Furin Protein Research Articles

Diesel exhaust particle inhalation in conjunction with high-fat diet consumption alters the expression of pulmonary SARS-COV-2 infection pathways, which is mitigated by probiotic treatment in C57BL/6 male mice

BackgroundBoth exposure to air pollutants and obesity are associated with increased incidence and severity of COVID-19 infection; however, the mechanistic pathways involved are not well-characterized. After being primed by the transmembrane protease serine 2 (TMPRSS2) or furin protease, SARS-CoV-2 uses the angiotensin-converting enzyme (ACE)-2 receptor to enter respiratory epithelial cells. The androgen receptor (AR) is known to regulate both TMPRSS2 and ACE2 expression, and neuropilin-1 (NRP1) is a proposed coreceptor for SARS-CoV-2; thus, altered expression of these factors may promote susceptibility to infection. As such, this study investigated the hypothesis that inhalational exposure to traffic-generated particulate matter (diesel exhaust particulate; DEP) increases the expression of those pathways that mediate SARS-CoV-2 infection and susceptibility, which is exacerbated by the consumption of a high-fat (HF) diet.MethodsFour- to six-week-old male C57BL/6 mice fed either regular chow or a HF diet (HF, 45% kcal from fat) were randomly assigned to be exposed via oropharyngeal aspiration to 35 µg DEP suspended in 35 µl 0.9% sterile saline or sterile saline only (control) twice a week for 30 days. Furthermore, as previous studies have shown that probiotic treatment can protect against exposure-related inflammatory outcomes in the lungs, a subset of study animals fed a HF diet were concurrently treated with 0.3 g/day Winclove Ecologic® Barrier probiotics in their drinking water throughout the study.ResultsOur results revealed that the expression of ACE2 protein increased with DEP exposure and that TMPRSS2, AR, NRP1, and furin protein expression increased with DEP exposure in conjunction with a HF diet. These DEP ± HF diet-mediated increases in expression were mitigated with probiotic treatment.ConclusionThese findings suggest that inhalational exposure to air pollutants in conjunction with the consumption of a HF diet contributes to a more susceptible lung environment to SARS-CoV-2 infection and that probiotic treatment could be beneficial as a preventative measure.

Structure-based virtual screening methods for the identification of novel phytochemical inhibitors targeting furin protease for the management of COVID-19.

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, is a highly contagious respiratory disease with widespread societal impact. The symptoms range from cough, fever, and pneumonia to complications affecting various organs, including the heart, kidneys, and nervous system. Despite various ongoing efforts, no effective drug has been developed to stop the spread of the virus. Although various types of medications used to treat bacterial and viral diseases have previously been employed to treat COVID-19 patients, their side effects have also been observed. The way SARS-CoV-2 infects the human body is very specific, as its spike protein plays an important role. The S subunit of virus spike protein cleaved by human proteases, such as furin protein, is an initial and important step for its internalization into a human host. Keeping this context, we attempted to inhibit the furin using phytochemicals that could produce minimal side effects. For this, we screened 408 natural phytochemicals from various plants having antiviral properties, against furin protein, and molecular docking and dynamics simulations were performed. Based on the binding score, the top three compounds (robustaflavone, withanolide, and amentoflavone) were selected for further validation. MM/GBSA energy calculations revealed that withanolide has the lowest binding energy of -57.2 kcal/mol followed by robustaflavone and amentoflavone with a binding energy of -45.2 kcal/mol and -39.68 kcal/mol, respectively. Additionally, ADME analysis showed drug-like properties for these three lead compounds. Hence, these natural compounds robustaflavone, withanolide, and amentoflavone, may have therapeutic potential for the management of SARS-CoV-2 by targeting furin.

Olive Leaf Extract Downregulates the Protein Expression of Key SARS-CoV-2 Entry Enzyme ACE-2, TMPRSS2, and Furin.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses ongoing global health challenges due to its propensity for mutations, which can undermine vaccine efficacy. With no definitive treatment available, urgent research into affordable and biocompatible therapeutic agents is extremely urgent. Angiotensin converting enzyme-2 (ACE-2), transmembrane protease serine subtype 2 (TMPRSS2), and Furin enzymes, which allow the virus to enter cells, are particularly important as potential drug targets among scientists. Olive leaf extract (OLE) has garnered attention for its potential against Coronavirus Disease-9 (COVID-19), yet its mechanism remains understudied. In this study, we aimed to investigate the effects of OLE on ACE-2, TMPRSS2, and Furin protein expressions by cell culture study. Total phenol, flavonoid content, and antioxidant capacity were measured by photometric methods, and oleuropein levels were measured by liquid LC-HR-MS. Cell viability was analyzed by ATP levels using a luminometric method. ACE-2, TMPRSS2, and Furin expressions were analyzed by the Western Blotting method. ACE-2, TMPRSS2, and Furin protein expression levels were significantly lower in a dose dependent manner and the highest inhibition was seen at 100 μg/ml OLE. The results showed that OLE may be a promising treatment candidate for COVID-19 disease. However, further studies need to be conducted in cells co-infected with the virus.

