<b>OBJECTIVEb>To investigate the cleavage of HBV core protein in vivo by proprotein convertase furin or its family members and observe the intracellular localization of the putative cleaved product.

<b>METHODSb>Recombinant HBV core protein was incubated with furin under different conditions in vitro, and the reaction was checked with Western blotting. The recombinant vectors expressed the putative cleaved fragment and intact core protein (serves as control) were constructed. The stable expression cell lines were established by transfecting constructs into HepG2 cell line, for which indirect immunofluorescence staining was used by monoclonal anti-HBc against the region shared by core protein and its cleaved product .The confocal microscopy was carried out to observe the intracellular distribution.

<b>RESULTSb>HBV core protein was cleaved by furin in vitro under different tested conditions. The molecular weight of the major cleaved product just about 15,000 was in concordance with the expectation. The expressed cleaved fragment could react to the monoclonal antibody against core protein, and mainly located in cytosol in particle style just like the intact core protein.

<b>CONCLUSIONb>HBV core protein can be cleaved by furin in vitro. The major cleaved product has similar antigenicity and subcellular distribution to core protein. These data suggest that proprotein convertase furin or its family members play important roles in HBV replication regulation, and the cleaved product may be involved in antiviral immunity of HBV infection. Further investigations are imperative.

Furin ; metabolism ; Genetic Vectors ; Hep G2 Cells ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B virus ; metabolism ; physiology ; Humans ; Microdissection ; Microscopy, Confocal ; Proprotein Convertases ; metabolism ; Transfection ; Virus Replication

<b>OBJECTIVEb>To study the effects of hepatitis B virus (HBV) infection on the expression of Furin, an important proprotein convertase, in liver cells to provide insights towards its potential as a therapeutic target for improved antiviral efficacy.

<b>METHODSb>Furin expression was measured in human liver specimens (infected tissues from patients with chronic HBV hepatitis vs. normal tissues from healthy donors) and in hepatoma cell lines (HBV-infected HepG2.2.15 cells vs. uninfected parental cell lines HepG2) using quantitative real-time RT-PCR (for mRNA), western blotting and immunohistochemistry (for protein).

<b>RESULTSb>Compared to the uninfected tissues and cells, the HBV-infected tissue and cells showed down-regulated expression of furin at both the mRNA and protein levels. In particular, the HepG2.2.15 cells showed -50% less furin mRNA expression than the HepG2 cells and the difference was statistically significant (P less than 0.05).

<b>CONCLUSIONb>HBV may suppress the host cell's expression of furin, possibly to benefit its survival and replication in the host cell.

Cell Line ; Furin ; metabolism ; Gene Expression Regulation ; Hep G2 Cells ; Hepatitis B virus ; physiology ; Hepatitis B, Chronic ; metabolism ; Host-Pathogen Interactions ; Humans ; Liver ; metabolism ; virology ; Proprotein Convertases ; metabolism ; Virus Replication

Based on evidence from numerous research studies, increased low-density lipoprotein cholesterol (LDL-C) is a clinically important factor for accelerated atherosclerosis and increased cardiovascular risk. The introduction of statin therapy has resulted in marked reductions in LDL-C and has proven to be clinically beneficial in cardiovascular events in patients with high cardiovascular risk. Nonetheless, many patients with elevated LDL-C do not achieve their LDL-C goals with current treatments. In addition, cardiovascular disease remains an important cause of mortality and morbidity worldwide. Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors further reduce LDL-C, and potentially reducing cardiovascular events. Several PCSK9 inhibitors are currently under clinical development and some of them have been studied in completed cardiovascular outcome trials, including evolocumab, alirocumab, and bococizumab. The results of the FOURIER trial, which was the first cardiovascular outcome trial to examine the impact of PCSK9 inhibition with evolocumab therapy on cardiovascular events, were reported in March of 2017. The results of the ODYSSEY Outcomes trial with alirocumab therapy were released in March of 2018 at the American College of Cardiology Annual Scientific Session. The SPIRE-1 and -2 trials which examined a PCSK9 inhibitor, bococizumab, were prematurely terminated because of discontinued development of bococizumab in November 2016. This review will discuss 3 recent cardiovascular outcome trials with PCSK9 inhibitors, with an emphasis on clinical implications and future therapeutic perspectives.
Atherosclerosis ; Cardiology ; Cardiovascular Diseases ; Cholesterol ; Humans ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; Lipoproteins ; Mortality ; Proprotein Convertases

