Functional and Multi-Omics Effects of an Optimized CRISPR-Mediated FURIN Depletion in U937 Monocytes.

The pro-protein convertase FURIN (PCSK3) is implicated in a wide range of normal and pathological biological processes such as infectious diseases, cancer and cardiovascular diseases. Previously, we performed a systemic inhibition of FURIN in a mouse model of atherosclerosis and demonstrated significant plaque reduction and alterations in macrophage function. To understand the cellular mechanisms affected by FURIN inhibition in myeloid cells, we optimized a CRISPR-mediated gene deletion protocol for successfully deriving hemizygous (HZ) and nullizygous (NZ) FURIN knockout clones in U937 monocytic cells using lipotransfection-based procedures and a dual guide RNA delivery strategy. We observed differences in monocyte and macrophage functions involving phagocytosis, lipid accumulation, cell migration, inflammatory gene expression, cytokine release patterns, secreted proteomics (cytokines) and whole-genome transcriptomics between wild-type, HZ and NZ FURIN clones. These studies provide a mechanistic basis on the possible roles of myeloid cell FURIN in cardiovascular disorders.

Effect of SARS-CoV-2 S protein on the proteolytic cleavage of the epithelial Na+ channel ENaC.

Severe cases of COVID-19 are characterized by development of acute respiratory distress syndrome (ARDS). Water accumulation in the lungs is thought to occur as consequence of an exaggerated inflammatory response. A possible mechanism could involve decreased activity of the epithelial Na+ channel, ENaC, expressed in type II pneumocytes. Reduced transepithelial Na+ reabsorption could contribute to lung edema due to reduced alveolar fluid clearance. This hypothesis is based on the observation of the presence of a novel furin cleavage site in the S protein of SARS-CoV-2 that is identical to the furin cleavage site present in the alpha subunit of ENaC. Proteolytic processing of αENaC by furin-like proteases is essential for channel activity. Thus, competition between S protein and αENaC for furin-mediated cleavage in SARS-CoV-2-infected cells may negatively affect channel activity. Here we present experimental evidence showing that coexpression of the S protein with ENaC in a cellular model reduces channel activity. In addition, we show that bidirectional competition for cleavage by furin-like proteases occurs between 〈ENaC and S protein. In transgenic mice sensitive to lethal SARS-CoV-2, however, a significant decrease in gamma ENaC expression was not observed by immunostaining of lungs infected as shown by SARS-CoV2 nucleoprotein staining.

Myelin Basic Protein Attenuates Furin-Mediated Bri2 Cleavage and Postpones Its Membrane Trafficking.

Myelin basic protein (MBP) is the second most abundant protein in the central nervous system and is responsible for structural maintenance of the myelin sheath covering axons. Previously, we showed that MBP has a more proactive role in the oligodendrocyte homeostasis, interacting with membrane-associated proteins, including integral membrane protein 2B (ITM2B or Bri2) that is associated with familial dementias. Here, we report that the molecular dynamics of the in silico-generated MBP-Bri2 complex revealed that MBP covers a significant portion of the Bri2 ectodomain, assumingly trapping the furin cleavage site, while the surface of the BRICHOS domain, which is responsible for the multimerization and activation of the Bri2 high-molecular-weight oligomer chaperone function, remains unmasked. These observations were supported by the co-expression of MBP with Bri2, its mature form, and disease-associated mutants, which showed that in mammalian cells, MBP indeed modulates the post-translational processing of Bri2 by restriction of the furin-catalyzed release of its C-terminal peptide. Moreover, we showed that the co-expression of MBP and Bri2 also leads to an altered cellular localization of Bri2, restricting its membrane trafficking independently of the MBP-mediated suppression of the Bri2 C-terminal peptide release. Further investigations should elucidate if these observations have physiological meaning in terms of Bri2 as a MBP chaperone activated by the MBP-dependent postponement of Bri2 membrane trafficking.

