Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.500
Filtrar
1.
Proc Natl Acad Sci U S A ; 118(47)2021 11 23.
Artículo en Inglés | MEDLINE | ID: mdl-34732583

RESUMEN

The SARS-CoV-2 coronavirus responsible for the global pandemic contains a novel furin cleavage site in the spike protein (S) that increases viral infectivity and syncytia formation in cells. Here, we show that O-glycosylation near the furin cleavage site is mediated by members of the GALNT enzyme family, resulting in decreased furin cleavage and decreased syncytia formation. Moreover, we show that O-glycosylation is dependent on the novel proline at position 681 (P681). Mutations of P681 seen in the highly transmissible alpha and delta variants abrogate O-glycosylation, increase furin cleavage, and increase syncytia formation. Finally, we show that GALNT family members capable of glycosylating S are expressed in human respiratory cells that are targets for SARS-CoV-2 infection. Our results suggest that host O-glycosylation may influence viral infectivity/tropism by modulating furin cleavage of S and provide mechanistic insight into the role of the P681 mutations found in the highly transmissible alpha and delta variants.


Asunto(s)
SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Animales , Fusión Celular , Línea Celular , Furina/metabolismo , Células Gigantes , Glicosilación , Humanos , N-Acetilgalactosaminiltransferasas/metabolismo , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética
2.
Anal Chem ; 93(42): 14036-14041, 2021 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-34633790

RESUMEN

Utilizing ionic current and fluorescent dual-signal-output nanochannels to achieve the detection of specific target species has received much attention. The introduction of an optical signal could not only improve the selectivity of the detection systems, but also make it possible to observe the reduction of the ionic current that originated from stimulus-triggered nanochannel changes. However, the resolution of an optical signal can only verify issues of the presence or absence and cannot precisely analyze the detailed chemical structural changes within nanochannels. Here, we employed a biocompatible condensation reaction between 2-cyanobenzothiazole (CBT) and d-cysteine, and synthesized molecules PCTC that can be polymerized by cutting off short peptide sequences in the presence of furin to realize the detection of furin with multiple signal outputs. Through the introduction of a UV light-sensitive DNA sequence to the capture probes (CPs) inside the nanochannels, the blocking of the nanochannels can be confirmed to the formed oligomers by mass spectrometry analysis.


Asunto(s)
Furina , Transporte Iónico
3.
Int J Biol Sci ; 17(14): 3954-3967, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34671211

RESUMEN

Furin is a proprotein convertase that activates different kinds of regulatory proteins, including SARS-CoV-2 spike protein which contains an additional furin-specific cleavage site. It is essential in predicting cancer patients' susceptibility to SARS-CoV-2 and the disease outcomes due to varying furin expressions in tumor tissues. In this study, we analyzed furin's expression, methylation, mutation rate, functional enrichment, survival rate and COVID-19 outcomes in normal and cancer tissues using online databases, and our IHC. As a result, furin presented with biased expression profiles in normal tissues, showing 12.25-fold higher than ACE2 in the lungs. The furin expression in tumors were significantly increased in ESCA and TGCT, and decreased in DLBC and THYM, indicating furin may play critical mechanistic functions in COVID-19 viral entry into cells in these cancer patients. Line with furin over/downexpression, furin promoter hypo-/hyper-methylation may be the regulatory cause of disease and lead to pathogenesis of ESCA and THYM. Furthermore, presence of FURIN-201 isoform with functional domains (P_proprotein, Peptidase_S8 and S8_pro-domain) is highest in all cancer types in comparison to other isoforms, demonstrating its use in tumorigenesis and SARS-Cov-2 entry into tumor tissues. Furin mutation frequency was highest in UCES, and its mutation might elevate ACE2 expression in LUAD and UCEC, reduce ACE2 expression in COAD, elevate HSPA5 expression in PAAD, and elevate TMPRSS2 expression in BRCA. These results showed that furin mutations mostly increased expression of ACE2, HSPA5, and TMPRSS2 in certain cancers, indicating furin mutations might facilitate COVID-19 cell entry in cancer patients. In addition, high expression of furin was significantly inversely correlated with long overall survival (OS) in LGG and correlated with long OS in COAD and KIRC, indicating that it could be used as a favorable prognostic marker for cancer patients' survival. GO and KEGG demonstrated that furin was mostly enriched in genes for metabolic and biosynthetic processes, retinal dehydrogenase activity, tRNA methyltransferase activity, and genes involving COVID-19, further supporting its role in COVID-19 and cancer metabolism. Moreover, Cordycepin (CD) inhibited furin expression in a dosage dependent manner. Altogether, furin's high expression might not only implies increased susceptibility to SARS-CoV-2 and higher severity of COVID-19 symptoms in cancer patients, but also it highlights the need for cancer treatment and therapy during the COVID-19 pandemic. CD might have a potential to develop an anti-SARS-CoV-2 drug through inhibiting furin expression.


