RESUMEN
The membrane fusion between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host cells is essential for the initial step of infection; therefore, the host cell membrane components, including sphingolipids, influence the viral infection. We assessed several inhibitors of the enzymes pertaining to sphingolipid metabolism, against SARS-CoV-2 spike protein (S)-mediated cell-cell fusion and viral infection. N-(4-Hydroxyphenyl) retinamide (4-HPR), an inhibitor of dihydroceramide Δ4-desaturase 1 (DES1), suppressed cell-cell fusion and viral infection. The analysis of sphingolipid levels revealed that the inhibition efficiencies of cell-cell fusion and viral infection in 4-HPR-treated cells were consistent with an increased ratio of saturated sphinganine-based lipids to total sphingolipids. We investigated the relationship of DES1 with the inhibition efficiencies of cell-cell fusion. The changes in the sphingolipid profile induced by 4-HPR were mitigated by the supplementation with exogenous cell-permeative ceramide; however, the reduced cell-cell fusion could not be reversed. The efficiency of cell-cell fusion in DES1 knockout (KO) cells was at a level comparable to that in wild-type (WT) cells; however, the ratio of saturated sphinganine-based lipids to the total sphingolipids was higher in DES1 KO cells than in WT cells. 4-HPR reduced cell membrane fluidity without any significant effects on the expression or localization of angiotensin-converting enzyme 2, the SARS-CoV-2 receptor. Therefore, 4-HPR suppresses SARS-CoV-2 S-mediated membrane fusion through a DES1-independent mechanism, and this decrease in membrane fluidity induced by 4-HPR could be the major cause for the inhibition of SARS-CoV-2 infection. IMPORTANCE Sphingolipids could play an important role in SARS-CoV-2 S-mediated membrane fusion with host cells. We studied the cell-cell fusion using SARS-CoV-2 S-expressing cells and sphingolipid-manipulated target cells, with an inhibitor of the sphingolipid metabolism. 4-HPR (also known as fenretinide) is an inhibitor of DES1, and it exhibits antitumor activity and suppresses cell-cell fusion and viral infection. 4-HPR suppresses membrane fusion through a decrease in membrane fluidity, which could possibly be the cause for the inhibition of SARS-CoV-2 infection. There is accumulating clinical data on the safety of 4-HPR. Therefore, it could be a potential candidate drug against COVID-19.
Asunto(s)
Membrana Celular/metabolismo , Fenretinida/farmacología , Fluidez de la Membrana/efectos de los fármacos , Oxidorreductasas/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Fusión Celular , Membrana Celular/genética , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Fluidez de la Membrana/genética , Oxidorreductasas/deficiencia , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Latency remains a barrier to achieving a sterilizing cure to HIV infection. It is thus important to find new host factor(s) to better understand maintenance of HIV latency and be exploited to develop new and more efficient latency reversing agents (LRAs). Here we employed RNA interference screening with a latently HIV-1-infected cell-line to identify Stathmin 1 (STMN1) as a host factor required for maintaining HIV-1 latency. Depletion of STMN1 significantly enhanced HIV-1 expression in a STMN1 depletion-dependent manner and forced expression of exogenous STMN1 suppressed it. We further showed that STMN1 depletion increases HIV-1 proviral transcriptional elongation. Moreover, chromatin immunoprecipitation (ChIP)-qPCR assays revealed STMN1 accumulation on/near the HIV-1 5' LTR region compared to other regions on the HIV-1 provirus, suggesting the possible contribution of STMN1 to HIV-1 transcription. These results suggest that STMN1 is required for the maintenance of HIV-1 latency and implicates STMN1 as a novel therapeutic target to eradicate HIV-1.
Asunto(s)
Infecciones por VIH/metabolismo , VIH-1/fisiología , Estatmina/metabolismo , Latencia del Virus , Infecciones por VIH/genética , Interacciones Huésped-Patógeno , Humanos , Interferencia de ARN , Estatmina/genética , Células THP-1RESUMEN
NF-κB was first identified in 1986 as a B cell-specific transcription factor inducing immunoglobulin κ light chain expression. Subsequent studies revealed that NF-κB plays important roles in development, organogenesis, immunity, inflammation, and neurological functions by spatiotemporally regulating cell proliferation, differentiation, and apoptosis in several cell types. Furthermore, studies on the signal pathways that activate NF-κB led to the discovery of TRAF family proteins with E3 ubiquitin ligase activity, which function downstream of the receptor. This discovery led to the proposal of an entirely new signaling mechanism concept, wherein K63-ubiquitin chains act as a scaffold for the signaling complex to activate downstream kinases. This concept has revolutionized ubiquitin studies by revealing the importance of the nonproteolytic functions of ubiquitin not only in NF-κB signaling but also in a variety of other biological systems. TRAF6 is the most diverged among the TRAF family proteins, and our studies uncovered its notable physiological and pathological functions.
