Effect of enzymatic hydrolysis with Alcalase or Protamex on technological and antioxidant properties of whey protein hydrolysates.

The aim of this study was to evaluate the effect of the enzymatic hydrolysis, performed using Alcalase and Protamex enzymes, on the technological functionalities and the antioxidant capacity of whey protein hydrolysates (WPHs) to identify the conditions allowing to obtain target functionality/ies. Samples were characterized for hydrolysis degree (DH), molecular weight distribution, structural properties, and food-related functionalities. Free sulfhydryl groups and surface hydrophobicity significantly decreased with the increase in DH, regardless of the used enzyme. The foaming and antioxidant properties of Alcalase WPHs were higher as compared to those of WPI, reaching the maximum value at DH = 18-20 %, while higher DH resulted in impaired functionality. Gelling properties were guaranteed when WPI was hydrolysed by Protamex at DH < 15 % while foaming and antioxidant abilities were fostered at 15 < DH < 21 %. These results were well correlated with MW distribution and were rationalized into a road map which represents a useful tool in the selection of proper hydrolysis conditions (time, DH, enzyme type) to obtain WPHs with tailored functionalities. Research outcomes highlighted the possibility to drive protein hydrolysis to optimize the desired functionality/ies.

Activities of proteases in deep eutectic solvents and removal of protein from chitin by subtilisin A in betaine/glycerol.

This research intended to remove residual protein from chitin with proteases in deep eutectic solvents (DESs). The activities of some proteases in several DESs, including choline chloride/p-toluenesulfonic acid, betaine/glycerol (Bet/G), choline chloride/malic acid, choline chloride/lactic acid, and choline chloride/urea, which are capable of dissolving chitin, were tested, and only in Bet/G some proteases were found to be active, with subtilisin A, ficin, and bromelain showing higher activity than other proteases. However, the latter two proteases caused degradation of chitin molecules. Further investigation revealed that subtilisin A in Bet/G did not exhibit "pH memory", which is a universal characteristic displayed by enzymes dispersed in organic phases, and the catalytic characteristics of subtilisin A in Bet/G differed significantly from those in aqueous phase. The conditions for protein removal from chitin by subtilisin A in Bet/G were determined: Chitin dissolved in Bet/G with 0.5 % subtilisin A (442.0 U/mg, based on the mass of chitin) was hydrolyzed at 45 °C for 30 min. The residual protein content in chitin decreased from 5.75 % ± 0.10 % to 1.01 % ± 0.12 %, improving protein removal by 57.20 % compared with protein removal obtained by Bet/G alone. The crystallinity and deacetylation degrees of chitin remained unchanged after the treatment.

PCSK9 plasma concentration is associated with epicardial adipose tissue volume and metabolic control in patients with type 1 diabetes.

Patients with type 1 diabetes (T1D) have a greater risk of cardiovascular disease. Proconvertase subtilisin-kexin 9 (PCSK9) is involved in the atherosclerosis process. This study aimed to determine the relationship between PCSK9 levels and epicardial adipose tissue (EAT) volume and cardiometabolic variables in patients with T1D. This was an observational cross-sectional study including 73 patients with T1D. Clinical, biochemical and imaging data were collected. We divided the patients into two groups according to their glycemic control and the EAT index (iEAT) percentile. We performed a correlation analysis between the collected variables and PCSK9 levels; subsequently, we performed a multiple regression analysis with the significant parameters. The mean age was 47.6 ± 8.5 years, 58.9% were men, and the BMI was 26.9 ± 4.6 kg/m2. A total of 31.5%, 49.3% and 34.2% of patients had hypertension, dyslipidemia and smoking habit, respectively. The PCSK9 concentration was 0.37 ± 0.12 mg/L, which was greater in patients with worse glycemic control (HbA1c > 7.5%), dyslipidemia and high EAT volume (iEAT > 75th percentile). The PCSK9 concentration was positively correlated with age (r = 0.259; p = 0.027), HbA1c (r = 0.300; p = 0.011), insulin dose (r = 0.275; p = 0.020), VLDL-C level (r = 0.331; p = 0.004), TG level (r = 0.328; p = 0.005), and iEAT (r = 0.438; p < 0.001). Multiple regression analysis revealed that 25% of the PCSK9 variability was explained by iEAT and HbA1c (p < 0.05). The PCSK9 concentration is associated with metabolic syndrome parameters, poor glycemic control and increased EAT volume in patients with T1D.