Asunto(s)
Antineoplásicos/uso terapéutico , COVID-19/virología , Desoxiadenosinas/uso terapéutico , Furina/metabolismo , Neoplasias/metabolismo , Antineoplásicos/farmacología , COVID-19/complicaciones , Línea Celular Tumoral , Desoxiadenosinas/farmacología , Susceptibilidad a Enfermedades , Furina/antagonistas & inhibidores , Furina/genética , Humanos , Neoplasias/complicaciones , Isoformas de Proteínas/metabolismo , Serina Endopeptidasas/metabolismo
4.
J Biol Chem ; 297(4): 101177, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34508778

RESUMEN

The hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote carriers of ASGR1 deletions exhibit ∼34% lower risk of coronary artery disease and ∼10% to 14% reduction of non-HDL cholesterol. Since the proprotein convertase PCSK9 is a major degrader of the low-density lipoprotein receptor (LDLR), we investigated the degradation and functionality of LDLR and/or PCSK9 by endogenous/overexpressed ASGR1 using Western blot and immunofluorescence in HepG2-naïve and HepG2-PCSK9-knockout cells. ASGR1, like PCSK9, targets LDLR, and both independently interact with/enhance the degradation of the receptor. This lack of cooperativity between PCSK9 and ASGR1 was confirmed in livers of wildtype (WT) and Pcsk9-/- mice. ASGR1 knockdown in HepG2-naïve cells significantly increased total (∼1.2-fold) and cell-surface (∼4-fold) LDLR protein. In HepG2-PCSK9-knockout cells, ASGR1 silencing led to ∼2-fold higher levels of LDLR protein and DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-LDL uptake associated with ∼9-fold increased cell-surface LDLR. Overexpression of WT-ASGR1/2 primarily reduced levels of immature non-O-glycosylated LDLR (∼110 kDa), whereas the triple Ala-mutant of Gln240/Trp244/Glu253 (characterized by loss of carbohydrate binding) reduced expression of the mature form of LDLR (∼150 kDa), suggesting that ASGR1 binds the LDLR in both a sugar-dependent and -independent fashion. The protease furin cleaves ASGR1 at the RKMK103↓ motif into a secreted form, likely resulting in a loss of function on LDLR. Altogether, we demonstrate that LDLR is the first example of a liver-receptor ligand of ASGR1. We conclude that silencing of ASGR1 and PCSK9 may lead to higher LDL uptake by hepatocytes, thereby providing a novel approach to further reduce LDL cholesterol levels.


Asunto(s)
Receptor de Asialoglicoproteína/metabolismo , Furina/metabolismo , Hígado/metabolismo , Proproteína Convertasa 9/metabolismo , Receptores de LDL/metabolismo , Animales , Receptor de Asialoglicoproteína/genética , Furina/genética , Células HEK293 , Células Hep G2 , Humanos , Ratones , Ratones Noqueados , Proproteína Convertasa 9/genética , Receptores de LDL/genética
5.
Cells ; 10(9)2021 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-34572076

RESUMEN

Coronavirus disease 19 (COVID-19) is caused by an enveloped, positive-sense, single-stranded RNA virus, referred to as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which belongs to the realm Riboviria, order Nidovirales, family Coronaviridae, genus Betacoronavirus and the species Severe acute respiratory syndrome-related coronavirus. This viral disease is characterized by a myriad of varying symptoms, such as pyrexia, cough, hemoptysis, dyspnoea, diarrhea, muscle soreness, dysosmia, lymphopenia and dysgeusia amongst others. The virus mainly infects humans, various other mammals, avian species and some other companion livestock. SARS-CoV-2 cellular entry is primarily accomplished by molecular interaction between the virus's spike (S) protein and the host cell surface receptor, angiotensin-converting enzyme 2 (ACE2), although other host cell-associated receptors/factors, such as neuropilin 1 (NRP-1) and neuropilin 2 (NRP-2), C-type lectin receptors (CLRs), as well as proteases such as TMPRSS2 (transmembrane serine protease 2) and furin, might also play a crucial role in infection, tropism, pathogenesis and clinical outcome. Furthermore, several structural and non-structural proteins of the virus themselves are very critical in determining the clinical outcome following infection. Considering such critical role(s) of the abovementioned host cell receptors, associated proteases/factors and virus structural/non-structural proteins (NSPs), it may be quite prudent to therapeutically target them through a multipronged clinical regimen to combat the disease.