Antibodies, Monoclonal, Humanized/therapeutic use* ; Humans ; Hyperlipidemias ; Hypertriglyceridemia/drug therapy* ; Proprotein Convertases ; Subtilisins

<b>BACKGROUNDb>Familial hypercholesterolemia (FH) is an autosomal disorder associated with elevated plasma low density lipoprotein (LDL) levels leading to premature coronary heart disease (CHD). As a result of long-term hyperlipemia, FH patients will present endarterium thickening and artherosclerosis. In the present study we scanned the related gene of a clinically diagnosed autosomal genetic hypercholesterolemia family for the possible mutations and established eukaryotic expression vector of mutation of proprotein convertase subtilisin/kexin type 9 (PCSK9) gene with gene recombination technique to investigate the contributions of the variation on low density lipoprotein receptor (LDL-R) metabolism and function alternation.

<b>METHODSb>Mutation detection was conducted for LDL-R, apolipoprotein B(100) (apoB(100)) and PCSK9 gene with nucleotide sequencing in a Chinese FH family. The full-length cDNA of wild type PCSK9 gene (WT-PCSK9) was obtained from Bel-7402. Site mutagenesis was used to establish the recombinant eukaryotic expression vector carrying pathogenic type of PCSK9 gene and the inserted fragment was sequenced. With the blank vector as control, liposome transfection method was used to transfect the Bel-7402 cells with recombinant plasmid. The expression of LDL-R mRNA was examined by RT-PCR. PCSK9 and the expression of LDL-R protein were determined by Western blotting.

<b>RESULTSb>The G-->T mutation at the 918 nucleotide of PCSK9 gene resulted in the substitution of the arginine by a serine at the codon 306 of exon 6. After sequencing, it was confirmed that the inserted fragment of established expression vector had correct size and sequence and the mutant was highly expressed in Bel-7402 cells. There was no significant variation in the levels of LDL-R mRNA. LDL-R mature protein was decreased by 57% after the cells were transfected by WT-PCSK9 plasmid. Mature LDL-R was significantly decreased by 12% after the cells were transfected by R306S mutant as evidenced by gray scale scanning, suggesting that the new mutant R306S can significantly decrease the expression of mature LDL-R protein.

<b>CONCLUSIONSb>A novel missense mutation of PCSK9 gene, R306S, was found and the eukaryotic expression vectors of mutant and wild-type of PCSK9 gene were established. There was no significant variation in the levels of LDL-R mRNA. The R306S mutation could significantly lead to the decrease of LDL-R mature protein expression, which might be the pathogenic gene of the FH family.

Adolescent ; Adult ; Female ; Humans ; Hyperlipoproteinemia Type II ; genetics ; Lipids ; blood ; Male ; Mutation ; Pedigree ; Proprotein Convertase 9 ; Proprotein Convertases ; Serine Endopeptidases ; genetics