Insights from structure-activity relationships and the binding mode of peptidic α-ketoamide inhibitors of the malaria drug target subtilisin-like SUB1.

Plasmodium multi-resistance, including against artemisinin, seriously threatens malaria treatment and control. Hence, new drugs are urgently needed, ideally targeting different parasitic stages, which are not yet targeted by current drugs. The SUB1 protease is involved in both hepatic and blood stages due to its essential role in the egress of parasites from host cells, and, as potential new target, it would meet the above criteria. We report here the synthesis as well as the biological and structural evaluation of substrate-based α-ketoamide SUB1 pseudopeptidic inhibitors encompassing positions P4-P2'. By individually substituting each position of the reference compound 1 (MAM-117, Ac-Ile-Thr-Ala-AlaCO-Asp-Glu (Oall)-NH2), we better characterized the structural determinants for SUB1 binding. We first identified compound 8 with IC50 values of 50 and 570 nM against Pv- and PfSUB1, respectively (about 3.5-fold higher potency compared to 1). Compound 8 inhibited P. falciparum merozoite egress in culture by 37% at 100 µM. By increasing the overall hydrophobicity of the compounds, we could improve the PfSUB1 inhibition level and antiparasitic activity, as shown with compound 40 (IC50 values of 12 and 10 nM against Pv- and PfSUB1, respectively, IC50 value of 23 µM on P. falciparum merozoite egress). We also found that 8 was highly selective towards SUB1 over three mammalian serine peptidases, supporting the promising value of this compound. Finally, several crystal 3D-structures of SUB1-inhibitor complexes, including with 8, were solved at high resolution to decipher the binding mode of these compounds.

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.

Subtilisin 3 production from Microsporum canis is independent of keratin substrate availability.

Dermatophytes are highly infectious fungi that cause superficial infections in keratinized tissues in humans and animals. This group of fungi is defined by their ability to digest keratin and encompasses a wide range of species. We investigated a critical adhesion protein, subtilisin 3, utilized by Microsporum canis during initial stages of infection, analyzing its production and expression under varying growth conditions. Additionally, as this protein must be expressed and produced for dermatophyte infections to occur, we developed and optimized a diagnostic antibody assay targeting this protein. Subtilisin 3 levels were increased in culture when grown in baffled flasks and supplemented with either l-cysteine or cat hair. As subtilisin 3 was also produced in cultures not supplemented with keratin or cysteine, this study demonstrated that subtilisin 3 production is not reliant on the presence of keratin or its derivatives. These findings could help direct future metabolic studies of dermatophytes, particularly during the adherence phase of infections.

Proprotein convertase subtilisin/kexin 9 inhibitor downregulates microRNA-130a-3p expression in hepatocytes to alleviates atherosclerosis progression.

Proprotein convertase subtilisin/kexin 9 (PCSK9) inhibitors have been shown to regulate lipid metabolism and reduce the risk of cardiovascular events. This study explores the effect and potential mechanism of PCSK9 inhibitors on lipid metabolism and coronary atherosclerosis. HepG2 cells were incubated with PCSK9 inhibitor. ApoE-/- mice were fed with a high fat to construct an atherosclerosis model, and then treated with PCSK9 inhibitor (8 mg/kg for 8 w). PCSK9 inhibitor downregulated microRNA (miRNA)-130a-3p expression in a dose-dependent manner. And, miR-130a-3p could bind directly to the 3' untranslated region (3'-UTR) region of LDLR to down-regulate LDLR expression in HepG2 cells, as confirmed by the luciferase reporter gene assay. In addition, miR-130a-3p overexpression significantly attenuated the promoting effect of PCSK9 inhibitor on LDLR and DiI-LDL uptake in HepG2 cells. More importantly, in vivo experiments confirmed that PCSK9 inhibitor could significantly inhibit miR-130a-3p levels and promote LDLR expression in liver tissues, thus regulating serum lipid profile and alleviating the progression of coronary atherosclerosis. PCSK9 inhibitor could moderately improve coronary atherosclerosis by regulating miR-130a-3p/LDLR axis, providing an exploitable strategy for the treatment of coronary atherosclerosis.

