular risk. However, a lack of PCSK9 could also lead to cholesterol accumulation in pancreatic islet beta cells, impairing insulin secretion. We reported earlier that 4-month-old male PCSK9-deficient (KO) C57BL/6 mice were hyperglycemic and insulin-insufficient relative to their wild-type (WT) counterparts. Here, we examined how gender and diet affect lipid and glucose homeostasis in these mice at 8 months of age. METHODS: After being fed a normal diet or a Western diet for over 6 months, KO mice were compared with same-gender WT mice for fasting plasma levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), glucose, and insulin; for glucose disposal and glucose-stimulated insulin secretion (GSIS); and for pancreatic islet morphology. RESULTS: A. Females: On normal diet, KO mice showed lower plasma TC, HDL-C, and LDL-C, higher plasma glucose, and normal glucose disposal despite impaired GSIS. On Western diet, they showed comparable plasma TC and HDL-C, but lower LDL-C, higher plasma glucose, and normal glucose disposal despite impaired GSIS. B. Males: On normal and Western diets, KO mice showed lower plasma TC, HDL-C, and LDL-C, similarly elevated plasma glucose, glucose intolerance, and impaired GSIS. C. Both: KO mice on either diet showed pancreatic islet dysmorphism, with larger, possibly immature secretory granules. CONCLUSIONS: Lower LDL-C and impaired GSIS are two major phenotypes in aged PCSK9-deficient C57BL/6 mice. These phenotypes are modulated by gender and diet.

Quercetin-3-glucoside increases low-density lipoprotein receptor (LDLR) expression, attenuates proprotein convertase subtilisin/kexin 9 (PCSK9) secretion, and stimulates LDL uptake by Huh7 human hepatocytes in culture.

Low-density lipoprotein receptor (LDLR) mediates hepatic clearance of plasma cholesterol; proprotein convertase subtilisin/kexin 9 (PCSK9) opposes this clearance by promoting LDLR degradation. The plant flavonoid quercetin-3-ß-d-glucoside (Q3G) has been shown to reduce hypercholesterolemia in experimental animals. Here, we examined how it affects LDLR and PCSK9 expression as well as LDL uptake by human Huh7 hepatocytes. At low micromolar concentrations, Q3G increased LDLR expression, reduced PCSK9 secretion, and stimulated LDL uptake. It also diminished intracellular sortilin, a sorting receptor known to facilitate PCSK9 secretion. Thus, as an LDLR inducer and a PCSK9 anti-secretagogue, Q3G may represent an effective anti-cholesterolemic agent.

PCSK9: a key modulator of cardiovascular health.

Since the discovery of proprotein convertase subtilisin kexin 9 (PCSK9) in 2003, this PC has attracted a lot of attention from the scientific community and pharmaceutical companies. Secreted into the plasma by the liver, the proteinase K-like serine protease PCSK9 binds the low-density lipoprotein (LDL) receptor at the surface of hepatocytes, thereby preventing its recycling and enhancing its degradation in endosomes/lysosomes, resulting in reduced LDL-cholesterol clearance. Surprisingly, in a nonenzymatic fashion, PCSK9 enhances the intracellular degradation of all its target proteins. Rare gain-of-function PCSK9 variants lead to higher levels of LDL-cholesterol and increased risk of cardiovascular disease; more common loss-of-function PCSK9 variants are associated with reductions in both LDL-cholesterol and risk of cardiovascular disease. It took 9 years to elaborate powerful new PCSK9-based therapeutic approaches to reduce circulating levels of LDL-cholesterol. Presently, PCSK9 monoclonal antibodies that inhibit its function on the LDL receptor are evaluated in phase III clinical trials. This review will address the biochemical, genetic, and clinical aspects associated with PCSK9's biology and pathophysiology in cells, rodent and human, with emphasis on the clinical benefits of silencing the expression/activity of PCSK9 as a new modality in the treatment of hypercholesterolemia and associated pathologies.

The multifaceted proprotein convertases: their unique, redundant, complementary, and opposite functions.

The secretory proprotein convertase (PC) family comprises nine members: PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, SKI-1/S1P, and PCSK9. The first seven PCs cleave their substrates at single or paired basic residues, and SKI-1/S1P cleaves its substrates at non-basic residues in the Golgi. PCSK9 cleaves itself once, and the secreted inactive protease escorts specific receptors for lysosomal degradation. It regulates the levels of circulating LDL cholesterol and is considered a major therapeutic target in phase III clinical trials. In vivo, PCs exhibit unique and often essential functions during development and/or in adulthood, but certain convertases also exhibit complementary, redundant, or opposite functions.

Differential effects of PCSK9 loss of function variants on serum lipid and PCSK9 levels in Caucasian and African Canadian populations.

OBJECTIVES: Variants of the secreted glycoprotein, proprotein convertase subtilisin/kexin 9 (PCSK9), associate with both hypo- and hyper-cholesterolemic phenotypes. Herein, we carried out full exonic sequencing of PCSK9 documenting the frequency of single and multiple PCSK9 variations and their effects on serum lipoprotein and PCSK9 levels in Caucasian Canadians. METHODS: The 12 exons of PCSK9 were sequenced in 207 unrelated Caucasian Canadians. Minor allele frequencies of PCSK9 variants were compared amongst LDL cholesterol (LDLC) quintiles. Serum PCSK9 levels were measured by ELISA and lipoproteins by enzymatic methods. Comparisons were made with a Caucasian family cohort (n=51) and first generation African Canadians (n=31). RESULTS: In Caucasians, but not African Canadians, the c.61_63insCTG (denoted L10Ins) and A53V PCSK9 variations were linked and their frequency was significantly higher among Caucasian Canadians with LDLC levels in the <25th percentile. In both the unrelated and family Caucasian cohorts those carrying the L10A53V PCSK9 variant had significantly lower LDLC without reduction in plasma PCSK9. The I474V PCSK9 variant associated with significantly lower serum PCSK9 and LDLC. A novel PCSK9 variant was identified; E206K. We found that the frequency of multiple PCSK9 variations was higher in first generation African Canadians. CONCLUSIONS: We showed that the L10A53V and I474V PCSK9 variants were significantly associated with lower LDLC levels in Caucasian Canadians but differed in their effect on serum PCSK9 concentrations, illuminating differences in their mechanism of inaction and indicating that that PCSK9 measurement alone may not always be a good indicator of PCSK9 function.Full exonic sequencing of PCSK9 pointed to factors that may contribute to L10Ins PCSK9 variant loss of function in Canadians of Caucasian but not those of African descent. These included; (1) its tight linkage with the A53V variant in Caucasians and/or (2) for both the L10 and I474V, the combined (and negating) effect of multiple, differing phenotypic PCSK9 variants within individuals of African ancestry for which combinations of PCSK9 variations and their overall frequency was higher. No population studies, to our knowledge, have addressed or accessed the effect of multiple PCSK9 variants on cholesterol profiles. Our results indicate that this should be considered.