Vero cell-adapted SARS-CoV-2 strain shows increased viral growth through furin-mediated efficient spike cleavage.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes several host proteases to cleave the spike (S) protein to enter host cells. SARS-CoV-2 S protein is cleaved into S1 and S2 subunits by furin, which is closely involved in the pathogenicity of SARS-CoV-2. However, the effects of the modulated protease cleavage activity due to S protein mutations on viral replication and pathogenesis remain unclear. Herein, we serially passaged two SARS-CoV-2 strains in Vero cells and characterized the cell-adapted SARS-CoV-2 strains in vitro and in vivo. The adapted strains showed high viral growth, effective S1/S2 cleavage of the S protein, and low pathogenicity compared with the wild-type strain. Furthermore, the viral growth and S1/S2 cleavage were enhanced by the combination of the Δ68-76 and H655Y mutations using recombinant SARS-CoV-2 strains generated by the circular polymerase extension reaction. The recombinant SARS-CoV-2 strain, which contained the mutation of the adapted strain, showed increased susceptibility to the furin inhibitor, suggesting that the adapted SARS-CoV-2 strain utilized furin more effectively than the wild-type strain. Pathogenicity was attenuated by infection with effectively cleaved recombinant SARS-CoV-2 strains, suggesting that the excessive cleavage of the S proteins decreases virulence. Finally, the high-growth-adapted SARS-CoV-2 strain could be used as the seed for a low-cost inactivated vaccine; immunization with this vaccine can effectively protect the host from SARS-CoV-2 variants. Our findings provide novel insights into the growth and pathogenicity of SARS-CoV-2 in the evolution of cell-cell transmission. IMPORTANCE: The efficacy of the S protein cleavage generally differs among the SARS-CoV-2 variants, resulting in distinct viral characteristics. The relationship between a mutation and the entry of SARS-CoV-2 into host cells remains unclear. In this study, we analyzed the sequence of high-growth Vero cell-adapted SARS-CoV-2 and factors determining the enhancement of the growth of the adapted virus and confirmed the characteristics of the adapted strain by analyzing the recombinant SARS-CoV-2 strain. We successfully identified mutations Δ68-76 and H655Y, which enhance viral growth and the S protein cleavage by furin. Using recombinant viruses enabled us to conduct a virus challenge experiment in vivo. The pathogenicity of SARS-CoV-2 introduced with the mutations Δ68-76, H655Y, P812L, and Q853L was attenuated in hamsters, indicating the possibility of the attenuation of excessive cleaved SARS-CoV-2. These findings provide novel insights into the infectivity and pathogenesis of SARS-CoV-2 strains, thereby significantly contributing to the field of virology.

Human MARCH1, 2, and 8 block Ebola virus envelope glycoprotein cleavage via targeting furin P domain.

Membrane-associated RING-CH (MARCH) family proteins were recently reported to inhibit viral replication through multiple modes. Previous work showed that human MARCH8 blocked Ebola virus (EBOV) glycoprotein (GP) maturation. Our study here demonstrates that human MARCH1 and MARCH2 share a similar pattern to MARCH8 in restricting EBOV GP-pseudotyped viral infection. Human MARCH1 and MARCH2 retain EBOV GP at the trans-Golgi network, reduce its cell surface display, and impair EBOV GP-pseudotyped virions infectivity. Furthermore, we uncover that the host proprotein convertase furin could interact with human MARCH1/2 and EBOV GP intracellularly. Importantly, the furin P domain is verified to be recognized by MARCH1/2/8, which is critical for their blocking activities. Besides, bovine MARCH2 and murine MARCH1 also impair EBOV GP proteolytic processing. Altogether, our findings confirm that MARCH1/2 proteins of different mammalian origins showed a relatively conserved feature in blocking EBOV GP cleavage, which could provide clues for subsequent MARCHs antiviral studies and may facilitate the development of novel strategies to antagonize enveloped virus infection.

The subtilisin-like protease furin regulates hemin-induced CD63 surface expression on platelets.