Impact of Bariatric Surgery on Subtilisin/Kexin Type 9 (PCSK9) Gene Expression and Inflammation in the Adipose Tissue of Obese Diabetic Rats.

Bariatric surgery improves dyslipidaemia and reduces body weight, but it remains unclear how bariatric surgery modulates gene expression in fat cells to influence the proprotein convertase subtilisin/kexin type 9 (PCSK-9) and low-density lipoprotein receptor (LDLR) gene expression. The expression of the PCSK9/LDLR/tumor necrosis factor-alpha (TNFα) gene in adipose tissue was measured in two groups of Zucker Diabetic Sprague Dawley (ZDSD) rats after Roux-en-Y gastric bypass (RYGB) surgery or 'SHAM' operation. There was lower PCSK9 (p = 0.02) and higher LDLR gene expression (p = 0.02) in adipose tissue in rats after RYGB. Weight change did not correlate with PCSK9 gene expression (r = -0.5, p = 0.08) or TNFα gene expression (r = -0.4, p = 0.1). TNFα gene expression was positively correlated with PCSK9 gene expression (r = 0.7, p = 0.001) but not correlated with LDLR expression (r = -0.3, p = 0.3). Circulating triglyceride levels were lower in RYGB compared to the SHAM group (1.1 (0.8-1.4) vs. 1.5 (1.0-4.2), p = 0.038) mmol/L with no difference in cholesterol levels. LDLR gene expression was increased post-bariatric surgery with the potential to reduce the number of circulating LDL particles. PCSK9 gene expression and TNFα gene expression were positively correlated after RYGB in ZDSD rats, suggesting that the modulation of pro-inflammatory pathways in adipose tissue after RYGB may partly relate to PCSK9 and LDLR gene expression.

The effect of resistance training on PCSK9 levels in patients undergoing cardiac rehabilitation after coronary artery bypass grafting: a randomized study.

BACKGROUND: Resistance training is commonly recommended as part of secondary prevention for post-coronary artery bypass graft (CABG) patients in conjunction with aerobic exercise. Despite its potential benefits, there is currently a lack of studies investigating the impact of resistance training on proprotein convertase subtilisin kexin 9 (PCSK9). AIM: To evaluate the effect of intensive resistance training on proprotein convertase subtilisin kexin 9 (PCSK9) levels among post-CABG patients undergoing cardiac rehabilitation (CR). METHODS: In this prospective, open-label, randomized trial, 87 post-coronary artery bypass graft (CABG) patients were randomly assigned into two groups: moderate to high intensity resistance training and aerobic training (n = 44) or aerobic training alone (n = 43) for a total of 12 sessions. Changes in PCSK9 levels was determined as a primary endpoint, while secondary endpoints included changes in the six-minute walk test (6-MWT) results, aerobic capacity, WHO-5 well-being index, fasting blood glucose, and lipid profile. Both groups underwent intention-to-treat analysis. RESULTS: Following completion of cardiac rehabilitation program, the intervention group demonstrated a significant decrease in mean PCSK9 levels when compared to the control group (ß = -55 ng/ml, 95% CI -6.7 to -103.3, p = 0.026), as well as significant improvements in the 6-MWT result (ß = 28.2 m, 95% CI 2.4-53.9, p = 0.033), aerobic capacity (ß = 0.9 Mets, 95% CI 0.1-1.7, p = 0.021), and WHO-5 well-being index (ß = 8.1, 95% CI 2.0-14.4, p = 0.011) in patients who received resistance and aerobic training. No statistically significant changes were observed in fasting blood glucose, cholesterol, LDL-C, HDL-C, and triglyceride levels. CONCLUSION: Resistance training in CR significantly reduced PCSK-9 levels and increases patient's functional capacity and quality of life. (NCT02674659 04/02/2016).