Novel genetic association of the Furin gene polymorphism rs1981458 with COVID-19 severity among Indian populations

SARS CoV-2, the causative agent for the ongoing COVID-19 pandemic, it enters the host cell by activating the ACE2 receptor with the help of two proteasesi.e., Furin and TMPRSS2. Therefore, variations in these genes may account for differential susceptibility and severity between populations. Previous studies have shown that the role of ACE2 and TMPRSS2 gene variants in understanding COVID-19 susceptibility among Indian populations. Nevertheless, a knowledge gap exists concerning the COVID-19 susceptibility of Furin gene variants among diverse South Asian ethnic groups. Investigating the role of Furin gene variants and their global phylogeographic structure is essential to comprehensively understanding COVID-19 susceptibility in these populations. We have used 450 samples from diverse Indian states and performed linear regression to analyse the Furin gene variant's with COVID-19 Case Fatality Rate (CFR) that could be epidemiologically associated with disease severity outcomes. Associated genetic variants were further evaluated for their expression and regulatory potential through various Insilco analyses. Additionally, we examined the Furin gene using next-generation sequencing (NGS) data from 393 diverse global samples, with a particular emphasis on South Asia, to investigate its Phylogeographic structure among diverse world populations. We found a significant positive association for the SNP rs1981458 with COVID-19 CFR (p < 0.05) among diverse Indian populations at different timelines of the first and second waves. Further, QTL and other regulatory analyses showed various significant associations for positive regulatory roles of rs1981458 and Furin gene, mainly in Immune cells and virus infection process, highlighting their role in host immunity and viral assembly and processing. The Furin protein–protein interaction suggested that COVID-19 may contribute to Pulmonary arterial hypertension via a typical inflammation mechanism. The phylogeographic architecture of the Furin gene demonstrated a closer genetic affinity of South Asia with West Eurasian populations. Therefore, it is worth proposing that for the Furin gene, the COVID-19 susceptibility of South Asians will be more similar to the West Eurasian population. Our previous studies on the ACE2 and TMPRSS2 genes showed genetic affinity of South Asian with East Eurasians and West Eurasians, respectively. Therefore, with the collective information from these three important genes (ACE2, TMPRSS2 and Furin) we modelled COVID-19 susceptibilityof South Asia in between these two major ancestries with an inclination towards West Eurasia. In conclusion, this study, for the first time, concluded the role of rs1981458 in COVID-19 severity among the Indian population and outlined its regulatory potential.This study also highlights that the genetic structure for COVID-19 susceptibilityof South Asia is distinct, however, inclined to the West Eurasian population. We believe this insight may be utilised as a genetic biomarker to identify vulnerable populations, which might be directly relevant for developing policies and allocating resources more effectively during an epidemic.

Identification of potential biomarkers for SLE through mRNA expression profiling

ABSTRACT Background: Systemic lupus erythematosus (SLE) is an autoimmune disease that influences numerous body systems. Furin, tristetraprolin (TTP), and NOD, LRR, and pyrin domain-containing protein 3 (NLRP3) contribute in developing autoimmune illnesses. Aim: Understandthe role of furin, TTP, and NLRP3 mRNA gene expression in SLE pathogenesis and prognosis. Methods: Total 210 individuals were enrolled, divided in two group: cases and control; 105 participants in each group. Real-time quantitative PCR for furin, TTP,and NLRP3 mRNA gene expression were determined for each subject. Results: SLE patients showed significantly higher serum furin [median 20.10 (0.0–162.88) in comparison with control group [median 1.10 (0.33–8.64)] with significant pvalue (p < 0.001), for NLRP3 expression [median 7.03 (0.0–282.97) compared to control group [median 1.0 (0.44–9.48)] with significant p value (p = 0.006)but lower TTP [median 2.37 (0.0–30.13)] in comparison with control group [median 7.90 (1.0–29.29)] with significant p value (p < 0.001) . Elevated levels of Furin and NLRP3 and low levels of TTP were linked to increased illness activity. Conclusion: Furin and NLRP increase in SLE and higher with illness activity. TTP is lowerin SLE and negatively correlates with disease activity.

