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.

Since the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), its genetic and geographical origins remain unclear, resulting in suspicions about its natural origin. In one of our previous studies, we reported the presence of a furin cleavage site RRAR in the junction region between S1 and S2 subunits of the spike protein, which was discovered as the first crucial clue for the origin tracing of SARS-CoV-2. In the present study, we conducted an integrative analysis of new genome data from bat Sarbecovirus strains reported after the COVID-19 outbreak. The primary results included the identification of BANAL-20-52, Rp22DB159, and S18CXBatR24 as three close relatives of SARS-CoV-2 and the successful detection of seven out of nine key genomic features (designated as RC0-7 and ORF8) observed in wild types of SARS-CoV-2 in the three close relatives from Laos, Vietnam, and Yunnan province of China, respectively. The most significant contribution of the present study lies in the detection of RC1 in wild genotype in a bat Sarbecovirus population BANAL-20-52 belonging to. Encoding a segment of the NSP3 protein, RC1 was discovered as the second crucial clue for the origin tracing of SARS-CoV-2. Although RC0, encoding the junction furin cleavage site, remains undetected outside of the SARS-CoV-2 genome, Feuang of Laos is the sole place where eight of the nine wild-type features (RC1-7 and ORF8) have been detected.

Epithelial Na+ channels (ENaCs) are activated by proteolysis of the α and γ subunits at specific sites flanking embedded inhibitory tracts. To examine the role of α subunit proteolysis in channel activation in vivo, we generated mice lacking the distal furin cleavage site in the α subunit (αF2M mice). On a normal Na+ control diet, no differences in ENaC protein abundance in kidney or distal colon were noted between wild-type (WT) and αF2M mice. Patch-clamp analyses revealed similar levels of ENaC activity in kidney tubules, while no physiologically relevant differences in blood chemistry or aldosterone levels were detected. Male αF2M mice did exhibit diminished ENaC activity in the distal colon, as measured by amiloride-sensitive short-circuit current (ISC). Following dietary Na+ restriction, WT and αF2M mice had similar natriuretic and colonic ISC responses to amiloride. However, single-channel activity was significantly lower in kidney tubules from Na+-restricted αF2M mice compared with WT littermates. ENaC α and γ subunit expression in kidney and distal colon were also enhanced in Na+-restricted αF2M vs. WT mice, in association with higher aldosterone levels. These data provide evidence that disrupting α subunit proteolysis impairs ENaC activity in vivo, requiring compensation in response to Na+ restriction. KEY POINTS: The epithelial Na+ channel (ENaC) is activated by proteolytic cleavage in vitro, but key questions regarding the role of ENaC proteolysis in terms of whole-animal physiology remain to be addressed. We studied the in vivo importance of this mechanism by generating a mouse model with a genetic disruption to a key cleavage site in the ENaC\'s α subunit (αF2M mice). We found that αF2M mice did not exhibit a physiologically relevant phenotype under normal dietary conditions, but have impaired ENaC activation (channel open probability) in the kidney during salt restriction. ENaC function at the organ level was preserved in salt-restricted αF2M mice, but this was associated with higher aldosterone levels and increased expression of ENaC subunits, suggesting compensation was required to maintain homeostasis. These results provide the first evidence that ENaC α subunit proteolysis is a key regulator of channel activity in vivo.

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.

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.

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.

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.
CONCLUSIONS: 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.

Severe coronavirus disease 2019 and post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are associated with neurological complications that may be linked to direct infection of the central nervous system (CNS), but the selective pressures ruling neuroinvasion are poorly defined. Here we assessed SARS-CoV-2 evolution in the lung versus CNS of infected mice. Higher levels of viral divergence were observed in the CNS than the lung after intranasal challenge with a high frequency of mutations in the spike furin cleavage site (FCS). Deletion of the FCS significantly attenuated virulence after intranasal challenge, with lower viral titres and decreased morbidity compared with the wild-type virus. Intracranial inoculation of the FCS-deleted virus, however, was sufficient to restore virulence. After intracranial inoculation, both viruses established infection in the lung, but dissemination from the CNS to the lung required the intact FCS. Cumulatively, these data suggest a critical role for the FCS in determining SARS-CoV-2 tropism and compartmentalization.

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.

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.