Structural, Functional, and Mutational Studies of a Potent Subtilisin Inhibitor from Budgett's Frog, Lepidobatrachus laevis.

Subtilases play a significant role in microbial pathogen infections by degrading the host proteins. Subtilisin inhibitors are crucial in fighting against these harmful microorganisms. LL-TIL, from skin secretions of Lepidobatrachus laevis, is a cysteine-rich peptide belonging to the I8 family of inhibitors. Protease inhibitory assays demonstrated that LL-TIL acts as a slow-tight binding inhibitor of subtilisin Carlsberg and proteinase K with inhibition constants of 91 pM and 2.4 nM, respectively. The solution structures of LL-TIL and a mutant peptide reveal that they adopt a typical TIL-type fold with a canonical conformation of a reactive site loop (RSL). The structure of the LL-TIL-subtilisin complex and molecular dynamics (MD) simulations provided an in-depth view of the structural basis of inhibition. NMR relaxation data and molecular dynamics simulations indicated a rigid conformation of RSL, which does not alter significantly upon subtilisin binding. The energy calculation for subtilisin inhibition predicted Ile31 as the highest contributor to the binding energy, which was confirmed experimentally by site-directed mutagenesis. A chimeric mutant of LL-TIL broadened the inhibitory profile and attenuated subtilisin inhibition by 2 orders of magnitude. These results provide a template to engineer more specific and potent TIL-type subtilisin inhibitors.

Effects of PCSK9 inhibitors on coronary microcirculation, inflammation and cardiac function in patients with CHD after PCI: a protocol for systematic review and meta-analysis.

INTRODUCTION: Coronary heart disease (CHD) is one of the common cardiovascular diseases that seriously jeopardise human health, and endothelial inflammation and dyslipidaemia are the initiating links leading to its occurrence. Percutaneous coronary intervention (PCI) is one of the most effective surgical treatments for CHD with narrowed or blocked blood vessels, which can quickly unblock the blocked vessels and restore coronary blood supply. However, most patients may experience coronary microcirculation disorders (CMDs) and decreased cardiac function after PCI treatment, which directly affects the efficacy of PCI and the prognosis of patients. Preprotein converting enzyme subtilisin/Kexin 9 (PCSK9) inhibitors are novel pleiotropy lipid-lowering drug with dual anti-inflammation and lipid-lowering effects, and represent a new clinical pathway for rapid correction of dyslipidaemia. Therefore, we designed this protocol to systematically evaluate the effects of PCSK9 inhibitors on coronary microcirculation and cardiac function in patients with CHD after PCI, and to provide high-quality evidence-based evidence for the clinical application of PCSK9 inhibitors. METHODS AND ANALYSIS: This protocol is reported strictly in accordance with the 2020 Preferred Reporting Items for Systematic Reviews and Meta-analyses Protocols Guidelines. We will search PubMed, EMBASE, Web of Science and three Chinese databases (CNKI, Wanfang and VIP database) according to preset search strategies, without language and publication data restrictions. We will work with manual retrieval to screen references that have been included in the literature. Google Scholar will be used to search for grey literature. The final included literature must meet the established inclusion criteria. Titles, abstracts and full text will be extracted independently by two reviewers, and disagreements will be resolved through discussion or the involvement of a third reviewer. Extracted data will be analysed using Review Manager V.5.3. The Cochrane Risk of Bias Tool will be used to evaluate the risk of bias. Publication bias will be assessed by funnel plots. Heterogeneity will be assessed by I2 test and subgroup analyses will be used to further investigate potential sources of heterogeneity. The quality of the literature will be assessed by GRADE score. This protocol will start in January 2026 and end in December 2030. ETHICS AND DISSEMINATION: This study is a systematic review of published literature data and no special ethical approval was required. PROSPERO REGISTRATION NUMBER: CRD42022346189.