Accumulation of free heme B in the plasma can be the result of severe hemolytic events, when the scavenger system for free hemoglobin and heme B is overwhelmed. Free heme B can be oxidized into toxic hemin, which has been proven to activate platelet degranulation and aggregation and promote thrombosis. In the present study we analyzed the effect of hemin on the activation-mediated lysosomal degranulation and CD63 surface expression on platelets using classic flow cytometry and fluorescence microscopy techniques. Classical platelet activators were used as control to distinguish the novel effects of hemin from known activation pathways. CD63 is a tetraspanin protein, also known as lysosomal-associated membrane protein 3 or LAMP-3. In resting platelets CD63 is located within the membrane of delta granules and lysosomes of platelet, from where it is integrated into the platelet outer membrane upon stimulation. We were able to show that hemin like the endogenous platelet activators ADP, collagen or thrombin does provoke CD63 re-localization. Interestingly, only hemin-induced CD63 externalization is dependent on the subtilisin-like pro-protein convertase furin as shown by inhibitor experiments. Furthermore, we were able to demonstrate that hemin induces lysosome secretion, a source of the hemin-mediated CD63 presentation. Again, only the hemin-induced lysosome degranulation is furin dependent. In summary we have shown that the pro-protein convertase furin plays an important role in hemin-mediated lysosomal degranulation and CD63 externalization.

Genome-wide bioinformatics analysis of human protease capacity for proteolytic cleavage of the SARS-CoV-2 spike glycoprotein.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) primarily enters the cell by binding the virus's spike (S) glycoprotein to the angiotensin-converting enzyme 2 receptor on the cell surface, followed by proteolytic cleavage by host proteases. Studies have identified furin and transmembrane protease serine 2 proteases in priming and triggering cleavages of the S glycoprotein, converting it into a fusion-competent form and initiating membrane fusion, respectively. Alternatively, SARS-CoV-2 can enter the cell through the endocytic pathway, where activation is triggered by lysosomal cathepsin L. However, other proteases are also suspected to be involved in both entry routes. In this study, we conducted a genome-wide bioinformatics analysis to explore the capacity of human proteases in hydrolyzing peptide bonds of the S glycoprotein. Predictive models of sequence specificity for 169 human proteases were constructed and applied to the S glycoprotein together with the method for predicting structural susceptibility to proteolysis of protein regions. After validating our approach on extensively studied S2' and S1/S2 cleavage sites, we applied our method to each peptide bond of the S glycoprotein across all 169 proteases. Our results indicate that various members of the proprotein convertase subtilisin/kexin type, type II transmembrane family serine protease, and kallikrein families, as well as specific coagulation factors, are capable of cleaving S2' or S1/S2 sites. We have also identified a potential cleavage site of cathepsin L at the K790 position within the S2' loop. Structural analysis suggests that cleavage of this site induces conformational changes similar to the cleavage at the R815 (S2') position, leading to the exposure of the fusion peptide and subsequent fusion with the membrane. Other potential cleavage sites and the influence of mutations in common SARS-CoV-2 variants on proteolytic efficiency are discussed.IMPORTANCEThe entry of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) into the cell, activated by host proteases, is considerably more complex in coronaviruses than in most other viruses and is not fully understood. There is evidence that other proteases beyond the known furin and transmembrane protease serine 2 can activate the spike protein. Another example of uncertainty is the cleavage site for the alternative endocytic route of SARS-CoV-2 entrance, which is still unknown. Bioinformatics methods, modeling protease specificity and estimating the structural susceptibility of protein regions to proteolysis, can aid in studying this topic by predicting the involved proteases and their cleavage sites, thereby substantially reducing the amount of experimental work. Elucidating the mechanisms of spike protein activation is crucial for preventing possible future coronavirus pandemics and developing antiviral drugs.

Analysis of the molecular determinants for furin cleavage of the spike protein S1/S2 site in defined strains of the prototype coronavirus murine hepatitis virus (MHV).