The basis of complications in the context of SARS-CoV-2 infection: Pathological activation of ADAM17

The virulence of SARS-CoV-2 decreases with increasing infectivity, the primary approaches for antiviral treatments will be preventing or minimizing the complications resulting from virus infection. ADAM metallopeptidase domain 17 (ADAM17) activation by SARS-CoV-2 infection has a dual effect on the development of the disease: increased release of inflammatory cytokines and dysregulation of Angiotensin converting enzyme II (ACE2) on cell surfaces, inflammatory cytokine infiltration and loss of ACE2 protective function lead to a significant increase in the incidence of related complications. Importantly, pathologically activated ADAM17 showed superior features than S protein in regulating ACE2 expression and participating in the intra cellular replication of SARS-CoV-2. In short, SARS-CoV-2 elicits only a limited immune response when it promotes its own replication and pathogenicity through ADAM17. Therefore, the pathological activation of ADAM17 may also represent a diminished innate antiviral defense and an altered strategy of SARS-CoV-2 infection. In this review, we summarized recent advances in our understanding of the pathophysiology of ADAM17, with a focus on the new findings that SARS-CoV-2 affects ADAM17 expression through Furin protein converting enzyme and Mitogen-activated protein kinase (MAPK) pathway, and raises the hypothesis that SARS-CoV-2 may mediates the pathological activation of ADAM17 by hijacking the actin regulatory pathway, and discussed the underlying biological principles.

Hypoxia modifies levels of the SARS-CoV-2 cell entry proteins, angiotensin-converting enzyme 2, and furin in fetal human brain endothelial cells

It is not known whether human fetal brain endothelial cells (hfBECs) that form the blood-brain barrier (BBB) express ACE2, TMPRSS2 and Furin; SARS-CoV-2 cell entry proteins. It is also unclear whether hypoxia, commonly observed during severe maternal COVID-19, can modify their level of expression. We hypothesized that hfBECs isolated from early- and mid-gestation brain microvessels express ACE2, TMPRSS2 and Furin. We also hypothesized that hypoxia modifies their expression levels in a gestational-age and time of exposure-dependent manner. To investigate whether early- and mid-gestation hfBECs express ACE2, TMPRSS2 and Furin SARS-CoV-2 associated cell entry proteins. We also determined the effects of hypoxia on ACE2, TMPRSS2 and Furin expression levels in hfBECs. This is a prospective study where hfBECs isolated from early- (12.4 ± 0.7 weeks), and mid-gestation (17.9 ± 0.5) fetal brain microvessels (N=6/group) were exposed to different oxygen tensions (20-, 5- and 1%-oxygen) for 6-, 24- and 48-h. ACE2, TMPRSS2 and Furin mRNA and protein levels and localization were assessed using qPCR, Western blot and immunofluorescence. ACE2, TMPRSS2 and Furin co-localize with the endothelial cell marker von Willebrand factor (vWF) in hfBECS isolated from early- and mid-pregnancy. In early-gestation, TMPRSS2 mRNA expression was decreased by 5% oxygen compared to 20% oxygen after 6h of exposure (p<0.05). In mid-gestation, 5% oxygen downregulated ACE2 mRNA compared to 20% oxygen after 24h (p<0.05). Furin mRNA expression was decreased under 5% and 1% oxygen compared to 20% oxygen (p<0.05) after 24h. In mid-gestation, ACE2 protein levels were decreased under 5% and 1% oxygen (p<0.001) after 24h. In contrast, Furin protein levels were increased under 1% oxygen compared to 20% oxygen after 24h (p<0.05). At 48h, 1% oxygen increased ACE2 protein levels compared to 20% oxygen (p<0.01). Hypoxia modifies the expression of selected SARS-CoV-2 cell entry proteins in hfBECs, in a gestational-age- and time of exposure-dependent manner. Since severe COVID-19 illness may lead to maternal hypoxia, an altered expression of these proteins in the developing human BBB could potentially lead to altered SARS-CoV-2 brain invasion, and neurological sequelae in neonates born to pregnancies complicated by SARS-CoV-2 infection.