Gene expression and molecular characterization of recombinant subtilisin from Bacillus subtilis with antibacterial, antioxidant and anticancer properties.

This study investigated the multifunctional attributes such as, antibacterial, antioxidant and anticancer potential of recombinant subtilisin. A codon-optimized subtilisin gene was synthesized from Bacillus subtilis and was successfully transformed into E. coli DH5α cells which was further induced for high level expression in E. coli BL21 (DE3). An affinity purified ~40 kDa recombinant subtilisin was obtained that revealed to be highly alkali-thermostable based on the thermodynamic parameters. The kinetic parameters were deduced that indicated higher affinity of N-Suc-F-A-A-F-pNA substrate towards subtilisin. Recombinant subtilisin demonstrated strong antibacterial activity against several pathogens and showed minimum inhibitory concentration of 0.06 µg/mL against B. licheniformis and also revealed high stability under the influence of several biochemical factors. It also displayed antioxidant potential in a dose dependent manner and exhibited cell cytotoxicity against A549 and MCF-7 cancerous cell lines with IC50 of 5 µM and 12 µM respectively. The identity of recombinant subtilisin was established by MALDI-TOF mass spectrum depicting desired mass peaks and N-terminal sequence as MRSK by MALDI-TOF-MS. The deduced N- terminal amino acid sequence by Edman degradation revealed high sequence similarity with subtilisins from Bacillus strains. The structural and functional analysis of recombinant antibacterial subtilisin was elucidated by Raman, circular dichroism and nuclear magnetic resonance spectroscopy and thermogravimetric analysis. The results contribute to the development of highly efficient subtilisin with enhanced catalytic properties making it a promising candidate for therapeutic applications in healthcare industries.

The Effect of 4-Methylcatechol on Platelets in Familial Hypercholesterolemic Patients Treated with Lipid Apheresis and/or Proprotein Convertase Subtilisin Kexin 9 Monoclonal Antibodies.

Elevated low-density lipoprotein (LDL) cholesterol levels lead to atherosclerosis and platelet hyperaggregability, both of which are known culprits of arterial thrombosis. Normalization of LDL cholesterol in familial hypercholesterolemia (FH) is not an easy task and frequently requires specific treatment, such as regularly performed lipid apheresis and/or novel drugs such as proprotein convertase subtilisin kexin 9 monoclonal antibodies (PCSK9Ab). Moreover, a high resistance rate to the first-line antiplatelet drug acetylsalicylic acid (ASA) stimulated research of novel antiplatelet drugs. 4-methylcatechol (4-MC), a known metabolite of several dietary flavonoids, may be a suitable candidate. The aim of this study was to analyse the antiplatelet effect of 4-MC in FH patients and to compare its impact on two FH treatment modalities via whole-blood impedance aggregometry. When compared to age-matched, generally healthy controls, the antiplatelet effect of 4-MC against collagen-induced aggregation was higher in FH patients. Apheresis itself improved the effect of 4-MC on platelet aggregation and blood from patients treated with this procedure and pretreated with 4-MC had lower platelet aggregability when compared to those solely treated with PCKS9Ab. Although this study had some inherent limitations, e.g., a low number of patients and possible impact of administered drugs, it confirmed the suitability of 4-MC as a promising antiplatelet agent and also demonstrated the effect of 4-MC in patients with a genetic metabolic disease for the first time.

Resveratrol protects against postmenopausal atherosclerosis progression through reducing PCSK9 expression via the regulation of the ERα-mediated signaling pathway.

