Non-canonical olfactory pathway activation induces cell fusion of cervical cancer cells.

Multinucleation occurs in various types of advanced cancers and contributes to their malignant characteristics, including anticancer drug resistance. Therefore, inhibiting multinucleation can improve cancer prognosis; however, the molecular mechanisms underlying multinucleation remain elusive. Here, we introduced a genetic mutation in cervical cancer cells to induce cell fusion-mediated multinucleation. The olfactory receptor OR1N2 was heterozygously mutated in these fused cells; the same OR1N2 mutation was detected in multinucleated cells from clinical cervical cancer specimens. The mutation-induced structural change in the OR1N2 protein activated protein kinase A (PKA), which, in turn, mediated the non-canonical olfactory pathway. PKA phosphorylated and activated furin protease, resulting in the cleavage of the fusogenic protein syncytin-1. Because this cleaved form of syncytin-1, processed by furin, participates in cell fusion, furin inhibitors could suppress multinucleation and reduce surviving cell numbers after anticancer drug treatment. The improved anticancer drug efficacy indicates a promising therapeutic approach for advanced cervical cancers.

Social defeat stress impairs systemic iron metabolism by activating the hepcidin-ferroportin axis.

Chronic psychological stress has been reported to decrease circulating iron concentrations and impair hematopoiesis. However, the underlying mechanisms remain unclear. This study aimed to investigate the effects of psychological stress on biological iron metabolism by using the social defeat stress (SDS) model, a widely used model of depression. Compared with control mice, mice subjected to SDS (SDS mice) had lower social interaction (SI) behavior. The SDS mice also showed impaired hematopoiesis, as evidenced by reduced circulating red blood cell counts, elevated reticulocyte counts, and decreased plasma iron levels. In the SDS mice, the iron contents in the bone marrow decreased, whereas those in the spleen increased, suggesting dysregulation in systemic iron metabolism. The concentrations of plasma hepcidin, an important regulator of systemic iron homeostasis, increased in the SDS mice. Meanwhile, the concentrations of ferroportin, an iron transport protein negatively regulated by hepcidin, were lower in the spleen and duodenum of the SDS mice than in those of the control mice. Treatment with dalteparin, a hepcidin inhibitor, prevented the decrease in plasma iron levels in the SDS mice. The gene expression and enzyme activity of furin, which converts the precursor hepcidin to active hepcidin, were high and positively correlated with plasma hepcidin concentration. Thus, furin activation might be responsible for the increased plasma hepcidin concentration. This study is the first to show that psychological stress disrupts systemic iron homeostasis by activating the hepcidin-ferroportin axis. Consideration of psychological stressors might be beneficial in the treatment of diseases with iron-refractory anemia.

Colorimetric detection of furin based on enhanced catalytic activity of G-quadruplex/hemin DNAzyme.

BACKGROUND: Rapid and sensitive colorimetric detection methods are crucial for diseases diagnosis, particularly those involving proteases like furin, which are implicated in various conditions, including cancer. Traditional detection methods for furin suffer from limitations in sensitivity and practicality for on-site detection, motivating the development of novel detection strategies. Therefore, developing a simple, enzyme-free, and rapid colorimetric analysis method with high sensitivity for furin detection is imperative. RESULTS: Herein, we have proposed a colorimetric method in this work for the first time to detect furin, leveraging the assembly of G-quadruplex/hemin DNAzyme with enhanced catalytic activity. Specifically, a peptide-DNA conjugate (PDC) comprising a furin-recognition peptide and flanking DNA sequences for signal amplification is designed to facilitate the DNAzyme assembly. Upon furin treatment, PDC cleavage triggers a cyclic catalytic hairpin assembly reaction to form the complementary double-stranded structures by hairpin 1 (HP1) and hairpin 2 (HP2), bringing the G-quadruplex sequence in HP1 closer to hemin on HP2. Moreover, the resulting G-quadruplex/hemin DNAzymes exhibit robust peroxidase-like activity, enabling the catalysis of the colorimetric reaction of ABTS2- for furin detection. Our method demonstrates high sensitivity, rapid response, and compatibility with complex sample matrices, achieving a detection limit as low as 1.1 pM. SIGNIFICANCE: The DNAzyme reported in this work exhibits robust catalytic activity, enabling high sensitivity and good efficiency for the detection. By eliminating the requirement for exogenous enzymes, our approach enables visual furin detection without expensive instrumentation and reagents, promising significant utility in biomedical and clinical diagnostic applications. Given the various design of peptide sequence and the programmability of DNA, it can be readily applied to analyzing other useful tumor biomarkers.