We analyzed the spike protein S1/S2 cleavage of selected strains of a prototype coronavirus, mouse hepatitis virus (MHV) by the cellular protease furin, in order to understand the structural requirements underlying the sequence selectivity of the scissile segment. The probability of cleavage of selected MHV strains was first evaluated from furin cleavage scores predicted by the ProP computer software, and then cleavage was measured experimentally with a fluorogenic peptide cleavage assay consisting of S1/S2 peptide mimics and purified furin. We found that in vitro cleavability varied across MHV strains in line with predicted results-but with the notable exception of MHV-A59, which was not cleaved despite a high score predicted for its sequence. Using the known X-Ray structure of furin in complex with a substrate-like inhibitor as an initial structural reference, we carried out molecular dynamics (MD) simulations to learn the modes of binding of the peptides in the furin active site, and the suitability of the complex for initiation of the enzymatic cleavage. We identified the 3D structural requirements of the furin active site configuration that enable bound peptides to undergo cleavage, and the way in which the various strains tested experimentally are fulfilling these requirements. We find that despite some flexibility in the organization of the peptide bound to the active site of the enzyme, the presence of a histidine at P2 of MHV-A59 fails to properly orient the sidechain of His194 of the furin catalytic triad and therefore produces a distortion that renders the peptide/complex structural configuration in the active site incompatible with requirements for cleavage initiation. The Ser/Thr in P1 of MHV-2 and MHV-S has a similar effect of distorting the conformation of the furin active site residues produced by the elimination of the canonical salt-bridge formed by arginine in P1 position. This work informs a study of coronavirus infection and pathogenesis with respect to the function of the viral spike protein, and suggests an important process of viral adaptation and evolution within the spike S1/S2 structural loop.

Cleavage-Activation of Respiratory Viruses - Half a Century of History from Sendai Virus to SARS-CoV-2.

Many viruses require the cleavage-activation of membrane fusion proteins by host proteases in the course of infection. This knowledge is based on historical studies of Sendai virus in the 1970s. From the 1970s to the 1990s, avian influenza virus and Newcastle disease virus were studied, showing a clear link between virulence and the cleavage-activation of viral membrane fusion proteins (hemagglutinin and fusion proteins) by host proteases. In these viruses, cleavage of viral membrane fusion proteins by furin is the basis for their high virulence. Subsequently, from the 2000s to the 2010s, the importance of TMPRSS2 in activating the membrane fusion proteins of various respiratory viruses, including seasonal influenza viruses, was demonstrated. In late 2019, severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) emerged and caused a pandemic. The virus continues to mutate, producing variants that have caused global pandemics. The spike protein of SARS-CoV-2 is characterized by two cleavage sites, each of which is cleaved by furin and TMPRSS2 to achieve membrane fusion. SARS-CoV-2 variants exhibit altered sensitivity to these proteases. Thus, studying the cleavage-activation of membrane fusion proteins by host proteases is critical for understanding the ongoing pandemic and developing countermeasures against it.

The possible role furin and furin inhibitors in endometrial adenocarcinoma: A narrative review.

BACKGROUND: Endometrial adenocarcinoma (EAC) is a malignant tumor of the endometrium. EAC is the most common female malignancy following the menopause period. About 40% of patients with EAC are linked with obesity and interrelated with hypertension, diabetes mellitus, and high circulating estrogen levels. Proprotein convertase (PC) furin was involved in the progression of EAC. RECENT FINDINGS: Furin is a protease enzyme belonging to the subtilisin PC family called PC subtilisin/kexin type 3 that converts precursor proteins to biologically active forms and products. Aberrant activation of furin promotes abnormal cell proliferation and the development of cancer. Furin promotes angiogenesis, malignant cell proliferation, and tissue invasion by malignant cells through its pro-metastatic and oncogenic activities. Furin activity is correlated with the malignant proliferation of EAC. Higher expression of furin may increase the development of EAC through overexpression of pro-renin receptors and disintegrin and metalloprotease 17 (ADAM17). As well, inflammatory signaling in EAC promotes the expression of furin with further propagation of malignant transformation. CONCLUSION: Furin is associated with the development and progression of EAC through the induction of proliferation, invasion, and metastasis of malignant cells of EAC. Furin induces ontogenesis in EAC through activation expression of ADAM17, pro-renin receptor, CD109, and TGF-ß. As well, EAC-mediated inflammation promotes the expression of furin with further propagation of neoplastic growth and invasion.