Elevated circulating proprotein convertase subtilisin/kexin 9 (PCSK9) levels are an important contributor to postmenopausal atherosclerosis (AS). We have previously reported that resveratrol (RSV), as a phytoestrogen, reduces hepatocyte steatosis and PCSK9 expression in L02 cells. This study aimed to investigate how RSV reduces PCSK9 expression to inhibit postmenopausal AS progression. Here, we found that treatment of Ovx/ApoE -/- mice with RSV significantly reduced dyslipidemia, plasma PCSK9 concentration and aortic plaque area. In addition, RSV significantly inhibited liver fat accumulation and improved the hepatocyte ultrastructure. Further studies showed that RSV upregulated estrogen receptor α (ERα) expression, while reduced the liver X receptor α (LXRα) expression and sterol regulatory-element-binding protein-1c (SREBP-1c) transcriptional activity. In vitro, RSV inhibited insulin-induced elevated intracellular/extracellular PCSK9 levels, enhanced receptor-mediated uptake of low-density lipoproteins in HepG2 cells. Furthermore, RSV attenuated the activity of the SRE-dependent PCSK9 promoter. However, these effects can be partially reversed by the antiestrogen ICI 182,780. Attenuation of these changes with ERα inhibition suggest that RSV may prevent the progression of postmenopausal AS by reducing PCSK9 expression in hepatocytes through ERα-mediated signaling.

Crystal structure of a subtilisin-like autotransporter passenger domain reveals insights into its cytotoxic function.

Autotransporters (ATs) are a large family of bacterial secreted and outer membrane proteins that encompass a wide range of enzymatic activities frequently associated with pathogenic phenotypes. We present the structural and functional characterisation of a subtilase autotransporter, Ssp, from the opportunistic pathogen Serratia marcescens. Although the structures of subtilases have been well documented, this subtilisin-like protein is associated with a 248 residue ß-helix and itself includes three finger-like protrusions around its active site involved in substrate interactions. We further reveal that the activity of the subtilase AT is required for entry into epithelial cells as well as causing cellular toxicity. The Ssp structure not only provides details about the subtilase ATs, but also reveals a common framework and function to more distantly related ATs. As such these findings also represent a significant step forward toward understanding the molecular mechanisms underlying the functional divergence in the large AT superfamily.

Functional Characterization of p.(Arg160Gln) PCSK9 Variant Accidentally Found in a Hypercholesterolemic Subject.

Familial hypercholesterolaemia (FH) is an autosomal dominant dyslipidaemia, characterised by elevated LDL cholesterol (LDL-C) levels in the blood. Three main genes are involved in FH diagnosis: LDL receptor (LDLr), Apolipoprotein B (APOB) and Protein convertase subtilisin/kexin type 9 (PCSK9) with genetic mutations that led to reduced plasma LDL-C clearance. To date, several PCSK9 gain-of-function (GOF) variants causing FH have been described based on their increased ability to degrade LDLr. On the other hand, mutations that reduce the activity of PCSK9 on LDLr degradation have been described as loss-of-function (LOF) variants. It is therefore important to functionally characterise PCSK9 variants in order to support the genetic diagnosis of FH. The aim of this work is to functionally characterise the p.(Arg160Gln) PCSK9 variant found in a subject suspected to have FH. Different techniques have been combined to determine efficiency of the autocatalytic cleavage, protein expression, effect of the variant on LDLr activity and affinity of the PCSK9 variant for the LDLr. Expression and processing of the p.(Arg160Gln) variant had a result similar to that of WT PCSK9. The effect of p.(Arg160Gln) PCSK9 on LDLr activity is lower than WT PCSK9, with higher values of LDL internalisation (13%) and p.(Arg160Gln) PCSK9 affinity for the LDLr is lower than WT, EC50 8.6 ± 0.8 and 25.9 ± 0.7, respectively. The p.(Arg160Gln) PCSK9 variant is a LOF PCSK9 whose loss of activity is caused by a displacement of the PCSK9 P' helix, which reduces the stability of the LDLr-PCSK9 complex.

Novel Target Study to Cure Cardiovascular Disease regarding Proprotein Converse Subtilisin/Kexin Type 9.