Dyslipidemia, highly elevated, low-density lipoprotein (LDL) cholesterol, is a major cardiovascular risk factor. Statins have been proven to effectively reduce the risk of atherosclerotic cardiovascular disease (ASCVD) and are recommended as a first-line therapy for the primary and secondary prevention of ASCVD. However, statins may not be sufficient in decreasing LDL cholesterol levels and pose a significant on-treatment residual risk of major cardiovascular events (i.e., residual cholesterol risk) according to meta-analyses of statin trials. Current guidelines for cholesterol management to achieve additional LDL cholesterol reduction and reduce ASCVD risk recommend two hyperlipidemic agents besides statins. Use of ezetimibe, a cholesterol absorption inhibitor, leads to additional LCL cholesterol reduction and decreased ASCVD risk, when added to statin therapy, without raising significant safety concerns. Furthermore, in combination with a mild-to-moderate statin intensity, ezetimibe is used in situations of statin-associated adverse effects such as myalgia and the combination therapy is relatively safer. Monoclonal antibody of proprotein convertase subtilisin/kexin type 9 (PCSK9), alirocumab, and evolocumab, have been approved to lower LDL cholesterol level. While there are drawbacks to the use of PCSK9 inhibitors, including high cost and adverse events such as injection site reaction, they significantly decreased serum LDL cholesterol levels and thereby ASCVD risks when added to maximally tolerated statin therapy.
Absorption ; Cardiovascular Diseases ; Cholesterol ; Cholesterol, LDL ; Dyslipidemias ; Ezetimibe ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; Lipoproteins ; Myalgia ; Proprotein Convertases ; Risk Factors ; Secondary Prevention

PURPOSE: To investigate associations for polymorphisms in β-carotene 9′,10′-oxygenase (BCO2, rs10431036 and rs11214109), proprotein convertase subtilisin kexin type 9 (PCSK9, rs11583680), and tribbles pseudokinase 1 (TRIB1, rs17321515 and rs2954029), as well as lifestyle factors, with ischemic stroke (IS). MATERIALS AND METHODS: This nested case-control study included 161 patients with IS and 483 matched control individuals. We collected medical reports, lifestyle details, and blood samples from individuals and used the PCR-ligase detection reaction method to genotype single nucleotide polymorphisms (SNPs). RESULTS: The GA+AA genotype of rs10431036 (p<0.001) and rs17321515 (p=0.003), the CT+TT genotype of rs11214109 (p=0.005), and the TA+AA genotype of rs2954029 (p=0.006) in dominant models increased the risk of IS. In additive models, the GG genotype of rs17321515 (p=0.005) and the TT genotype of rs2954029 (p=0.008) increased the risk of IS. Adequate intake of fruits/vegetables reduced the risk of IS (p=0.005). Although there was no interaction between genes and fruits/vegetables, people with inadequate intake of fruits/vegetables who carried a risk genotype had a higher risk of IS than those only having inadequate fruits/vegetables intake or those only carrying a risk genotype. Also, the haplotypes AC, AT, and GT (comprising rs10431036 and rs11214109) and GT (comprising rs2954029 and rs17321515) were found to be associated with an increased risk of IS (p<0.05). CONCLUSION: Polymorphisms in BCO2 and TRIB1 and fruits/vegetables intake were associated with IS. These results provide the theoretical basis for gene screening to prevent chronic cerebrovascular diseases.
Case-Control Studies ; Cerebrovascular Disorders ; Genotype ; Haplotypes ; Humans ; Life Style ; Mass Screening ; Methods ; Polymorphism, Single Nucleotide ; Proprotein Convertases ; Stroke ; Subtilisin