Structural insights into Furin enzyme inhibition to block SARS-CoV-2 spike protein cleavage: an in-silico approach.

This study investigates the binding affinity and interactions of the Furin enzyme with two inhibitors, Naphthofluorescein and decanoyl-RVKR-chloromethylketone (CMK), using molecular docking and molecular dynamics (MD) simulations. Molecular docking results showed binding affinities of - 9.18 kcal/mol for CMK and - 5.39 kcal/mol for Naphthofluorescein. To further understand the stability and conformational changes of these complexes, MD simulations were performed. Despite CMK's favorable docking score, MD simulations revealed that its binding interactions at the Furin-active site were unstable, with significant changes observed during the simulation. In contrast, Naphthofluorescein maintained strong and stable interactions throughout the MD simulation, as confirmed by RMSD and RMSF analyses. The binding-free-energy analysis also supported the stability of Naphthofluorescein. These findings indicate that Naphthofluorescein exhibits greater stability and binding affinity as a Furin inhibitor compared to CMK. The results of this in-silico study suggest that Naphthofluorescein, along with CMK, holds the potential for repurposing as a treatment for COVID-19, subject to further validation through clinical studies.

Docking, MD Simulations, and DFT Calculations: Assessing W254's Function and Sartan Binding in Furin.

Furins are serine endoproteases that are involved in many biological processes, where they play important roles in normal metabolism, in the activation of various pathogens, while they are a target for therapeutic intervention. Dichlorophenyl-pyridine "BOS" compounds are well known drugs that are used as inhibitors of human furin by an induced-fit mechanism, in which tryptophan W254 in the furin catalytic cleft acts as a molecular transition energy gate. The binding of "BOS" drug into the active center of furin has been computationally studied using the density functional theory (DFT) and ONIOM multiscaling methodologies. The binding enthalpies of the W254 with the furin-BOS is -32.8 kcal/mol ("open") and -18.8 kcal/mol ("closed"), while the calculated torsion barrier was found at 30 kcal/mol. It is significantly smaller than the value of previous MD calculations due to the relaxation of the environment, i.e., nearby groups of the W254, leading to the reduction of the energy demands. The significant lower barrier explains the experimental finding that the dihedral barrier of W254 is overcome. Furthermore, sartans were studied to evaluate their potential as furin inhibitors. Sartans are AT1 antagonists, and they effectively inhibit the hypertensive effects induced by the peptide hormone Angiotensin II. Here, they have been docked into the cavity to evaluate their effect on the BOS ligand via docking and molecular dynamics simulations. A consistent binding of sartans within the cavity during the simulation was found, suggesting that they could act as furin inhibitors. Finally, sartans interact with the same amino acids as W254, leading to a competitive binding that may influence the pharmacological efficacy and potential drug interactions of sartans.

Evaluation of Potential Furin Protease Inhibitory Properties of Pioglitazone, Rosiglitazone, and Pirfenidone: An In Silico Docking and Molecular Dynamics Simulation Approach.

Furin (Fur) is a member of the protease convertase family; its expression is crucial for cleaving and maturing many proteins. Fur also represents a therapeutic target in cancer, autoimmune diseases, and viral infections. Pioglitazone (PGZ) and rosiglitazone (RGZ) are thiazolidinediones prescribed to type 2 diabetes patients and are structurally similar to the known Fur inhibitors naphthofluorescein (NPF) and pirfenidone (PFD). Thus, this study used molecular docking and molecular dynamics to assess and compare the affinities and the molecular interactions of these four ligands with the Fur active site (FurAct) and the recently described Fur allosteric site (FurAll). The 7QXZ Fur structure was used for molecular dockings, and for the best pose complexes, molecular dynamics were run for 100 ns. The best affinities of the ligand/FurAct and ligand/FurAll complexes were with NPF, PGZ, and RGZ, while PFD presented the lowest affinity. Asp154 was the central residue involved in FurAct complex formation, while Glu488 and Asn310 were the central residues involved in FurAll complex formation. This study shows the potential of RGZ, PGZ, and PFD as Fur competitive (FurAct) and non-competitive (FurAll) inhibitors. Therefore, they are candidates for repurposing in response to future emerging diseases through the modulation of Fur activity.