Asunto(s)
COVID-19 , Interacciones Microbiota-Huesped , SARS-CoV-2/patogenicidad , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , COVID-19/patología , COVID-19/virología , Sistemas de Liberación de Medicamentos , Furina/química , Furina/metabolismo , Humanos , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Estructura Molecular , Neuropilinas/química , Neuropilinas/metabolismo , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Unión Proteica , Receptores Virales/química , Receptores Virales/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Resultado del Tratamiento , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Internalización del Virus
7.
Physiol Rep ; 9(17): e15014, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34523264

RESUMEN

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is threatening people's lives and impacting their health. It is still unclear whether people engaged in physical activity are at an increased risk of SARS-CoV-2 infection and severe forms of COVID-19. In order to provide data to help answer this question, we, therefore, investigated the effects of endurance training on the levels of host proteins involved in SARS-CoV-2 infection in mice. Eight-week-old C57BL/6J mice were subjected to treadmill running (17-25 m/min, 60-90 min, 5 sessions/week, 8 weeks). After the intervention, the levels of angiotensin-converting enzyme 2 (ACE2; host receptor for SARS-CoV-2), transmembrane protease serine 2 (TMPRSS2; host protease priming fusion of SARS-CoV-2 to host cell membranes), FURIN (host protease that promotes binding of SARS-CoV-2 to host receptors), and Neuropilin-1 (host coreceptor for SARS-CoV-2) were measured in 10 organs that SARS-CoV-2 can infect (larynx, trachea, lung, heart, jejunum, ileum, colon, liver, kidney, and testis). Six organs (heart, lung, jejunum, liver, trachea, and ileum) showed changes in the levels of at least one of the proteins. Endurance training increased ACE2 levels in heart (+66.4%), lung (+37.1%), jejunum (+24.7%) and liver (+27.4%), and FURIN in liver (+17.9%) tissue. In contrast, endurance training decreased Neuropilin-1 levels in liver (-39.7%), trachea (-41.2%), and ileum (-39.7%), and TMPRSS2 in lung (-11.3%). Taken together, endurance training altered the levels of host proteins involved in SARS-CoV-2 cell entry in an organ-dependent manner.


Asunto(s)
COVID-19/virología , Condicionamiento Físico Animal , Resistencia Física , Receptores Virales/metabolismo , SARS-CoV-2/patogenicidad , Internalización del Virus , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , COVID-19/enzimología , Furina/metabolismo , Interacciones Huésped-Patógeno , Masculino , Ratones Endogámicos C57BL , Neuropilina-1/metabolismo , Carrera , Serina Endopeptidasas/metabolismo
8.
Viruses ; 13(9)2021 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-34578428

RESUMEN

Many viruses that cause serious diseases in humans and animals, including the betacoronaviruses (beta-CoVs), such as SARS-CoV, MERS-CoV, and the recently identified SARS-CoV-2, have natural reservoirs in bats. Because these viruses rely entirely on the host cellular machinery for survival, their evolution is likely to be guided by the link between the codon usage of the virus and that of its host. As a result, specific cellular microenvironments of the diverse hosts and/or host tissues imprint peculiar molecular signatures in virus genomes. Our study is aimed at deciphering some of these signatures. Using a variety of genetic methods we demonstrated that trends in codon usage across chiroptera-hosted CoVs are collaboratively driven by geographically different host-species and temporal-spatial distribution. We not only found that chiroptera-hosted CoVs are the ancestors of SARS-CoV-2, but we also revealed that SARS-CoV-2 has the codon usage characteristics similar to those seen in CoVs infecting the Rhinolophus sp. Surprisingly, the envelope gene of beta-CoVs infecting Rhinolophus sp., including SARS-CoV-2, had extremely high CpG levels, which appears to be an evolutionarily conserved trait. The dissection of the furin cleavage site of various CoVs infecting hosts revealed host-specific preferences for arginine codons; however, arginine is encoded by a wider variety of synonymous codons in the murine CoV (MHV-A59) furin cleavage site. Our findings also highlight the latent diversity of CoVs in mammals that has yet to be fully explored.


