Diagnostic ability of Peptidase S8 gene in the Arthrodermataceae causing dermatophytoses: A metadata analysis.

An unambiguous identification of dermatophytes causing dermatophytoses is necessary for accurate clinical diagnosis and epidemiological implications. In the current taxonomy of the Arthrodermataceae, the etiological agents of dermatophytoses consist of seven genera and members of the genera Trichophyton are the most prevalent etiological agents at present. The genera Trichophyton consists of 16 species that are grouped as clades, but the species borderlines are not clearly delimited. The aim of the present study was to determine the discriminative power of subtilisin gene variants (SUB1-SUB12) in family Arthrodermataceae, particularly in Trichophyton. Partial and complete reads from 288 subtilisin gene sequences of 12 species were retrieved and a stringent filtering following two different approaches for analysis (probability of correct identification (PCI) and gene gap analysis) conducted to determine the uniqueness of the subtilisin gene subtypes. SUB1 with mean PCI value of 60% was the most suitable subtilisin subtype for specific detection of T.rubrum complex, however this subtype is not reported in members of T. mentagrophytes complex which is one of the most prevalent etiological agent at present. Hence, SUB7 with 40% PCI value was selected for testing its discriminative power in Trichophyton species. SUB7 specific PCR based detection of dermatophytes was tested for sensitivity and specificity. Sequences of SUB7 from 42 isolates and comparison with the ITS region showed that differences within the subtilisin gene can further be used to differentiate members of the T. mentagrophytes complex. Further, subtilisin cannot be used for the differentiation of T. benhamiae complex since all SUB subtypes show low PCI scores. Studies on the efficiency and limitations of the subtilisin gene as a diagnostic tool are currently limited. Our study provides information that will guide researchers in considering this gene for identifying dermatophytes causing dermatophytoses in human and animals.

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.

Atherosclerosis and Toll-Like Receptor4 (TLR4), Lectin-Like Oxidized Low-Density Lipoprotein-1 (LOX-1), and Proprotein Convertase Subtilisin/Kexin Type9 (PCSK9).

Atherosclerosis is a leading cause of death in the world. A significant body of evidence suggests that inflammation and various players are implicated and have pivotal roles in the formation of atherosclerotic plaques. Toll-like receptor 4 (TLR4) is linked with different stages of atherosclerosis. This receptor is highly expressed in the endothelial cells (ECs) and atherosclerotic plaques. TLR4 activation can lead to the production of inflammatory cytokines and related responses. Lectin-like oxidized low-density lipoprotein-1 (LOX-1), an integral membrane glycoprotein with widespread expression on the ECs, is involved in atherosclerosis and has some common pathways with TLR4 in atherosclerotic lesions. In addition, proprotein convertase subtilisin/kexin type9 (PCSK9), which is a regulatory enzyme with different roles in cholesterol uptake, is implicated in atherosclerosis. At present, TLR4, PCSK9, and LOX-1 are increasingly acknowledged as key players in the pathogenesis of atherosclerotic cardiovascular diseases. Herein, we presented the current evidence on the structure, functions, and roles of TLR4, PCSK9, and LOX-1 in atherosclerosis.

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.

Causal relationship between PCSK9 inhibitor and primary glomerular disease: a drug target Mendelian randomization study.

Background: Successive observational studies have highlighted low-density lipoprotein cholesterol (LDL-C) as a standalone risk factor for the progression of chronic kidney disease (CKD) to end-stage renal disease. Lowering LDL-C levels significantly reduces the incidence of atherosclerotic events in patients with progressive CKD. Recent research indicates that proprotein convertase subtilisin kexin 9 (PCSK9) inhibitors not only effectively lower LDL-C levels in CKD patients but also exhibit therapeutic potential for autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and ulcerative colitis. However, the role of PCSK9 inhibitors (PCSK9i) in treating CKD beyond lowering LDL-C levels remains uncertain. Therefore, this study employs drug-targeted Mendelian randomization (MR) to investigate the causal impact of PCSK9i on primary glomerular diseases such as IgA nephropathy (IgAN), membranous nephropathy (MN), and nephrotic syndrome (NS). Methods: Single-nucleotide polymorphisms (SNPs) linked to LDL-C were sourced from the Global Lipids Genetics Consortium genome-wide association study (GWAS). Genes situated in proximity to 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), and PCSK9 served as proxies for therapeutic inhibition of these targets. The causal link between PCSK9i and the risk of primary glomerular disorders was discovered using drug-target MR studies. The HMGCR inhibitor, a drug target of statins, was utilized for comparative analysis with PCSK9i. Primary outcomes included the risk assessment for IgAN, MN, and NS, using the risk of coronary heart disease as a positive control. Results: The inhibition of PCSK9, as proxied genetically, was found to significantly reduce the risk of IgAN [odds ratio, OR (95% confidence interval, CI) = 0.05 (-1.82 to 1.93), p = 2.10 × 10-3]. Conversely, this inhibition was associated with an increased risk of NS [OR (95% CI) = 1.78 (1.34-2.22), p = 0.01]. Similarly, HMGCR inhibitors (HMGCRi) demonstrated a potential reduction in the risk of IgAN [OR (95%CI) = 0.0032 (-3.58 to 3.59), p = 1.60 × 10-3). Conclusions: PCSK9i markedly decreased the risk of IgAN, suggesting a potential mechanism beyond their primary effect on LDL-C. However, these inhibitors were also associated with an increased risk of NS. On the other hand, HMGCRi appears to serve as a protective factor against IgAN. Conversely, PCSK9i may pose a risk factor for NS, suggesting the necessity for cautious application and further research into their impacts on various glomerular diseases.