Furin, ADAM, and γ-secretase: Core regulatory targets in the Notch pathway and the therapeutic potential for breast cancer.

The activation of the Notch pathway promotes the occurrence and progression of breast cancer. The Notch signal plays different roles in different molecular subtypes of breast cancer. In estrogen receptor-positive (ER+) breast cancer, the Notch pathway regulates the activity of estrogen receptors. In human epidermal growth factor receptor 2-positive (HER2+) breast cancer, crosstalk between Notch and HER2 enhances HER2 signal expression. In triple-negative breast cancer (TNBC), Notch pathway activation is closely linked to tumor invasion and drug resistance. This article offers a comprehensive review of the structural domains, biological functions, and key targets of Notch with a specific focus on the roles of Furin protease, ADAM metalloprotease, and γ-secretase in breast cancer and their potential as therapeutic targets. We discuss the functions and mutual regulatory mechanisms of these proteinases in the Notch pathway as well as other potential targets in the Notch pathway, such as the glycosylation process and key transcription factors. This article also introduces new approaches in the treatment of breast cancer, with a special focus on the molecular characteristics and treatment response differences of different subtypes. We propose that the core regulatory molecules of the Notch pathway may become key targets for development of personalized treatment, which may significantly improve treatment outcomes and prognosis for patients with breast cancer.

Furin Egress from the TGN is Regulated by Membrane-Associated RING-CH Finger (MARCHF) Proteins and Ubiquitin-Specific Protease 32 (USP32) via Nondegradable K33-Polyubiquitination.

Furin primarily localizes to the trans-Golgi network (TGN), where it cleaves and activates a broad range of immature proproteins that play critical roles in cellular homeostasis, disease progression, and infection. Furin is retrieved from endosomes to the TGN after being phosphorylated, but it is still unclear how furin exits the TGN to initiate the post-Golgi trafficking and how its activity is regulated in the TGN. Here three membrane-associated RING-CH finger (MARCHF) proteins (2, 8, 9) are identified as furin E3 ubiquitin ligases, which catalyze furin K33-polyubiquitination. Polyubiquitination prevents furin from maturation by blocking its ectodomain cleavage inside cells but promotes its egress from the TGN and shedding. Further ubiquitin-specific protease 32 (USP32) is identified as the furin deubiquitinase in the TGN that counteracts the MARCHF inhibitory activity on furin. Thus, the furin post-Golgi trafficking is regulated by an interplay between polyubiquitination and phosphorylation. Polyubiquitination is required for furin anterograde transport but inhibits its proprotein convertase activity, and phosphorylation is required for furin retrograde transport to produce fully active furin inside cells.

PACS-1 variant protein is aberrantly localized in C. elegans model of PACS1/PACS2 syndromes.

PACS (Phosphofurin Acidic Cluster Sorting Protein) proteins are known for their roles in sorting cargo proteins to organelles and can physically interact with WD40 repeat-containing protein WDR37. PACS1, PACS2, and WDR37 variants are associated with multisystemic syndromes and neurodevelopmental disorders characterized by intellectual disability, seizures, developmental delays, craniofacial abnormalities, and autism spectrum disorder. However, the functional effects of syndromic variants at the cellular level remain unknown. Here, we report the expression pattern of C. elegans orthologs of PACS and WDR37 and their interaction. We show that cePACS-1 and ceWDR-37 co-localize to somatic cytoplasm of many types of cells, and are mutually required for expression, supporting a conclusion that the intermolecular dependence of PACS1/PACS2/PACS-1 and WDR37/WDR-37 is evolutionarily conserved. We further show that editing in PACS1 and PACS2 variants in cePACS-1 changes protein localization in multiple cell types, including neurons. Moreover, expression of human PACS1 can functionally complement C. elegans PACS-1 in neurons, demonstrating conserved functions of the PACS-WDR37 axis in an invertebrate model system. Our findings reveal effects of human variants and suggest potential strategies to identify regulatory network components that may contribute to understanding molecular underpinnings of PACS/WDR37 syndromes.

Novel proteolytic activation of Ebolavirus glycoprotein GP by TMPRSS2 and cathepsin L at an uncharted position can compensate for furin cleavage.