Structural insights into the effect of mutations in the spike protein of SARS-CoV-2 on the binding with human furin protein

The SARS COV-2 and its variants are spreading around the world at an alarming speed, due to its higher transmissibility and the conformational changes caused by mutations. The resulting COVID-19 pandemic has imposed severe health consequences on human health. Several countries of the world including Pakistan have studied its genome extensively and provided productive findings. In the current study, the mCSM, DynaMut2, and I-Mutant servers were used to analyze the effect of identified mutations on the structural stability of spike protein however, the molecular docking and simulations approaches were used to evaluate the dynamics of the bonding network between the wild-type and mutant spike proteins with furin. We addressed the mutational modifications that have occurred in the spike protein of SARS-COV-2 that were found in 215 Pakistani's isolates of COVID-19 patients to study the influence of mutations on the stability of the protein and its interaction with the host cell. We found 7 single amino acid substitute mutations in various domains that reside in spike protein. The H49Y, N74K, G181V, and G446V were found in the S1 domain while the D614A, V622F, and Q677H mutations were found in the central helices of the spike protein. Based on the observation, G181V, G446V, D614A, and V622F mutants were found highly destabilizing and responsible for structural perturbation. Protein-protein docking and molecular simulation analysis with that of furin have predicted that all the mutants enhanced the binding efficiency however, the V622F mutant has greatly altered the binding capacity which is further verified by the KD value (7.1 E−14) and therefore may enhance the spike protein cleavage by Furin and increase the rate of infectivity by SARS-CoV-2. On the other hand, the total binding energy for each complex was calculated which revealed −50.57 kcal/mol for the wild type, for G181V −52.69 kcal/mol, for G446V −56.44 kcal/mol, for D614A −59.78 kcal/mol while for V622F the TBE was calculated to be −85.84 kcal/mol. Overall, the current finding shows that these mutations have increased the binding of Furin for spike protein and shows that D614A and V622F have significant effects on the binding and infectivity.

Diabetes Mellitus and COVID-19: Possible Interactions and Mechanisms in Comorbid Patients

Beginning in December 2019 and still ongoing, coronavirus disease 2019 (COVID-19) infections have posed a public health challenge worldwide. There have been reports of diabetes mellitus (DM) as one of the most prevalent comorbidities in patients with COVID-19. Although the interactions and possible mechanisms of this association have not been fully established, the existence of DM is believed to aggravate the adverse effects of COVID-19 infection. Hence, the need for this paper. Findings from other studies have shown different possible mechanisms of how COVID-19 and DM aggravate the severity of each other. Among the hypothetical mechanisms reported between COVID-19 and DM in this paper are: COVID-19 causes complications of DM through the following: (1) Destruction of β-cells in the pancreas by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. (2) Cytokine storm generation which mediates tissue inflammation resulting in organ damage and (3) The use of corticosteroid drugs which have been found to be highly diabetogenic. Similarly, DM facilitates internalizing of SARS-CoV-2 symptoms through increasing expression of angiotensin-converting enzyme 2 (ACE2) and the furin protein, viral load, entrance and replication of SARS-CoV-2, glycosylation, and compromising of the immune response that worsens COVID-19. Having a clear understanding of the biochemical mechanisms of interactions between COVID-19 and DM may be useful for future research of agents targeted as therapeutic remedies for managing patients with diabetes infected with COVID-19 and vice versa.

Inhibition of Glypican-3 Cleavage Results in Reduced Cell Proliferation in a Liver Cancer Cell Line

IntroductionGlypican-3 (GPC3) is a surface-bound proteoglycan overexpressed in pediatric liver cancer and utilized clinically as an immunohistochemical tumor marker. Furin is a proprotein convertase that is ubiquitously expressed and shown to modify GPC3 post-translationally. In experimental models of epithelial-based cancers, furin inhibition decreased tumor cell migration and proliferation representing a potential therapeutic target. MethodsUsing a synthetic furin inhibitor, we evaluated proliferation, migration, protein, and RNA expression in two liver cancer cell lines, HepG2 (GPC3-positive) and SKHep1 cells (GPC3-negative). Total furin protein and GPC3 protein expression were assessed to evaluate functional levels of furin. ResultsThere was a reduction in HepG2 proliferation with addition of furin inhibitor at the 48-h timepoint, however there was an increase in HepG2 migration. ConclusionsGPC3 cleavage in hepatoblastoma (HB) has a role in cell proliferation with therapeutic potential, however furin inhibition is not an appropriate target for GPC3-expressing HB due to increased migration which may enhance metastatic potential.