Asunto(s)
Quirópteros/virología , Uso de Codones , Coronavirus/genética , Evolución Molecular , Animales , Furina/metabolismo , Variación Genética , Genoma Viral
9.
J Virol ; 95(21): e0135721, 2021 10 13.
Artículo en Inglés | MEDLINE | ID: mdl-34406867

RESUMEN

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include (i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increases the positive charge of the surface of this domain, (ii) insertions into the NTD of heterologous peptides containing positively charged amino acids, and (iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide, and makes viruses less capable of syncytium formation. These viral adaptations result in higher affinity of viral particles to heparin, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers, and 2 orders of magnitude higher infectivity. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA-positive (RNA+) viruses, evolution to HS binding may result in virus attenuation in vivo. IMPORTANCE The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to the ACE2 receptor and, later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the NTD and in the P1' position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells, and decrease the genome equivalent to PFU (GE/PFU) ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high-affinity interaction of the spikes with the ACE2 receptor.


Asunto(s)
Evolución Molecular , Heparitina Sulfato/metabolismo , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/metabolismo , Adaptación Biológica , Animales , Sitios de Unión , Chlorocebus aethiops , Efecto Citopatogénico Viral , ADN Complementario , Furina/metabolismo , Heparina/metabolismo , Interacciones Huésped-Patógeno , Unión Proteica , Dominios Proteicos , Procesamiento Proteico-Postraduccional , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Pase Seriado , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Células Vero , Ensayo de Placa Viral , Acoplamiento Viral
11.
mBio ; 12(4): e0141521, 2021 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-34425707

RESUMEN

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) possesses a discriminative polybasic cleavage motif in its spike protein that is recognized by the host furin protease. Proteolytic cleavage activates the spike protein, thereby affecting both the cellular entry pathway and cell tropism of SARS-CoV-2. Here, we investigated the impact of the furin cleavage site on viral growth and pathogenesis using a hamster animal model infected with SARS-CoV-2 variants bearing mutations at the furin cleavage site (S gene mutants). In the airway tissues of hamsters, the S gene mutants exhibited low growth properties. In contrast to parental pathogenic SARS-CoV-2, hamsters infected with the S gene mutants showed no body weight loss and only a mild inflammatory response, thereby indicating the attenuated variant nature of S gene mutants. This transient infection was sufficient for inducing protective neutralizing antibodies that cross-react with different SARS-CoV-2 lineages. Consequently, hamsters inoculated with S gene mutants showed resistance to subsequent infection with both the parental strain and the currently emerging SARS-CoV-2 variants belonging to lineages B.1.1.7 and P.1. Taken together, our findings revealed that the loss of the furin cleavage site causes attenuation in the airway tissues of hamsters and highlighted the potential benefits of S gene mutants as potential immunogens. IMPORTANCE SARS-CoV-2 uses its spike protein to enter target cells. The spike protein is cleaved by a host protease, and this event facilitates viral entry and broadens cell tropism. In this study, we employed SARS-CoV-2 mutants lacking the S protein cleavage site and characterized their growth and pathogenicity using hamsters, a laboratory animal model for SARS-CoV-2 infection. These mutants exerted low pathogenicity but induced sufficient levels of neutralizing antibodies in hamsters, which protected hamsters from rechallenge with pathogenic clinical SARS-CoV-2 strains. These virus mutants may be used as protective immunogens against SARS-CoV-2 infection.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/patología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética , Animales , Línea Celular , Chlorocebus aethiops , Reacciones Cruzadas/inmunología , Furina/metabolismo , Humanos , SARS-CoV-2/genética , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Vacunas Atenuadas/inmunología , Células Vero , Virulencia/genética
12.
Genes (Basel) ; 12(7)2021 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-34356057