Diminazene aceturate-induced cytotoxicity is associated with the deregulation of cell cycle signaling and downregulation of oncogenes Furin, c-MYC, and FOXM1 in human cervical carcinoma Hela cells.

Diminazene aceturate (DIZE) is an FDA-listed small molecule known for the treatment of African sleeping sickness. In vivo studies showed that DIZE may be beneficial for a range of human ailments. However, there is very limited information on the effects of DIZE on human cancer cells. The current study aimed to investigate the cytotoxic responses of DIZE, using the human carcinoma Hela cell line. WST-1 cell proliferation assay showed that DIZE inhibited the viability of Hela cells in a dose-dependent manner and the observed response was associated with the downregulation of Ki67 and PCNA cell proliferation markers. DIZE-treated cells stained with acridine orange-ethidium and JC-10 dye revealed cell death and loss of mitochondrial membrane potential (Ψm), compared with DMSO (vehicle) control, respectively. Cellular immunofluorescence staining of DIZE-treated cells showed upregulation of caspase 3 activities. DIZE-treated cells showed downregulation of mRNA for G1/S genes CCNA2 and CDC25A, S-phase genes MCM3 and PLK4, and G2/S phase transition/mitosis genes Aurka and PLK1. These effects were associated with decreased mRNA expression of Furin, c-Myc, and FOXM1 oncogenes. These results suggested that DIZE may be considered for its effects on other cancer types. To the best of our knowledge, this is the first study to evaluate the effect of DIZE on human cervical cancer cells.

Iron overload suppresses hippocampal neurogenesis in adult mice: Implication for iron dysregulation-linked neurological diseases.

AIMS: Adult hippocampal neurogenesis is an important player in brain homeostasis and its impairment participates in neurological diseases. Iron overload has emerged as an irreversible factor of brain aging, and is also closely related to degenerative disorders, including cognitive dysfunction. However, whether brain iron overload alters hippocampal neurogenesis has not been reported. We investigated the effect of elevated iron content on adult hippocampal neurogenesis and explored the underlying mechanism. METHODS: Mouse models with hippocampal iron overload were generated. Neurogenesis in hippocampus and expression levels of related molecules were assessed. RESULTS: Iron accumulation in hippocampus remarkably impaired the differentiation of neural stem cells, resulting in a significant decrease in newborn neurons. The damage was possibly attributed to iron-induced downregulation of proprotein convertase furin and subsequently decreased maturation of brain-derived neurotrophic factor (BDNF), thus contributing to memory decline and anxiety-like behavior of mice. Supportively, knockdown of furin indeed suppressed hippocampal neurogenesis, while furin overexpression restored the impairment. CONCLUSION: These findings demonstrated that iron overload damaged hippocampal neurogenesis likely via iron-furin-BDNF pathway. This study provides new insights into potential mechanisms on iron-induced neurotoxicity and the causes of neurogenesis injury and renders modulating iron homeostasis and furin expression as novel therapeutic strategies for treatment of neurological diseases.

Cryptococcal proteases exhibit the potential to activate the latent SARS-CoV-2 spike protein.

BACKGROUND: The COVID-19 pandemic has affected more than 650 million people and resulted in over 6.8 million deaths. Notably, the disease could co-manifest with microbial infections, like cryptococcosis, which also presents as a primary lung infection. OBJECTIVE: In this contribution, we sought to determine if cryptococcal supernatant (which contains secreted furin-like proteases) could activate the SARS-CoV-2 spike protein. METHODS: Molecular docking of the crystal structures of the SARS-CoV-2 spike protein (target) and selected cryptococcal proteases (ligands) was executed using the high ambiguity driven protein-protein docking (HADDOCK) server, with the furin protease serving as a reference ligand. The furin protease is found in human cells and typically activates the SARS-CoV-2 spike protein. Importantly, in order to provide experimental evidence for enzymatic activity, we also assessed the biochemical efficiency of cryptococcal proteases to initiate viral entry into HEK-293 T cells by SARS-CoV-2 spike pseudotyped Lentivirus. RESULTS: We show that the selected cryptococcal proteases could interact with the spike protein, and some had a better or comparable binding affinity for the spike protein than furin protease following an in silico comparative analysis of the molecular docking parameters. Furthermore, it was noted that the biochemical efficiency of the cryptococcal supernatant to transduce HEK-293 T cells with SARS-CoV-2 pseudovirions was comparable (p > 0.05) to that of recombinant furin. CONCLUSIONS: Taken together, these data show that cryptococcal proteases could activate the SARS-CoV-2 spike protein. In practice, it may be critical to determine if patients have an underlying cryptococcal infection, as this microbe could secrete proteases that may further activate the SARS-CoV-2 viral particles, thus undermining COVID-19 intervention measures.