Natural furin inhibitor(s) as potent therapeutic molecule to mitigate SARS-CoV-2 infection

The COVID 2019 has had been going pandemic as per WHO situation reports. The major differentiating point in this virus is the presence of a unique furin cleavage site. Our insilico study points out to the effectiveness of a potent plant origin furin inhibitor. We exploited the aspect of the cleavage machinery of furin subunits which is critical and indispensible to the entry of SARS-CoV-2 in human cells and subsequent massive contagion. In-silico analysis was done to observe the interactions of proposed analogs of protease inhibitor of plant origin against furin protein as well as the furin spike glycoprotein (SGP) binding machinery. This was done by docking protocols using Hex 6.0 software followed by molecular Dynamic simulation (MDS) analysis in 100 ns scale using Amber94. Further, the images were analysed with PyMol software. The analogs I, II and III included in our study showed strong interactivity against furin individually, as well as the furin-Spike Glycoprotein 1 binding machinery. The findings were confirmed using molecular dynamic simulation analysis which indicated good structural stability and ability to neutralise furin and furin-spike glycoprotein 1. Analog II was found to be the best interactive molecule against furin, showing the least deviation (1.484 ± 0.0064). Also, it was the most effective against furin + Spike glycoprotein I machinery [1.575 ± 0.01]. We report the first of its kind of natural furin inhibitor(s) which would disrupt the furin machinery of SARS-CoV-2 and help in controlling the COVID-19 contagion. Communicated by Ramaswamy H. Sarma

Loss of the Schizophrenia-Linked Furin Protein from Drosophila Mushroom Body Neurons Results in Antipsychotic-Reversible Habituation Deficits.

Habituation is a conserved adaptive process essential for incoming information assessment, which drives the behavioral response decrement to recurrent inconsequential stimuli and does not involve sensory adaptation or fatigue. Although the molecular mechanisms underlying the process are not well understood, habituation has been reported to be defective in a number of disorders including schizophrenia. We demonstrate that loss of furin1, the Drosophila homolog of a gene whose transcriptional downregulation has been linked to schizophrenia, results in defective habituation to recurrent footshocks in mixed sex populations. The deficit is reversible by transgenic expression of the Drosophila or human Furin in adult α'/β' mushroom body neurons and by acute oral delivery of the typical antipsychotic haloperidol and the atypical clozapine, which are commonly used to treat schizophrenic patients. The results validate the proposed contribution of Furin downregulation in schizophrenia and suggest that defective footshock habituation is a Drosophila protophenotype of the human disorder.SIGNIFICANCE STATEMENT Genome-wide association studies have revealed a number of loci linked to schizophrenia, but most have not been verified experimentally in a relevant behavioral task. Habituation deficits constitute a schizophrenia endophenotype. Drosophila with attenuated expression of the schizophrenia-linked highly conserved Furin gene present delayed habituation reversible with acute exposure to antipsychotics. This strongly suggests that footshock habituation defects constitute a schizophrenia protophenotype in Drosophila Furthermore, determination of the neurons whose regulated activity is required for footshock habituation provides a facile metazoan system to expediently validate putative schizophrenia genes and variants in a well understood simple brain.

Expression of SARS-CoV-2-Related Surface Proteins in Non-Small-Cell Lung Cancer Patients and the Influence of Standard of Care Therapy.