Asunto(s)
FN-kappa B/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Ubiquitinación , Animales , Humanos , Transducción de SeñalRESUMEN
microRNA-146a (miR-146a) has been previously implicated as an essential molecular brake, preventing immune overreaction and malignant transformation by attenuating NF-κB signaling, putatively via repression of the Traf6 and Irak1 genes. The exact contribution of miR-146a-mediated silencing of these genes to the control of immune activation is currently unknown. Therefore, we defined the role of the miR-146a-Traf6 signaling axis in the regulation of immune homeostasis using a genetic epistasis analysis in miR-146a-/- mice. We have uncovered a surprising separation of functions at the level of miR-146a targets. Lowering the Traf6 gene dose and consequent attenuation of NF-κB activation rescued several significant miR-146a-/- phenotypes, such as splenomegaly, aberrant myeloproliferation, and excessive inflammatory responses. In contrast, decreasing Traf6 expression had no effect on the development of the progressive bone marrow failure phenotype, as well as lymphomagenesis in miR-146a-/- mice, indicating that miR-146a controls these biological processes through different molecular mechanisms.
Asunto(s)
Autoinmunidad , Células Madre Hematopoyéticas/citología , Inflamación/inmunología , MicroARNs/inmunología , Mielopoyesis , Neoplasias/inmunología , Factor 6 Asociado a Receptor de TNF/inmunología , Animales , Femenino , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/inmunología , Homeostasis , Humanos , Inflamación/genética , Inflamación/fisiopatología , Masculino , Ratones , MicroARNs/genética , Células Mieloides/citología , Células Mieloides/inmunología , Neoplasias/genética , Neoplasias/fisiopatología , Factor 6 Asociado a Receptor de TNF/genéticaRESUMEN
The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is crucial for the development of adult T-cell leukemia (ATL), a highly malignant CD4+ T cell neoplasm. Among the multiple aberrant Tax-induced effects on cellular processes, persistent activation of transcription factor NF-κB, which is activated only transiently upon physiological stimulation, is essential for leukemogenesis. We and others have shown that Tax induces activation of the IκB kinase (IKK) complex, which is a critical step in NF-κB activation, by generating Lys63-linked polyubiquitin chains. However, the molecular mechanism underlying Tax-induced IKK activation is controversial and not fully understood. Here, we demonstrate that Tax recruits linear (Met1-linked) ubiquitin chain assembly complex (LUBAC) to the IKK complex and that Tax fails to induce IKK activation in cells that lack LUBAC activity. Mass spectrometric analyses revealed that both Lys63-linked and Met1-linked polyubiquitin chains are associated with the IKK complex. Furthermore, treatment of the IKK-associated polyubiquitin chains with Met1-linked-chain-specific deubiquitinase (OTULIN) resulted in the reduction of high molecular weight polyubiquitin chains and the generation of short Lys63-linked ubiquitin chains, indicating that Tax can induce the generation of Lys63- and Met1-linked hybrid polyubiquitin chains. We also demonstrate that Tax induces formation of the active macromolecular IKK complex and that the blocking of Tax-induced polyubiquitin chain synthesis inhibited formation of the macromolecular complex. Taken together, these results lead us to propose a novel model in which the hybrid-chain-dependent oligomerization of the IKK complex triggered by Tax leads to trans-autophosphorylation-mediated IKK activation.