Luminescent reporter cells enable the identification of broad-spectrum antivirals against emerging viruses.

The emerging viruses SARS-CoV-2 and arenaviruses cause severe respiratory and hemorrhagic diseases, respectively. The production of infectious particles of both viruses and virus spread in tissues requires cleavage of surface glycoproteins (GPs) by host proprotein convertases (PCs). SARS-CoV-2 and arenaviruses rely on GP cleavage by PCs furin and subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P), respectively. We report improved luciferase-based reporter cell lines, named luminescent inducible proprotein convertase reporter cells that we employ to monitor PC activity in its authentic subcellular compartment. Using these sensor lines we screened a small compound library in high-throughput manner. We identified 23 FDA-approved small molecules, among them monensin which displayed broad activity against furin and SKI-1/S1P. Monensin inhibited arenaviruses and SARS-CoV-2 in a dose-dependent manner. We observed a strong reduction in infectious particle release upon monensin treatment with little effect on released genome copies. This was reflected by inhibition of SARS-CoV-2 spike processing suggesting the release of immature particles. In a proof of concept experiment using human precision cut lung slices, monensin potently inhibited SARS-CoV-2 infection, evidenced by reduced infectious particle release. We propose that our PC sensor pipeline is a suitable tool to identify broad-spectrum antivirals with therapeutic potential to combat current and future emerging viruses.

Furin Regulates the Alveolarization of Neonatal Lungs in a Mouse Model of Hyperoxic Lung Injury.

Despite advances in treatment options, such as corticosteroid administration and less invasive respiratory support, bronchopulmonary dysplasia (BPD) remains an important prognostic factor in preterm infants. We previously reported that furin regulates changes in lung smooth muscle cell phenotypes, suggesting that it plays a critical role in BPD pathogenesis. Therefore, in this study, we aimed to evaluate whether it regulates the alveolarization of immature lungs through activating alveolarization-driving proteins. We first examined furin expression levels, and its functions, using an established hyperoxia-induced BPD mouse model. Thereafter, we treated mice pups, as well as primary myofibroblast cell cultures, with furin inhibitors. Finally, we administered the hyperoxia-exposed mice pups with recombinant furin. Immunofluorescence revealed the co-expression of furin with alpha-smooth muscle actin. Hyperoxia exposure for 10 d decreased alveolar formation, as well as the expression of furin and its target, IGF-1R. Hexa-D-arginine administration also significantly inhibited alveolar formation. Another furin inhibitor, decanoyl-RVKR-chloromethylketone, accumulated pro-IGF-1R, and decreased IGF-1R phosphorylation in myofibroblast primary cultures. Finally, recombinant furin treatment significantly improved alveolarization in hyperoxia-exposed mice pups. Furin regulates alveolarization in immature lungs. Therefore, this study provides novel insights regarding the involvement of furin in BPD pathogenesis, and highlights a potential treatment target for ameliorating the impact of BPD.

Probable human origin of the SARS-CoV-2 polybasic furin cleavage motif.