Simple SummarySARS-CoV-2 is a respiratory virus that uses ACE2 for host cell entry and the spike protein is primed by, among others, TMPRSS2 and FURIN. The goal of this study was to determine in which non-small-cell lung cancer (NSCLC) patients these proteins are expressed on the membrane of the lung cancer cells and in which patients this increased ACE2 expression results in higher levels of soluble (s)ACE2 in their serum. In addition, we studied the influence of standard of care (SOC) therapies on sACE2 levels. Membranous (m)ACE2 was co-expressed with mFURIN and/or mTMPRSS2 in 16% of the NSCLC patients, and mACE2 and sACE2 were more frequently expressed in mutant EGFR patients but not mutant-KRAS patients. Importantly, systemic SOC therapies did not result in increased sACE2 levels. This indicates that cancer cells can be infected by SARS-CoV-2 in these patients, as well as that soluble ACE2 could impact the course of COVID-19.In this study, we aimed to study the expression of SARS-CoV-2-related surface proteins in non-small-cell lung cancer (NSCLC) cells and identify clinicopathological characteristics that are related to increased membranous (m)ACE2 protein expression and soluble (s)ACE2 levels, with a particular focus on standard of care (SOC) therapies. ACE2 (n = 107), TMPRSS2, and FURIN (n = 38) protein expression was determined by immunohistochemical (IHC) analysis in NSCLC patients. sACE2 levels (n = 64) were determined in the serum of lung cancer patients collected before, during, or after treatment with SOC therapies. Finally, the TCGA lung adenocarcinoma (LUAD) database was consulted to study the expression of ACE2 in EGFR- and KRAS-mutant samples and ACE2 expression was correlated with EGFR/HER, RAS, BRAF, ROS1, ALK, and MET mRNA expression. Membranous (m)ACE2 was found to be co-expressed with mFURIN and/or mTMPRSS2 in 16% of the NSCLC samples and limited to the adenocarcinoma subtype. TMPRSS2 showed predominantly atypical cytoplasmic expression. mACE2 and sACE2 were more frequently expressed in mutant EGFR patients, but not mutant-KRAS patients. A significant difference was observed in sACE2 for patients treated with targeted therapies, but not for chemo- and immunotherapy. In the TCGA LUAD cohort, ACE2 expression was significantly higher in EGFR-mutant patients and significantly lower in KRAS-mutant patients. Finally, ACE2 expression was positively correlated with ERBB2-4 and ROS1 expression and inversely correlated with KRAS, NRAS, HRAS, and MET mRNA expression. We identified a role for EGFR pathway activation in the expression of mACE2 in NSCLC cells, associated with increased sACE2 levels in patients. Therefore, it is of great interest to study SARS-CoV-2-infected EGFR-mutated NSCLC patients in greater depth in order to obtain a better understanding of how mACE2, sACE2, and SOC TKIs can affect the course of COVID-19.

The Effects of Asprosin on Exercise-Intervention in Metabolic Diseases.

Fibrillin is the major constituent of extracellular microfibrils, which are distributed throughout connective tissues. Asprosin is derived from the C-terminal region of the FBN1 gene, which encodes profibrillin that undergoes cleavage by furin protein. In response to fasting with low dietary glucose, asprosin is released as a secreted factor from white adipose tissue, and is transported to the liver for the mediation of glucose release into the blood circulation. Through binding to OLFR734, an olfactory G-protein-coupled receptor in liver cells, asprosin induces a glucogenic effect to regulate glucose homeostasis. Bioinformatics analyses revealed that the FBN1 gene is abundantly expressed in human skeletal muscle-derived mesoangioblasts, osteoblast-like cells, and mesenchymal stem cells, indicating that the musculoskeletal system might play a role in the regulation of asprosin expression. Interestingly, recent studies suggest that asprosin is regulated by exercise. This timely review discusses the role of asprosin in metabolism, its receptor signalling, as well as the exercise regulation of asprosin. Collectively, asprosin may have a vital regulatory effect on the improvement of metabolic disorders such as diabetes mellitus and obesity via exercise.

Abstract 14408: An Angiotensin Receptor Blocker in the Presence of SARS CoV2 Pseudovirus Does Not Change Protein Expression of ACE2 in Human Heart Cell Models

Introduction: ACE inhibitor (ACEI) or angiotensin receptor blocker (ARB) use in COVID-19 raised concern due to reported increases in ACE2, the cell receptor for SARS-CoV2. We tested the hypothesis that ARB (Losartan) and ACEI (Captopril) do not impact SARS-CoV2 associated ACE2 expression in cardiomyocytes (CMs). Methods: Beating monolayer ventricular CMs were generated from human iPSCs and grown as engineered heart tissue constructs (EHTs), which mimic a more mature phenotype than monolayer CMs. Drug treatments (24hrs) without pseudovirus were applied to EHTs: (A) Vehicle, 1μM Losartan (ARB), 1μM Angiotensin II (AngII), 1μM Losartan+1μM Ang II; (B) Vehicle, 1μM Captopril, 1μM Ang I, or 1μM AngI+1μM Captopril with 2-3 replicates for each combination and RNAseq as outcome. Drug treatment A only was repeated in monolayer CMs with a 5-6 day to rVSV-SARS-Cov2Spike-Luciferase-FLAG tagged pseudovirus (2-4 replicates) with immunoblotting for ACE2, Cathepsin B (CTSB) and Furin proteins. Results: Immunoblot densitometry showed abundant ACE2 protein in untreated CMs and EHTs. A focused mRNA analysis of 12 genes associated with SARS-Cov2 entry and processing (EHTs) revealed no significant changes in expression of ACE2, NRP1, CTSB, CTSL and FURIN due to Losartan or Captopril treatment. There was a nonsignificant trend towards Losartan-induced increase in AGTR1 with attenuation when AngII was administered, while ITGA5 trended upwards with Losartan+AngII. AGTRI also trended upwards with Captopril and AngI+Captopril. Upon pseudoviral challenge, CMs demonstrated increased ACE2 (55%) and slightly decreased Furin (24%) protein, with unchanged CTSB, although results were not statistically significant. Losartan addition, regardless of AngII, did not alter SARS-CoV2Spike pseudovirus mediated changes of ACE2, Furin or CTSB proteins. Conclusions: Losartan or Captopril did not substantially alter gene expression of ACE2, CTSB, CTSL, FURIN and NRP1 in EHTs without pseudovirus. Losartan did not show convincing evidence for pseudoviral mediated changes of proteins important for viral entry and processing in CM cell models.