RESUMEN

The virus responsible for the COVID-19 global health crisis, SARS-CoV-2, has been shown to utilize the ACE2 protein as an entry point to its target cells. The virus has been shown to rely on the actions of TMPRSS2 (a serine protease), as well as FURIN (a peptidase), for the critical priming of its spike protein. It has been postulated that variations in the sequence and expression of SARS-CoV-2's receptor (ACE2) and the two priming proteases (TMPRSS2 and FURIN) may be critical in contributing to SARS-CoV-2 infectivity. This study aims to examine the different expression levels of FURIN in various tissues and age ranges in light of ACE2 and TMPRSS2 expression levels using the LungMAP database. Furthermore, we retrieved expression quantitative trait loci (eQTLs) of the three genes and their annotation. We analyzed the frequency of the retrieved variants in data from various populations and compared it to the Egyptian population. We highlight FURIN's potential interplay with the immune response to SARS-CoV-2 and showcase a myriad of variants of the three genes that are differentially expressed across populations. Our findings provide insights into potential genetic factors that impact SARS-CoV-2 infectivity in different populations and shed light on the varying expression patterns of FURIN.


Asunto(s)
Alelos , Enzima Convertidora de Angiotensina 2 , COVID-19 , Bases de Datos de Ácidos Nucleicos , Furina , Regulación Enzimológica de la Expresión Génica , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , SARS-CoV-2/metabolismo , Serina Endopeptidasas , Enzima Convertidora de Angiotensina 2/biosíntesis , Enzima Convertidora de Angiotensina 2/genética , COVID-19/enzimología , COVID-19/genética , Biología Computacional , Femenino , Furina/biosíntesis , Furina/genética , Humanos , Masculino , SARS-CoV-2/genética , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/genética
13.
Mol Biol Rep ; 48(9): 6655-6661, 2021 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-34392451

RESUMEN

Severe acute respiratory syndrome coronaviruses 2 (SARS-CoV-2) is the causative agent of current coronavirus disease 2019 (COVID-19) pandemic. Electrolyte disorders particularly potassium abnormalities have been repeatedly reported as common clinical manifestations of COVID-19. Here, we discuss how SARS-CoV-2 may affect potassium balance by impairing the activity of epithelial sodium channels (ENaC). The first hypothesis could justify the incidence of hypokalemia. SARS-CoV-2 cell entry through angiotensin-converting enzyme 2 (ACE2) may enhance the activity of renin-angiotensin-aldosterone system (RAAS) classical axis and further leading to over production of aldosterone. Aldosterone is capable of enhancing the activity of ENaC and resulting in potassium loss from epithelial cells. However, type II transmembrane serine protease (TMPRSS2) is able to inhibit the ENaC, but it is utilized in the case of SARS-CoV-2 cell entry, therefore the ENaC remains activated. The second hypothesis describe the incidence of hyperkalemia based on the key role of furin. Furin is necessary for cleaving both SARS-CoV-2 spike protein and ENaC subunits. While the furin is hijacked by the virus, the decreased activity of ENaC would be expected, which causes retention of potassium ions and hyperkalemia. Given that the occurrence of hypokalemia is higher than hyperkalemia in COVID-19 patients, the first hypothesis may have greater impact on potassium levels. Further investigations are warranted to determine the exact role of ENaC in SARS-CoV-2 pathogenesis.


Asunto(s)
COVID-19/metabolismo , Células Epiteliales/metabolismo , Canales Epiteliales de Sodio/metabolismo , Potasio/metabolismo , SARS-CoV-2/metabolismo , COVID-19/virología , Células Epiteliales/virología , Furina/metabolismo , Humanos , Pandemias/prevención & control , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo
14.
ACS Chem Biol ; 16(9): 1692-1700, 2021 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-34415722

RESUMEN

The pro-protein convertase furin is a highly specific serine protease involved in the proteolytic maturation of many proteins in the secretory pathway. It also activates surface proteins of many viruses including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furin inhibitors effectively suppress viral replication and thus are promising antiviral therapeutics with broad application potential. Polybasic substrate-like ligands typically trigger conformational changes shifting furin's active site cleft from the OFF-state to the ON-state. Here, we solved the X-ray structures of furin in complex with four different arginine mimetic compounds with reduced basicity. These guanylhydrazone-based inhibitor complexes showed for the first time an active site-directed binding mode to furin's OFF-state conformation. The compounds undergo unique interactions within the S1 pocket, largely different compared to substrate-like ligands. A second binding site was identified at the S4/S5 pocket of furin. Crystallography-based titration experiments confirmed the S1 site as the primary binding pocket. We also tested the proprotein convertases PC5/6 and PC7 for inhibition by guanylhydrazones and found an up to 7-fold lower potency for PC7. Interestingly, the observed differences in the Ki values correlated with the sequence conservation of the PCs at the allosteric sodium binding site. Therefore, OFF-state-specific targeting of furin can serve as a valuable strategy for structure-based development of PC-selective small-molecule inhibitors.