BACKGROUND: The key evolutionary step leading to the pandemic virus was the acquisition of the PRRA furin cleavage motif at the spike glycoprotein S1/S2 junction by a progenitor of SARS-CoV-2. Two of its features draw attention: (i) it is absent in other known lineage B beta-coronaviruses, including the newly discovered coronaviruses in bats from Laos and Vietnam, which are the closest known relatives of the covid virus; and, (ii) it introduced the pair of arginine codons (CGG-CGG), whose usage is extremely rare in coronaviruses. With an occurrence rate of only 3%, the arginine CGG codon is considered a minority in SARS CoV-2. On the other hand, Laos and Vietnam bat coronaviruses contain receptor-binding domains that are almost identical to that of SARS-CoV-2 and can therefore infect human cells despite the absence of the furin cleavage motif. RESULTS: Based on these data, the aim of this work is to provide a detailed sequence analysis between the SARS-CoV-2 S gene insert encoding PRRA and the human mRNA transcripts. The result showed a 100% match to several mRNA transcripts. The set of human genes whose mRNAs match this S gene insert are ubiquitous and highly expressed, e.g., the ATPase F1 (ATP5F1) and the ubiquitin specific peptidase 21 (USP21) genes; or specific genes of target organs or tissues of the SARS-CoV-2 infection (e.g., MEMO1, SALL3, TRIM17, CWC15, CCDC187, FAM71E2, GAB4, PRDM13). Results suggest that a recombination between the genome of a SARS-CoV-2 progenitor and human mRNA transcripts could be the origin of the S gene 12-nucleotide insert encoding the S protein PRRA motif. CONCLUSIONS: The hypothesis of probable human origin of the SARS-CoV-2 polybasic furin cleavage motif is supported by: (i) the nature of human genes whose mRNA sequence 100% match the S gene insert; (ii) the synonymous base substitution in the arginine codons (CGG-CGG); and (iii) further spike glycoprotein PRRA-like insertions suggesting that the acquisition of PRRA may not have been a single recombination event.

Expression of key SARS-CoV-2 entry molecules in surgically obtained human retinal biopsies.

PURPOSE: To investigate the presence of ACE2, TMPRSS2 and Furin, i.e., a key player in the ocular infection with SARS-COV-2, in surgically obtained human retinal tissue samples from SARS-CoV-2-negative patients, using gene expression analysis. METHODS: The mechanisms and entry paths of ocular infections have been ill-defined so far. To better understand the possible entry routes, we used surgically explanted retinal tissue from nine patients that were not infected with SARS-CoV-2 and analyzed the message expression of the three key molecules that confer viral entry into cells using polymerase chain reaction. RESULTS: The median age of the patients (n = 9) included in the study was 52 years (IQR 48, 55). Eight patients underwent surgery for rhegmatogenous retinal detachment and one patient for tractional retinal detachment. Gene expression for the proteins studied was detected in all nine patients. The results of analysis by Livak's method (2001) demonstrated a median TMPRSS2 gene expression value of 20.9 (IQR 11.7, 33.7), a median ACE2 gene expression value of 2.09 (IQR 1.14, 2.79) and a median Furin gene expression value of 8.33 (IQR 5.90, 11.8). CONCLUSION: In conclusion, TMPRSS2, Furin and ACE2 are expressed in the retina and may contribute to the retinal involvement in COVID-19 patients. Expression may vary among individuals, which may explain why some patients may be more prone to retinal involvement during SARS-CoV-2 infection COVID-19 patients than others. Variability in the expression of TMPRSS2, Furin and ACE2 proteins themselves may also explain the presence or development of retinal symptoms of varying severity.

An Overview of the Role of Furin in Type 2 Diabetes.

Post-translational modifications (PTMs) play important roles in regulating several human diseases, like cancer, neurodegenerative disorders, and metabolic disorders. Investigating PTMs' contribution to protein functions is critical for modern biology and medicine. Proprotein convertases (PCs) are irreversible post-translational modifiers that have been extensively studied and are considered as key targets for novel therapeutics. They cleave proteins at specific sites causing conformational changes affecting their functions. Furin is considered as a PC model in regulating growth factors and is involved in regulating many pro-proteins. The mammalian target of the rapamycin (mTOR) signaling pathway is another key player in regulating cellular processes and its dysregulation is linked to several diseases including type 2 diabetes (T2D). The role of furin in the context of diabetes has been rarely explored and is currently lacking. Moreover, furin variants have altered activity that could have implications on overall health. In this review, we aim to highlight the role of furin in T2D in relation to mTOR signaling. We will also address furin genetic variants and their potential effect on T2D and ß-cell functions. Understanding the role of furin in prediabetes and dissecting it from other confounding factors like obesity is crucial for future therapeutic interventions in metabolic disorders.