The Potential Role of Osteopontin and Furin in Worsening Disease Outcomes in COVID-19 Patients with Pre-Existing Diabetes.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the ongoing coronavirus disease 2019 (COVID-19) pandemic, with more than 50 million cases reported globally. Findings have consistently identified an increased severity of SARS-CoV-2 infection in individuals with diabetes. Osteopontin, a cytokine-like matrix-associated phosphoglycoprotein, is elevated in diabetes and drives the expression of furin, a proprotein convertase implicated in the proteolytic processing and activation of several precursors, including chemokines, growth factors, hormones, adhesion molecules, and receptors. Elevated serum furin is a signature of diabetes mellitus progression and is associated with a dysmetabolic phenotype and increased risk of diabetes-linked premature mortality. Additionally, furin plays an important role in enhancing the infectivity of SARS-CoV-2 by promoting its entry and replication in the host cell. Here, we hypothesize that diabetes-induced osteopontin and furin protein upregulation results in worse outcomes in diabetic patients with SARS-CoV-2 infection owing to the roles of these protein in promoting viral infection and increasing metabolic dysfunction. Thus, targeting the osteopontin-furin axis may be a plausible strategy for reducing mortality in SARS-CoV-2 patients with diabetes.

Increased FURIN expression in rheumatoid arthritis patients and its anti‐inflammatory effect

BackgroundFURIN belongs to the proprotein convertase family that processes proproteins and is involved in many diseases. However, the role of FURIN in rheumatoid arthritis (RA) remains unknown. In this study, we investigated the association between circulating FURIN and disease activity in patients with RA and the effect of FURIN in THP‐1‐derived macrophages.MethodsA total of 108 RA patients and 39 healthy controls participants were included in this study. RA patients were divided into four disease activity groups determined by the Disease Activity Score of 28 joints (DAS28). FURIN expression in peripheral blood mononuclear cells (PBMCs) and serum was detected by using quantitative real‐time polymerase chain reaction (qRT‐PCR) and enzyme‐linked immunosorbent assay (ELISA), respectively. Western blotting and qRT‐PCR were used to detect cytokines level after interfering FURIN expressed in THP‐1‐derived macrophages.ResultsBoth FURIN mRNA and protein levels were significantly higher in RA patients than in healthy controls participants (P < .001). No significant difference in FURIN expression was observed among the four RA groups (P > .05). Spearman correlation revealed that FURIN positively correlated with transforming growth factor‐β1(TGF‐β1), rheumatoid factor (RF), and anti‐cyclic citrullinated peptide (anti‐CCP). Moreover, the inhibition of FURIN in THP‐1‐derived macrophages promoted the caspase‐1 and IL‐1β expression (P < .05).ConclusionFURIN levels were significantly increased in the peripheral blood of RA patients and were not associated with disease activity. The inhibition of FURIN in THP‐1‐derived macrophages with elevated IL‐1β levels shows that FURIN may have an anti‐inflammatory effect.