A multistep priming process involving furin and endosomal cathepsin B and L (CatB/L) has been described for the Orthoebolavirus zairense (EBOV) glycoprotein GP. Inhibition or knockdown of either furin or endosomal cathepsins, however, did not prevent virus multiplication in cell cultures. Moreover, an EBOV mutant lacking the furin cleavage motif (RRTRR→AGTAA) was able to replicate and cause fatal disease in nonhuman primates, indicating that furin cleavage may be dispensable for virus infectivity. Here, by using protease inhibitors and EBOV GP-carrying recombinant vesicular stomatitis virus (VSV) and transcription and replication-competent virus-like particles (trVLPs) we found that processing of EBOV GP is mediated by different proteases in different cell lines depending on the protease repertoire available. Endosomal cathepsins were essential for EBOV GP entry in Huh-7 but not in Vero cells, in which trypsin-like proteases and stably expressed trypsin-like transmembrane serine protease 2 (TMPRSS2) supported wild-type EBOV GP and EBOV GP_AGTAA mutant entry. Furthermore, we show that the EBOV GP_AGTAA mutant is cleaved into fusion-competent GP2 by TMPRSS2 and by CatL at a so far unknown site. Fluorescence microscopy co-localization studies indicate that EBOV GP cleavage by TMPRSS2 may occur in the TGN prior to virus release or in the late endosome at the stage of virus entry into a new cell. Our data show that EBOV GP must be proteolytically activated to support virus entry but has even greater flexibility in terms of proteases and the precise cleavage site than previously assumed.

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.

MARCH1 and MARCH2 inhibit pseudorabies virus replication by trapping the viral cell-to-cell fusion complex in trans-Golgi network.

The membrane-associated RING-CH (MARCH) family of proteins are members of the E3 ubiquitin ligase family and are essential for a variety of biological functions. Currently, MARCH proteins are discovered to execute antiviral functions by directly triggering viral protein degradation or blocking the furin cleavage of viral class I fusion proteins. Here, we report a novel antiviral mechanism of MARCH1 and MARCH2 (MARCH1/2) in the replication of Pseudorabies virus (PRV), a member of the Herpesviridae family. We discovered MARCH1/2 restrict PRV replication at the cell-to-cell fusion step. Furthermore, MARCH1/2 block gB cleavage, and this is dependent on their E3 ligase activity. Interestingly, the blocking of gB cleavage by MARCH1/2 does not contribute to their antiviral activity in vitro. We discovered that MARCH1/2 are associated with the cell-to-cell fusion complex of gB, gD, gH, and gL and trap these viral proteins in the trans-Golgi network (TGN) rather than degrading them. Overall, we conclude that MARCH1/2 inhibit PRV by trapping the viral cell-to-cell fusion complex in TGN.

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.

Screening of Small-Molecule Libraries Using SARS-CoV-2-Derived Sequences Identifies Novel Furin Inhibitors.

SARS-CoV-2 is the pathogen responsible for the most recent global pandemic, which has claimed hundreds of thousands of victims worldwide. Despite remarkable efforts to develop an effective vaccine, concerns have been raised about the actual protection against novel variants. Thus, researchers are eager to identify alternative strategies to fight against this pathogen. Like other opportunistic entities, a key step in the SARS-CoV-2 lifecycle is the maturation of the envelope glycoprotein at the RARR685↓ motif by the cellular enzyme Furin. Inhibition of this cleavage greatly affects viral propagation, thus representing an ideal drug target to contain infection. Importantly, no Furin-escape variants have ever been detected, suggesting that the pathogen cannot replace this protease by any means. Here, we designed a novel fluorogenic SARS-CoV-2-derived substrate to screen commercially available and custom-made libraries of small molecules for the identification of new Furin inhibitors. We found that a peptide substrate mimicking the cleavage site of the envelope glycoprotein of the Omicron variant (QTQTKSHRRAR-AMC) is a superior tool for screening Furin activity when compared to the commercially available Pyr-RTKR-AMC substrate. Using this setting, we identified promising novel compounds able to modulate Furin activity in vitro and suitable for interfering with SARS-CoV-2 maturation. In particular, we showed that 3-((5-((5-bromothiophen-2-yl)methylene)-4-oxo-4,5 dihydrothiazol-2-yl)(3-chloro-4-methylphenyl)amino)propanoic acid (P3, IC50 = 35 µM) may represent an attractive chemical scaffold for the development of more effective antiviral drugs via a mechanism of action that possibly implies the targeting of Furin secondary sites (exosites) rather than its canonical catalytic pocket. Overall, a SARS-CoV-2-derived peptide was investigated as a new substrate for in vitro high-throughput screening (HTS) of Furin inhibitors and allowed the identification of compound P3 as a promising hit with an innovative chemical scaffold. Given the key role of Furin in infection and the lack of any Food and Drug Administration (FDA)-approved Furin inhibitor, P3 represents an interesting antiviral candidate.