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.

Inhibition of Proprotein Convertase Subtilisin/Kexin-9 After Kidney Transplant: Single-Center Experience Among Patients With High Cardiovascular Risk.

OBJECTIVES: The benefits of reduction in low-density lipoprotein cholesterol by evolocumab by nearly 60% has not been evaluated among kidney transplant recipients to our knowledge. We assessed the efficacy and safety of evolocumab, a proprotein convertase subtilisin/kexin-9 inhibitor, in reducing lipids and cardiovascular events among kidney transplant recipients in a randomized controlled study. MATERIALS AND METHODS: Between June 2017 and June 2019, we enrolled 197 kidney transplant recipients with high cardiovascular risk score (>20). Patients who received evolocumab (140 mg/2 weeks) comprised group 1 (n = 98), and patients maintained on statin therapy comprised group 2 (n = 99). We followed patients clinically and with necessary laboratory investigations over 24 months. RESULTS: The 2 groups had comparable demographic characteristics (P > .05). Before enrollment in the study, smokers were significantly more prevalent in group 1, whereas posttransplant diabetes mellitus was more prevalent in group 2 (P = .033). Moreover, baseline serum creatinine was higher in group 1, whereas immunosuppression was equivalent in both groups (P > .05). We found no significant differences between the 2 groups concerning cardiovascular events, and both graft and patient outcomes were comparable (P > .05). The higher baseline cholesterol in group 1 (5.5 vs 4.7 mmol/L; P < .001) decreased significantly after 3 months and thereafter (P = .031) compared with levels in group 2 and baseline values (P < .001). We reported 2 cases of acute myocardial infarction and 1 atrial fibrillation in group 2. CONCLUSIONS: Proprotein convertase subtilisin/kexin-9 inhibitors, as an added therapy to statins, are safe and effective in treating hypercholesterolemia after kidney transplant. Evolocumab can minimize cardiovascular events after kidney transplant in patients with high events at baseline. Longer-term trials with larger number of patients are needed to confirm its beneficial effects on cardiovascular complications and patient and graft survival.

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.

Prodomain-driven enzyme dimerization: a pH-dependent autoinhibition mechanism that controls Plasmodium Sub1 activity before merozoite egress.

Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBCs) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes. Over the last two decades, it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi-step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto-maturation in the endoplasmic reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergoes further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the PV. Here, we report the crystal structure of full-length P. falciparum Sub1 (PfS1FL) and demonstrate, through structural, biochemical, and biophysical studies, that the atypical Plasmodium-specific Sub1 PD directly promotes the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, explaining how PmX processing and full activation of Sub1 can occur in different cellular compartments, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition that plays a key role in P. falciparum merozoites egress from infected host cells.IMPORTANCEMalaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin-like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is required in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here, we show that the unusual, parasite-specific Sub1 prodomain is directly responsible for its acidic-dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites.

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.

Role of PCSK9 inhibitors in the management of dyslipidaemia.

Proprotein convertase subtilisin kexin9 (PCSK9) inhibitors are novel agents that lower LDL cholesterol and reduce cardio-vascular event rate. Being expensive, these agents are reserved for those with high risk or very high risk of CV events and with suboptimal response to statins and ezetimibe, with or without bempedoic acid or those intolerant to statins.

Protective effect of alirocumab, a PCSK9 inhibitor, on the sciatic nerve of rats with diabetic peripheral neuropathy.