Asunto(s)
Antivirales/metabolismo , Furina/antagonistas & inhibidores , Guanidinas/metabolismo , Hidrazonas/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Antivirales/química , Dominio Catalítico , Cristalografía por Rayos X , Pruebas de Enzimas , Furina/química , Furina/metabolismo , Guanidinas/química , Células HEK293 , Humanos , Hidrazonas/química , Cinética , Proproteína Convertasa 5/antagonistas & inhibidores , Proproteína Convertasa 5/química , Unión Proteica , Conformación Proteica , Inhibidores de Serina Proteinasa/química , Subtilisinas/antagonistas & inhibidores , Subtilisinas/química
15.
J Am Heart Assoc ; 10(16): e019862, 2021 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-34387094

RESUMEN

Background Thoracic aortic aneurysms (TAAs) occur because of abnormal remodeling of aortic extracellular matrix and are accompanied by the emergence of proteolytically active myofibroblasts. The microRNA miR-133a regulates cellular phenotypes and is reduced in clinical TAA specimens. This study tested the hypothesis that miR-133a modulates aortic fibroblast phenotype, and overexpression by lentivirus attenuates the development of TAA in a murine model. Methods and Results TAA was induced in mice. Copy number of miR-133a was reduced in TAA tissue and linear regression analysis confirmed an inverse correlation between aortic diameter and miR-133a. Analyses of phenotypic markers revealed an mRNA expression profile consistent with myofibroblasts in TAA tissue. Fibroblasts were isolated from the thoracic aortae of mice with/without TAA. When compared with controls, miR-133a was reduced, migration was increased, adhesion was reduced, and the ability to contract a collagen disk was increased. Overexpression/knockdown of miR-133a controlled these phenotypes. After TAA induction in mice, a single tail-vein injection of either miR-133a overexpression or scrambled sequence (control) lentivirus was performed. Overexpression of miR-133a attenuated TAA development. The pro-protein convertase furin was confirmed to be a target of miR-133a by luciferase reporter assay. Furin was elevated in this murine model of TAA and repressed by miR-133a replacement in vivo resulting in reduced proteolytic activation. Conclusions miR-133a regulates aortic fibroblast phenotype and over-expression prevented the development of TAA in a murine model. These findings suggest that stable alterations in aortic fibroblasts are associated with development of TAA and regulation by miR-133a may lead to a novel therapeutic strategy.


Asunto(s)
Aorta Torácica/metabolismo , Aneurisma de la Aorta Torácica/prevención & control , Fibroblastos/metabolismo , Terapia Genética , MicroARNs/genética , Remodelación Vascular , Animales , Aorta Torácica/patología , Aneurisma de la Aorta Torácica/inducido químicamente , Aneurisma de la Aorta Torácica/genética , Aneurisma de la Aorta Torácica/metabolismo , Cloruro de Calcio , Adhesión Celular , Movimiento Celular , Células Cultivadas , Dilatación Patológica , Modelos Animales de Enfermedad , Fibroblastos/patología , Furina/genética , Furina/metabolismo , Vectores Genéticos , Lentivirus/genética , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Fenotipo
16.
Arch Virol ; 166(9): 2541-2549, 2021 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-34258664