PACS-1 variant protein is aberrantly localized in C. elegans model of PACS1/PACS2 syndromes.

PACS (Phosphofurin Acidic Cluster Sorting Protein) proteins are known for their roles in sorting cargo proteins to organelles and can physically interact with WD40 repeat-containing protein WDR37. PACS1, PACS2, and WDR37 variants are associated with multisystemic syndromes and neurodevelopmental disorders characterized by intellectual disability, seizures, developmental delays, craniofacial abnormalities, and autism spectrum disorder. However, the effects of syndromic variants on function in vivo remains unknown. Here, we report the expression pattern of C. elegans orthologs of PACS and WDR37 and their interaction. We show that cePACS-1 and ceWDR-37 co-localize to somatic cytoplasm of many types of cells, and are mutually required for expression, supporting a conclusion that the intermolecular dependence of PACS1/PACS2/PACS-1 and WDR37/WDR-37 is evolutionarily conserved. We further show that editing in PACS1 and PACS2 variants in cePACS-1 changes protein localization in multiple cell types, including neurons. Moreover, expression of human PACS1 can functionally complement C. elegans PACS-1 in neurons, demonstrating conserved functions of the PACS-WDR37 axis in an invertebrate model system. Our findings reveal effects of human variants and suggest potential strategies to identify regulatory network components that may contribute to understanding molecular underpinnings of PACS/WDR37 syndromes.

Targeting furin, a cellular proprotein convertase, for COVID-19 prevention and therapeutics.

SARS-CoV-2 has triggered an international outbreak of the highly contagious acute respiratory disease known as COVID-19. Identifying key targets in the virus infection lifecycle is crucial for developing effective prevention and therapeutic strategies against it. Furin is a serine endoprotease that belongs to the family of proprotein convertases and plays a critical role in the entry of host cells by SARS-CoV-2. Furin can cleave a specific S1/S2 site, PRRAR, on the spike protein of SARS-CoV-2, which promotes viral transmission by facilitating membrane fusion. Hence, targeting furin could hold clinical implications for the prevention and treatment of COVID-19. This review offers an overview of furin's structure, substrates, function, and inhibitors, with a focus on its potential role in SARS-CoV-2 infection.

Furin knockdown inhibited EndMT and abnormal proliferation and migration of endothelial cells.

BACKGROUND: In the pathogenesis of atherosclerotic cardiovascular disorders, vascular endothelium is crucial. A critical step in the development of atherosclerosis is endothelial dysfunction. Furin may play a factor in vascular remodeling, inflammatory cell infiltration, regulation of plaque stability, and atherosclerosis by affecting the adhesion and migration of endothelial cells. It is yet unknown, though, how furin contributes to endothelial dysfunction. METHODS: We stimulated endothelial cells with oxidized modified lipoprotein (ox-LDL). Endothelial-to-mesenchymal transition (EndMT) was found using immunofluorescence (IF) and western blot (WB). Furin expression level and Hippo/YAP signal activation were found using reverse transcription-quantitative PCR (RT-qPCR) and WB, respectively. To achieve the goal of furin knockdown, we transfected siRNA using the RNA transmate reagent. Following furin knockdown, cell proliferation, and migration were assessed by the CCK-8, scratch assay, and transwell gold assay, respectively. WB and IF both picked up on EndMT. WB and RT-qPCR, respectively, were used to find furin's expression level. We chose the important micrornas that can regulate furin and we then confirmed them using RT-qPCR. RESULTS: EndMT was created by ox-LDL, evidenced by the up-regulation of mesenchymal cell markers and the down-regulation of endothelial cell markers. Furin expression levels in both protein and mRNA were increased, and the Hippo/YAP signaling pathway was turned on. Furin knockdown dramatically reduced the aberrant migration and proliferation of endothelial cells by ox-LDL stimulation. Furin knockdown can also suppress ox-LDL-induced EndMT, up-regulate indicators of endothelial cells, and down-regulate markers of mesenchymal cells. After ox-LDL stimulation and siRNA transfection, furin's expression level was up-regulated and down-regulated. CONCLUSION: Our study demonstrated that furin knockdown could affect ox-LDL-induced abnormal endothelial cell proliferation, migration, and EndMT. This implies that furin plays an important role in endothelial dysfunction.