Cleavage of the soluble (pro)renin receptor (sATP6AP2) in the placenta

IntroductionThe (pro)renin receptor (ATP6AP2) is cleaved and released as soluble ATP6AP2 (sATP6AP2). The sATP6AP2 is detected in plasma and urine and is elevated in women with gestational diabetes and preeclampsia. The source and cleavage pathway of sATP6AP2 in pregnancy is unknown. The syncytiotrophoblast is the major placental secretory layer and is in direct contact with maternal blood. Both FURIN and Site 1 protease (MBTPS1) cleave sATP6AP2 in non-placental cells. We postulated that ATP6AP2 was cleaved by FURIN and/or MBTPS1 and that sATP6AP2 is secreted by the placental syncytiotrophoblast. MethodsTerm primary trophoblast cells were transfected with FURIN siRNA, negative control siRNA or vehicle. In a separate experiment, primary trophoblasts were treated with a pro-protein convertase inhibitor (DEC-RVKR-CMK), an MBTPS1 inhibitor (PF 429242) or vehicle. Trophoblasts were left to spontaneously syncytialise before cells and supernatants were collected and intracellular and extracellular sATP6AP2 levels analysed by immunoblot.Results: sATP6AP2 is secreted by placental trophoblasts. Levels of intra and extra-cellular sATP6AP2 decrease with syncytialisation (P = 0.01 and P = 0.02, respectively), as do FURIN mRNA (P = 0.0003) and protein (P = 0.0007). FURIN siRNA decreased FURIN mRNA and protein levels (both P < 0.0001). Neither FURIN siRNA or PF 429242 affected sATP6AP2 levels. DEC-RVKR-CMK significantly decreased extracellular sATP6AP2 protein levels (P = 0.02). DiscussionSoluble ATP6AP2 is secreted by placental trophoblasts and levels decrease with syncytialisation. DEC-RVKR-CMK, a broad inhibitor of pro-protein convertases reduced extracellular sATP6AP2 levels, but FURIN siRNA and MBTPS1 inhibition had no effect. Hence, a convertase other than FURIN or MBTPS1 is most likely responsible for placental sATP6AP2 secretion.

Furin-responsive triterpenine-based liposomal complex enhances anticervical cancer therapy through size modulation

The accumulation and penetration of antitumor drugs in tumor tissues are directly related to their antitumor effects. The particle size of the nanodrug delivery system is one of the most important factors for the accumulation and penetration of antitumor drugs within tumor tissues. Generally, nanodelivery systems of intermediate size (100–120 nm) are capable of efficient accumulation owing to prolonged circulation and enhanced permeability and retention (EPR) effect; however, smaller ones (20–40 nm) are effective for deep penetration within tumor tissue. Currently a conventional drug delivery system cannot possess two types of optimal sizes, simultaneously. To solve this and to enhance cervical cancer treatment, a furin-responsive triterpenine-based liposomal complex (PEGcleavable Tf-CTM/L), with Tf-CTM (transferrin-modified tripterine-loaded coix seed oil microemulsion) in core, coated with a thermo-sensitive lipid and a kind of PEG shell modified with a furin-cleavable peptide was developed to improve tumor-specific accumulation and penetration. Herein, PEGcleavable Tf-CTM/L was capable of efficient accumulation because of EPR effect. The PEG shells could timely detach under stimulation of overexpressed furin protein to solve the problem of the steric hindrance dilemma. The small-sized Tf-CTM released under stimulation of tumor microthermal environment in cervical cancer, which was efficient with regards to deep penetration at tumor sites. Notably, compared to the use of triterpenine alone, PEGcleavable Tf-CTM/L promoted anticervical efficacy and displayed diminished systemic toxicity by efficient accumulation and deep penetration of antitumor drugs within tumor tissues. Our study provides a new strategy, and holds promising potential for anticervical cancer treatment.

BacMam production and crystal structure of nonglycosylated apo human furin at 1.89 Å resolution.

Furin, also called proprotein convertase subtilisin/kexin 3 (PCSK3), is a calcium-dependent serine endoprotease that processes a wide variety of proproteins involved in cell function and homeostasis. Dysregulation of furin has been implicated in numerous disease states, including cancer and fibrosis. Mammalian cell expression of the furin ectodomain typically produces a highly glycosylated, heterogeneous protein, which can make crystallographic studies difficult. Here, the expression and purification of nonglycosylated human furin using the BacMam technology and site-directed mutagenesis of the glycosylation sites is reported. Nonglycosylated furin produced using this system retains full proteolytic activity indistinguishable from that of the glycosylated protein. Importantly, the nonglycosylated furin protein reliably forms extremely durable apo crystals that diffract to high resolution. These crystals can be soaked with a wide variety of inhibitors to enable a structure-guided drug-discovery campaign.