SARS-CoV-2 Spike Protein Is Capable of Inducing Cell-Cell Fusions Independent from Its Receptor ACE2 and This Activity Can Be Impaired by Furin Inhibitors or a Subset of Monoclonal Antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was responsible for the COVID-19 pandemic, efficiently spreads cell-to-cell through mechanisms facilitated by its membrane glycoprotein spike. We established a dual split protein (DSP) assay based on the complementation of GFP and luciferase to quantify the fusogenic activity of the SARS-CoV-2 spike protein. We provide several lines of evidence that the spike protein of SARS-CoV-2, but not SARS-CoV-1, induced cell-cell fusion even in the absence of its receptor, angiotensin-converting enzyme 2 (ACE2). This poorly described ACE2-independent cell fusion activity of the spike protein was strictly dependent on the proteasomal cleavage of the spike by furin while TMPRSS2 was dispensable. Previous and current variants of concern (VOCs) differed significantly in their fusogenicity. The Delta spike was extremely potent compared to Alpha, Beta, Gamma and Kappa, while the Omicron spike was almost devoid of receptor-independent fusion activity. Nonetheless, for all analyzed variants, cell fusion was dependent on furin cleavage and could be pharmacologically inhibited with CMK. Mapping studies revealed that amino acids 652-1273 conferred the ACE2-independent fusion activity of the spike. Unexpectedly, residues proximal to the furin cleavage site were not of major relevance, whereas residue 655 critically regulated fusion. Finally, we found that the spike's fusion activity in the absence of ACE2 could be inhibited by antibodies directed against its N-terminal domain (NTD) but not by antibodies targeting its receptor-binding domain (RBD). In conclusion, our BSL-1-compatible DSP assay allowed us to screen for inhibitors or antibodies that interfere with the spike's fusogenic activity and may therefore contribute to both rational vaccine design and development of novel treatment options against SARS-CoV-2.

Natural fucoidans inhibit coronaviruses by targeting viral spike protein and host cell furin.

Fucoidans are a class of long chain sulfated polysaccharides and have multiple biological functions. Herein, four natural fucoidans extracted from Fucus vesiculosus, F. serratus, Laminaria japonica and Undaria pinnatifida, were tested for their HCoV-OC43 inhibition and found to demonstrate EC50 values ranging from 0.15 to 0.61 µg/mL. That from U. pinnatifida exhibited the most potent anti-HCoV-OC43 activity with an EC50 value of 0.15 ± 0.02 µg/mL, a potency largely independent of its sulfate content. Comparison of the gene expression profiles of fucoidan-treated and untreated cells infected with HCoV-OC43 revealed that fucoidan treatment effectively diminished HCoV-OC43 gene expressions associated with induced chemokines, cytokines and viral activities. Further studies using a highly fucoidan-resistant HCoV-OC43 determined that fucoidan inhibited HCoV-OC43 infection via interfering with viral entry and led to the identification of the specific site on the N-terminal region of spike protein, that located adjacent to the host cell receptor binding domain, targeted by the virus. Furthermore, in a SARS-CoV-2 pseudovirus neutralization assay, fucoidan also blocked SARS-CoV-2 entry. In vitro and in vivo, fucoidan decreased SARS-CoV-2 viral loads and inhibited viral infection in Calu-3 or Vero E6 cells and SARS-CoV-2 infected hamsters, respectively. Fucoidan was also found to inhibit furin activity, and reported furin inhibitors were found to inhibit viral infection by wild type HCoV-OC43 or SARS-CoV-2. Accordingly, we conclude that fucoidans inhibit coronaviral infection by targeting viral spike protein and host cell furin to interfere with viral entry.