FURIN, IFNL4, and TLR2 gene polymorphisms in relation to COVID-19 severity: a case-control study in Egyptian patients.

BACKGROUND AND AIM: A wide range of clinical manifestations and outcomes, including liver injury, have been reported in COVID-19 patients. We investigated the association of three substantial gene polymorphisms (FURIN, IFNL4, and TLR2) with COVID-19 disease susceptibility and severity to help predict prognosis. METHODS: 150 adult COVID-19-assured cases were categorized as follows: 78 patients with a non-severe presentation, 39 patients with severe disease, and 33 critically ill patients. In addition, 74 healthy controls were included. Clinical and laboratory evaluations were carried out, including complete and differential blood counts, D-dimer, lactate dehydrogenase (LDH), C-reactive protein (CRP), procalcitonin, ferritin, interleukin-6 (Il-6), and liver and kidney functions. FURIN (rs6226), IFNL4 (rs12979860), and TLR2 (rs3804099) genotyping allelic discrimination assays were conducted using real-time PCR. RESULTS: The FURIN, IFNL4, and TLR2 genotypes and their alleles differed significantly between COVID-19 patients and controls, as well as between patients with severe or critical illness and those with a non-severe presentation. According to a multivariable regression analysis, FURIN (C/T + T/T) and TLR2 (T/C + C/C) mutants were associated with COVID-19 susceptibility, with odds ratios of 3.293 and 2.839, respectively. FURIN C/C and IFNL4 T/T mutants were significantly linked to severe and critical illnesses. Multivariate regression analysis showed that FURIN (G/C + C/C) genotypes and IFNL4 T/T homozygosity were independent risk factors associated with increased mortality. CONCLUSION: FURIN, IFNL4, and TLR2 gene variants are associated with the risk of COVID-19 occurrence as well as increased severity and poor outcomes in Egyptian patients.

Vitamin A Status Modulates Epithelial Mesenchymal Transition in the Lung: The Role of Furin.

Vitamin A deficiency (VAD) induced TGF-ß hyperactivation and reduced expression of cell adhesion proteins in the lung, suggesting that the disruption of retinoic acid (RA) signaling leads to epithelial-mesenchymal transition (EMT). To elucidate the role of lung vitamin A status in EMT, several EMT markers and the expression of the proprotein convertase furin, which activates TGF-ß, were analyzed in two experimental models. Our in vivo model included control rats, VAD rats, and both control rats and VAD rats, treated with RA. For the in vitro studies, human bronchoalveolar epithelial cells treated with RA were used. Our data show that EMT and furin are induced in VAD rats. Furthermore, furin expression continues to increase much more markedly after treatment of VAD rats with RA. In control rats and cell lines, an acute RA treatment induced a significant increase in furin expression, concomitant with changes in EMT markers. A ChIP assay demonstrated that RA directly regulates furin transcription. These results emphasize the importance of maintaining vitamin A levels within the physiological range since both levels below and above this range can cause adverse effects that, paradoxically, could be similar. The role of furin in EMT is discussed.

Influence of proteolytic cleavage of ENaC's γ subunit upon Na+ and K+ handling.

The epithelial Na+ channel (ENaC) γ subunit is essential for homeostasis of Na+, K+, and body fluid. Dual γ subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146, in the mouse γ subunit). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (γQ4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short-circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of γQ4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γQ4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in γQ4/Q4 mice was not altered. γQ4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of γ subunit cleavage should not be used in isolation to evaluate ENaC activity. Furthermore, factors independent of γ subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo.NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) is activated in vitro by post-translational proteolysis. In vivo, low Na+ or high K+ diets enhance ENaC proteolysis, and proteolysis is hypothesized to contribute to channel activation in these settings. Using a mouse expressing ENaC with disruption of a key proteolytic cleavage site, this study demonstrates that impaired proteolytic activation of ENaC's γ subunit has little impact upon channel open probability or the ability of mice to adapt to low Na+ or high K+ diets.