Dyslipidemia has been considered a risk factor for diabetic peripheral neuropathy. Proprotein convertase subtilisin-like/Kexin 9 inhibitor (PCSK9) inhibitors are a new type of lipid-lowering drug currently in clinical use. The role of PCSK9 in diabetic peripheral neuropathy is still unclear. In this study, the effect of alirocumab, a PCSK9 inhibitor, on the sciatic nerve in rats with diabetic peripheral neuropathy and its underlying mechanisms were investigated. The diabetic peripheral neuropathy rat model was established by using a high-fat diet combined with streptozotocin injection, and experimental subjects were divided into normal, diabetic peripheral neuropathy, and alirocumab groups. The results showed that Alirocumab improved nerve conduction, morphological changes, and small fiber deficits in rats with DPN, possibly related to its amelioration of oxidative stress and the inflammatory response.

Assessing the Impact of PCSK9 and HMGCR Inhibition on Liver Function: Drug-Target Mendelian Randomization Analyses in Four Ancestries.

BACKGROUND & AIMS: Observational studies have linked lipid-lowering drug targets pro-protein convertase subtilisin/kexin 9 (PCSK9) and HMG-CoA reductase (HMGCR) with adverse liver outcomes; however, liver disease incidence varies across diverse populations, and the long-term hepatic impact of these lipid-lowering drugs among non-white Europeans remains largely unknown. METHODS: We use single nucleotide polymorphisms (SNPs) in PCSK9 and HMGCR loci from genome-wide association study data of low-density lipoprotein cholesterol in 4 populations (East Asian [EAS], South Asian [SAS], African [AFR], and European [EUR]) to perform drug-target Mendelian randomization investigating relationships between PCSK9 and HMGCR inhibition and alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), and bilirubin. RESULTS: Analyses of PCSK9 instruments, including functional variants R46L and E670G, failed to find evidence for relationships of low-density lipoprotein cholesterol lowering via PCSK9 variants and adverse effects on ALT, AST, GGT, or ALP among the cohorts. PCSK9 inhibition was associated with increased direct bilirubin levels in EUR (ß = 0.089; P value = 5.69 × 10-6) and, nominally, in AFR (ß = 0.181; P value = .044). HMGCR inhibition was associated with reduced AST in SAS (ß = -0.705; P value = .005) and, nominally, reduced AST in EAS (ß = -0.096; P value = .03), reduced ALP in EUR (ß = -2.078; P value = .014), and increased direct bilirubin in EUR (ß = 0.071; P value = .032). Sensitivity analyses using genetic instruments derived from circulating PCSK9 protein levels, tissue-specific PCSK9 expression, and HMGCR expression were in alignment, strengthening causal inference. CONCLUSIONS: We did not find ALT, AST, GGT, or ALP associated with genetically proxied PCSK9 and HMGCR inhibition across ancestries. We identified possible relationships in several ancestries between PCSK9 and increased direct and total bilirubin and between HMGCR and reduced AST. These findings support long-term safety profiles and low hepatotoxic risk of PCSK9 and HMGCR inhibition in diverse populations.

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.

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.

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.

PCSK7: A novel regulator of apolipoprotein B and a potential target against non-alcoholic fatty liver disease.

BACKGROUND: Epidemiological evidence links the proprotein convertase subtilisin/kexin 7 (PCSK7) to triglyceride (TG) metabolism. We associated the known PCSK7 gain-of-function non-coding SNP rs236918 with higher levels of plasma apolipoprotein B (apoB) and the loss-of-function coding variant p.Pro777Leu (SNP rs201598301) with lower apoB and TG. Herein, we aimed to unravel the in vivo role of liver PCSK7. METHODS: We biochemically defined the functional role of PCSK7 in lipid metabolism using hepatic cell lines and Pcsk7-/- mice. Our findings were validated following subcutaneous administration of hepatocyte-targeted N-acetylgalactosamine (GalNAc)-antisense oligonucleotides (ASOs) against Pcsk7. RESULTS: Independent of its proteolytic activity, membrane-bound PCSK7 binds apoB100 in the endoplasmic reticulum and enhances its secretion. Mechanistically, the loss of PCSK7/Pcsk7 leads to apoB100 degradation, triggering an unfolded protein response, autophagy, and ß-oxidation, eventually reducing lipid accumulation in hepatocytes. Non-alcoholic fatty liver disease (NAFLD) was induced by a 12-week high fat/fructose/cholesterol diet in wild type (WT) and Pcsk7-/- mice that were then allowed to recover on a 4-week control diet. Pcsk7-/- mice recovered more effectively than WT mice from all NAFLD-related liver phenotypes. Finally, subcutaneous administration of GalNAc-ASOs targeting hepatic Pcsk7 to WT mice validated the above results. CONCLUSIONS: Our data reveal hepatic PCSK7 as one of the major regulators of apoB, and its absence reduces apoB secretion from hepatocytes favoring its ubiquitination and degradation by the proteasome. This results in a cascade of events, eventually reducing hepatic lipid accumulation, thus supporting the notion of silencing PCSK7 mRNA in hepatocytes for targeting NAFLD.

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.