RESUMEN

The SARS-CoV-2 spike protein Q677P/H mutation and furin cleavage site (FCS) have been shown to affect cell tropism and virus transmissibility. Here, we analyzed the frequency of Q677P/H and FCS point mutations in 1,144,793 human and 1042 animal spike protein sequences and from those of the emergent variants B.1.1.7, B.1.351, P.1, B.1.429 + B.1.427, and B.1.525, which were deposited in the database of the GISAID Initiative. Different genetic polymorphisms, particularly P681H and A688V, were detected in the FCS, mainly in human isolates, and otherwise, only pangolin and bat sequences had these mutations. Multiple FCS amino acid deletions such as Δ680SPRRA684 and Δ685RSVA688 were only detected in eight and four human isolates, respectively. Surprisingly, deletion of the entire FCS motif as Δ680SPRRARSVA688 and Δ680SPRRARSVAS689 was detected only in three human isolates. On the other hand, analysis of FCS from emergent variants showed no deletions in the FCS except for spike P681del, which was detected in seven B.1.1.7 isolates from the USA. Spike Q677P was detected only once in variant, B.1.1.7, whereas Q677H was detected in all variants, i.e., B.1.1.7 (n = 1938), B.1.351 (n = 28), P.1 (n = 9), B.1.429 + B.1.427 (n = 132), and B.1.525 (n = 1584). Structural modeling predicted that mutations or deletions at or near the FCS significantly alter the cleavage loop structure and would presumably affect furin binding. Taken together, our results show that Q677H and FCS point mutations are prevalent and may have various biological effects on the circulating variants. Therefore, we recommend urgent monitoring and surveillance of the investigated mutations, as well as laboratory assessment of their pathogenicity and transmissibility.


Asunto(s)
COVID-19/epidemiología , Furina/metabolismo , Polimorfismo Genético , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , COVID-19/transmisión , COVID-19/virología , Quirópteros/virología , Monitoreo Epidemiológico , Euterios/virología , Evolución Molecular , Furina/química , Expresión Génica , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteolisis , SARS-CoV-2/química , SARS-CoV-2/clasificación , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo
17.
mBio ; 12(4): e0058721, 2021 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-34311586

RESUMEN

Since the D614G substitution in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, the variant strain has undergone a rapid expansion to become the most abundant strain worldwide. Therefore, this substitution may provide an advantage for viral spreading. To explore the mechanism, we analyzed 18 viral isolates containing S proteins with either G614 or D614 (S-G614 and S-D614, respectively). The plaque assay showed a significantly higher virus titer in S-G614 than in S-D614 isolates. We further found increased cleavage of the S protein at the furin substrate site, a key event that promotes syncytium formation, in S-G614 isolates. The enhancement of the D614G substitution in the cleavage of the S protein and in syncytium formation has been validated in cells expressing S protein. The effect on the syncytium was abolished by furin inhibitor treatment and mutation of the furin cleavage site, suggesting its dependence on cleavage by furin. Our study pointed to the impact of the D614G substitution on syncytium formation through enhanced furin-mediated S cleavage, which might increase the transmissibility and infectivity of SARS-CoV-2 strains containing S-G614. IMPORTANCE Analysis of viral genomes and monitoring of the evolutionary trajectory of SARS-CoV-2 over time has identified the D614G substitution in spike (S) as the most prevalent expanding variant worldwide, which might confer a selective advantage in transmission. Several studies showed that the D614G variant replicates and transmits more efficiently than the wild-type virus, but the mechanism is unclear. By comparing 18 virus isolates containing S with either D614 or G614, we found significantly higher virus titers in association with higher furin protease-mediated cleavage of S, an event that promotes syncytium formation and virus infectivity, in the S-G614 viruses. The effect of the D614G substitution on furin-mediated S cleavage and the resulting enhancement of the syncytium phenotype has been validated in S-expressing cells. This study suggests a possible effect of the D614G substitution on S of SARS-CoV-2; the antiviral effect through targeting furin protease is worthy of being investigated in proper animal models.


Asunto(s)
COVID-19/transmisión , Furina/metabolismo , Células Gigantes/virología , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Sustitución de Aminoácidos/genética , Animales , COVID-19/patología , Línea Celular , Chlorocebus aethiops , Furina/antagonistas & inhibidores , Aptitud Genética/genética , Genoma Viral/genética , Células HEK293 , Humanos , SARS-CoV-2/aislamiento & purificación , Células Vero , Carga Viral/genética , Replicación Viral/genética
18.
Virus Res ; 303: 198522, 2021 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-34314772

RESUMEN

The B.1.1.7 SARS-CoV-2 strain that has emerged in the UK in early December presents seven mutations and three deletions on S-protein structure that could lead to a more infective strain. The P681H mutation in the "PRRAR" furin cleavage site might affect the binding affinity to furin enzyme and hence its infectivity. Therefore, in this study, various structural bioinformatics approaches were used to model the S-protein structure with the B.1.1.7 variant amino acid substitutions and deletions. In addition to modelling the binding of furin to the cleavage site of the wild-type and the B.1.1.7 variant. Conclusively the B.1.1.7 variant resulted in dynamic stability, conformational changes and variations in binding energies in the S-protein structure, resulting in a more favourable binding of furin enzyme to the SARS-CoV-2 S-protein.