PURPOSE: Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) and promotes degradation of the LDLR. Inhibition of PCSK9 either by reducing its expression or by blocking its activity results in the upregulation of the LDLR and subsequently lowers the plasma concentration of LDL-cholesterol. As a modality to inhibit PCSK9 action, we searched the chemical library for small molecules that block the binding of PCSK9 to the LDLR. MATERIALS AND METHODS: We selected 100 chemicals that bind to PCSK9 where the EGF-AB fragment of the LDLR binds via in silico screening of the ChemBridge chemical library, using the computational GOLD algorithm analysis. Effects of chemicals were evaluated using the PCSK9-LDLR binding assay, immunoblot analysis, and the LDL-cholesterol uptake assay in vitro, as well as the fast performance liquid chromatography assay for plasma lipoproteins in vivo. RESULTS: A set of chemicals were found that decreased the binding of PCSK9 to the EGF-AB fragment of the LDLR in a dose-dependent manner. They also increased the amount of the LDLR significantly and subsequently increased the uptake of fluorescence-labeled LDL in HepG2 cells. Additionally, one particular molecule lowered the plasma concentration of total cholesterol and LDL-cholesterol significantly in wild-type mice, while such an effect was not observed in Pcsk9 knockout mice. CONCLUSION: Our findings strongly suggest that in silico screening of small molecules that inhibit the protein-protein interaction between PCSK9 and the LDLR is a potential modality for developing hypercholesterolemia therapeutics.
Animals ; Cholesterol/*blood ; Cholesterol, LDL/blood ; Hep G2 Cells ; Humans ; Mice ; Mice, Knockout ; Proprotein Convertases/*metabolism ; Receptors, LDL/*metabolism ; Serine Endopeptidases/*metabolism ; *Small Molecule Libraries

In recent studies, the reported prevalence of heterozygous familial hypercholesterolemia (FH) has been higher than in previous reports. Although cascade genetic screening is a good option for efficient identification of affected patients, diagnosis using only clinical criteria is more common in real clinical practice. Cardiovascular risk is much higher in FH patients due to longstanding low density lipoprotein cholesterol (LDL-C) burden and is also influenced by other risk factors. Although guidelines emphasize aggressive LDL-C reduction, the majority of patients cannot reach the LDL-C goal by conventional pharmacotherapy. Novel therapeutics such as proprotein convertase subtilisin/kexin type 9 inhibitors have shown strong lipid lowering efficacy and are expected to improve treatment results in FH patients.
Cholesterol, LDL ; Coronary Disease ; Diagnosis* ; Drug Therapy ; Genetic Testing ; Genetics ; Humans ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; Hyperlipoproteinemia Type II* ; Prevalence ; Proprotein Convertases ; Risk Factors

<b>BACKGROUNDb>In a previously identified locus linked to hypertension on chromosome 15q, we identified three blood pressure candidate genes: insulin-like growth factor 1 receptor gene (IGF1R), myocyte specific enhancer factor 2A gene (MEF2A), and paired basic amino acid cleaving enzyme 4 gene (PACE4). In this study, we tested their associations with hypertension using haplotype analysis.

<b>METHODSb>A total of 288 unrelated individuals, including 163 high diastolic blood pressure (DBP) subjects and 125 normal DBP subjects were enrolled in this case-control study. Twenty single nucleotide polymorphisms (SNPs) in the three genes were genotyped using polymerase chain reaction followed by restriction enzyme digestion. Haplotype analysis was accomplished in the following stages: (1) pair-wise linkage disequilibrium test among SNPs on the same gene was performed to explore blocks in which recombination is very unlikely to happen; (2) Estimation-Maximization algorithm was applied to estimate haplotype frequencies in each block; (3) the chi-square test was used to examine the specific haplotype difference, and a permutation test was used to examine the overall haplotype profile difference between cases and controls in each block.

<b>RESULTSb>An estimated haplotype "CCCCG" frequency in the haplotype block on the PACE4 gene was significantly higher in high DBP cases than in controls (P < 0.01). The overall estimated haplotype profile in this block was also significantly different between the cases and the controls (P < 0.001). This association indicates.

<b>CONCLUSIONSb>This study for the first time demonstrated that PACE4 gene may play an important role in the regulation of DBP. This association indicates that variations influencing DBP resides in or near this genomic region.

Adult ; Blood Pressure ; physiology ; Case-Control Studies ; Diastole ; physiology ; Female ; Haplotypes ; Humans ; Male ; Polymorphism, Single Nucleotide ; Proprotein Convertases ; Serine Endopeptidases ; genetics