Objective: This study is aimed at screening the potential ideal lead compounds from natural drug library (ZINC database), which had potential inhibition effects against proprotein converse subtilisin/kexin type 9 (PCSK9), and contributing to enrich the practical basis of PCSK9 inhibitor screening. Methods: A series of computer-aided virtual screening techniques were used to identify potential inhibitors of PCSK9. Structure-based virtual screening by LibDock was carried out to calculate the LibDock scores, followed by ADME (absorption, distribution, metabolism, and excretion) and toxicity predictions. Molecule docking was next employed to demonstrate the binding affinity and mechanism between the candidate ligands and PCSK9 macromolecule. Finally, molecular dynamics simulation was performed to evaluate the stability of ligand-PCSK9 complex under natural circumstance. Results: Two novel natural compounds ZINC000004099069 and ZINC000014952116 from the ZINC database were found to bind with PCSK9 with a higher binging affinity together with more favorable interaction energy. Also, they were predicted to be non-CYP2D6 inhibitors, together with low rodent carcinogenicity and AMES mutagenicity as well as hepatotoxicity. Molecular dynamics simulation analysis demonstrated that these two complex ZINC000004099069- and ZINC000014952116-PCSK9 had more favorable potential energy compared to the reference ligand, which could exist stably whether in vivo or in vitro. Conclusion: This study elucidated that ZINC000004099069 and ZINC000014952116 were finally screened as safe and potential drug candidates, which may have great significance in the development of PCSK9 inhibitor development.

Subtilisin from Bacillus amyloliquefaciens induces apoptosis in breast cancer cells through ubiquitin-proteasome-mediated tubulin degradation.

To search for novel proteases from environmental isolates which can induce apoptosis in cancer cells, we have purified subtilisin from Bacillus amyloliquefaciens and studied its anti-cancer properties. Subtilisin induced apoptosis in colon (HT29) and breast (MCF7) cancer cells but showed no effect on mouse peritoneal macrophages and normal breast cells (MCF10A). Western blot analysis showed that Bax, Bcl-2 level remained unchanged but tubulin level decreased significantly. Subtilisin does not induce the intrinsic pathway of apoptosis, rather it induced tubulin degradation in MCF-7 cells, whereas in normal cells (MCF-10A) tubulin degradation was not observed. Subtilisin activates ubiquitination and proteasomal-mediated tubulin degradation which was completely restored in presence of proteasome inhibitor MG-132. We further observed PARKIN, one of the known E3-ligase, is overexpressed and interacts with tubulin in subtilisin treated cells. Knockdown of PARKIN effectively downregulates ubiquitination and inhibits degradation of tubulin. PARKIN activation and tubulin degradation lead to ER-stress which in turn activates caspase-7 and PARP cleavage, thus guiding the subtilisin treated cells towards apoptosis. To our knowledge this is the first report of subtilisin induced apoptosis in cancer cells by proteasomal degradation of tubulin.

A Comprehensive Review of PCSK9 Inhibitors.

Cardiovascular disease (CVD) is the leading cause of death in the United States and worldwide. A major risk factor for this condition is increased serum low-density lipoprotein cholesterol (LDL-C) levels for which statins have been successful in reducing serum LDL-C to healthy concentrations. However, patients who are statin intolerant or those who do not achieve their treatment goals while on high-intensity statin therapy, such as those with familial hypercholesterolemia, remain at risk. With the discovery of PCSK9 inhibitors, the ability to provide more aggressive treatment for patients with homozygous and heterozygous familial hypercholesterolemia has increased. Ezetimibe reduces LDL-C by 15%-20% when combined with statin.2,3 Protein convertase subtilisin/kexin type 9 (PCSK9) inhibitors have been found to achieve profound reductions in LDL-C (54%-74%) when added to statins. They have shown dramatic effects at lowering major adverse cardiovascular events (MACE) in high-risk patients4 with LDL-C levels ≥70 mg/dL and can be used in populations that are statin intolerant or not at goal levels with maximally tolerated statin therapy. PCSK9 inhibitors also produce minimal side effects. Myopathy, a common side effect for patients on statins, has been rare in patients on PCSK9 inhibitors. Randomized trials have shown that reduction in LDL-C has translated to clinical benefits even in patients who have not achieved their LDL-C target.