Asunto(s)
Furina/metabolismo , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Sitios de Unión , Furina/química , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación , Unión Proteica , Conformación Proteica , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética
19.
Viruses ; 13(7)2021 06 25.
Artículo en Inglés | MEDLINE | ID: mdl-34202098

RESUMEN

Inhibition of the binding of enveloped viruses surface glycoproteins to host cell receptor(s) is a major target of vaccines and constitutes an efficient strategy to block viral entry and infection of various host cells and tissues. Cellular entry usually requires the fusion of the viral envelope with host plasma membranes. Such entry mechanism is often preceded by "priming" and/or "activation" steps requiring limited proteolysis of the viral surface glycoprotein to expose a fusogenic domain for efficient membrane juxtapositions. The 9-membered family of Proprotein Convertases related to Subtilisin/Kexin (PCSK) serine proteases (PC1, PC2, Furin, PC4, PC5, PACE4, PC7, SKI-1/S1P, and PCSK9) participate in post-translational cleavages and/or regulation of multiple secretory proteins. The type-I membrane-bound Furin and SKI-1/S1P are the major convertases responsible for the processing of surface glycoproteins of enveloped viruses. Stefan Kunz has considerably contributed to define the role of SKI-1/S1P in the activation of arenaviruses causing hemorrhagic fever. Furin was recently implicated in the activation of the spike S-protein of SARS-CoV-2 and Furin-inhibitors are being tested as antivirals in COVID-19. Other members of the PCSK-family are also implicated in some viral infections, such as PCSK9 in Dengue. Herein, we summarize the various functions of the PCSKs and present arguments whereby their inhibition could represent a powerful arsenal to limit viral infections causing the present and future pandemics.


Asunto(s)
Regulación Viral de la Expresión Génica , Proproteína Convertasas/metabolismo , Virosis/virología , Internalización del Virus , Virus/genética , Transporte Biológico , Furina/metabolismo , Humanos , Proproteína Convertasa 9/metabolismo , Proproteína Convertasas/genética , Proteolisis , SARS-CoV-2/enzimología , SARS-CoV-2/metabolismo , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Envoltura Viral/metabolismo , Virus/metabolismo
20.
Sci Rep ; 11(1): 14390, 2021 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-34257394

RESUMEN

The coronavirus disease 2019 (COVID-19) pandemic affected people at all ages. Whereas pregnant women seemed to have a worse course of disease than age-matched non-pregnant women, the risk of feto-placental infection is low. Using a cohort of 66 COVID-19-positive women in late pregnancy, we correlated clinical parameters with disease severity, placental histopathology, and the expression of viral entry and Interferon-induced transmembrane (IFITM) antiviral transcripts. All newborns were negative for SARS-CoV-2. None of the demographic parameters or placental histopathological characteristics were associated with disease severity. The fetal-maternal transfer ratio for IgG against the N or S viral proteins was commonly less than one, as recently reported. We found that the expression level of placental ACE2, but not TMPRSS2 or Furin, was higher in women with severe COVID-19. Placental expression of IFITM1 and IFITM3, which have been implicated in antiviral response, was higher in participants with severe disease. We also showed that IFITM3 protein expression, which localized to early and late endosomes, was enhanced in severe COVID-19. Our data suggest an association between disease severity and placental SARS-CoV-2 processing and antiviral pathways, implying a role for these proteins in placental response to SARS-CoV-2.


Asunto(s)
COVID-19/metabolismo , Placenta/metabolismo , SARS-CoV-2/patogenicidad , Adulto , Enzima Convertidora de Angiotensina 2/metabolismo , Femenino , Furina/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Transmisión Vertical de Enfermedad Infecciosa , Masculino , Proteínas de la Nucleocápside/metabolismo , Embarazo , Complicaciones Infecciosas del Embarazo/metabolismo , Complicaciones Infecciosas del Embarazo/virología , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Adulto Joven
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...