Asunto(s)
Activación Enzimática/fisiología , Productos del Gen tax/metabolismo , Infecciones por HTLV-I/metabolismo , Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Electroforesis en Gel de Poliacrilamida , Células HEK293 , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Humanos , Immunoblotting , Inmunoprecipitación , Células Jurkat , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/fisiología , TransfecciónRESUMEN
Many bacterial pathogens can enter various host cells and then survive intracellularly, transiently evade humoral immunity, and further disseminate to other cells and tissues. When bacteria enter host cells and replicate intracellularly, the host cells sense the invading bacteria as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) by way of various pattern recognition receptors. As a result, the host cells induce alarm signals that activate the innate immune system. Therefore, bacteria must modulate host inflammatory signalling and dampen these alarm signals. How pathogens do this after invading epithelial cells remains unclear, however. Here we show that OspI, a Shigella flexneri effector encoded by ORF169b on the large plasmid and delivered by the type ΙΙΙ secretion system, dampens acute inflammatory responses during bacterial invasion by suppressing the tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6)-mediated signalling pathway. OspI is a glutamine deamidase that selectively deamidates the glutamine residue at position 100 in UBC13 to a glutamic acid residue. Consequently, the E2 ubiquitin-conjugating activity required for TRAF6 activation is inhibited, allowing S. flexneri OspI to modulate the diacylglycerol-CBM (CARD-BCL10-MALT1) complex-TRAF6-nuclear-factor-κB signalling pathway. We determined the 2.0 Å crystal structure of OspI, which contains a putative cysteine-histidine-aspartic acid catalytic triad. A mutational analysis showed this catalytic triad to be essential for the deamidation of UBC13. Our results suggest that S. flexneri inhibits acute inflammatory responses in the initial stage of infection by targeting the UBC13-TRAF6 complex.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Shigella flexneri/enzimología , Shigella flexneri/inmunología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Amidohidrolasas/genética , Secuencia de Aminoácidos , Animales , Ácido Aspártico/metabolismo , Proteína 10 de la LLC-Linfoma de Células B , Biocatálisis , Caspasas/metabolismo , Dominio Catalítico/genética , Cristalografía por Rayos X , Cisteína/metabolismo , Análisis Mutacional de ADN , Diglicéridos/antagonistas & inhibidores , Diglicéridos/metabolismo , Disentería Bacilar/microbiología , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Células HEK293 , Células HeLa , Histidina/metabolismo , Humanos , Inmunidad Innata , Inflamación/enzimología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Shigella flexneri/genética , Shigella flexneri/patogenicidad , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética , Factores de Virulencia/metabolismoRESUMEN
BACKGROUND: HIV-1 infected patients frequently have osteolytic bone disease, which is caused by the dysregulation of the bone remodeling system that involves the interaction between osteoblasts and osteoclasts, but the relationship between osteolytic disease and HIV-1 infection remains unclear. In this study we tested whether HIV-1 infection of osteoclasts affects their differentiation. RESULTS: We prepared human osteoclasts from CD14+ monocytes and examined them for their susceptibility to HIV-1. Furthermore, we investigated the effect of HIV-1 infection on osteoclast differentiation. CD14-derived osteoclasts were shown to express CD4, CCR5, and CXCR4 each at the similar level to that shown with macrophages. R5-tropic HIV-1 and X4-tropic HIV-1 were found to infect CD14-derived osteoclasts and replicate in them. Furthermore, HIV-1 infection induced formation of larger osteoclastst, enhanced the expression of mRNAs for three osteoclast specific marker molecules (tartrate-resistant acid phosphatase, cathepsin K, and the calcitonin receptor), and up-regulated osteoclast bone resorption activity. CONCLUSIONS: Our results suggest that osteoclasts serve as a novel target for HIV-1 infection, which may enhance the osteoclast differentiation contributing to the development of osteolytic disease in HIV-1-infected patients.
Asunto(s)
Diferenciación Celular , VIH-1/fisiología , Osteoclastos/fisiología , Osteoclastos/virología , Replicación Viral , Antígenos CD4/análisis , Células Cultivadas , Humanos , Receptores de Lipopolisacáridos/análisis , Osteoclastos/química , Receptores CCR5/análisis , Receptores CXCR4/análisisRESUMEN
Receptor activator of nuclear factor κB (RANK) is a member of the tumor necrosis factor receptor superfamily (TNFRSF) and triggers osteoclastogenesis by inducing the expression of NFATc1 through the activation of the NF-κB and MAPK pathways. Cellular inhibitors of apoptosis proteins 1 and 2 (cIAP1/2), which are ubiquitin E3 ligases, are involved in the activation of the NF-κB and MAPK pathways by various members of the TNFRSF. However, the involvement of cIAP1/2 in RANK signaling has remained largely unknown. In this study, we reveal the involvement of cIAP1/2 in RANK ligand (RANKL)-induced osteoclastogenesis. The over-expression of cIAP1 or cIAP2 in the mouse monocytic cell line Raw264.7 resulted in the significant suppression of RANKL-induced NFATc1 mRNA expression and osteoclastogenesis, whereas the activation of the NF-κB and MAPK pathways was barely changed by these over-expressions. The depletion of endogenous cIAP1/2 by their specific inhibitor MV1 or their siRNA-mediated knockdown resulted in enhanced RANKL-induced NFATc1 expression and osteoclastogenesis without affecting the activation of the NF-κB and MAPK pathways. In combination, these results indicate that cIAP1/2 negatively regulate osteoclastogenesis by inhibiting NFATc1 mRNA expression in a manner that is distinct from the previously identified functions of cIAP1/2.
Asunto(s)
Diferenciación Celular , Proteínas Inhibidoras de la Apoptosis/metabolismo , Factores de Transcripción NFATC/metabolismo , Osteoclastos/metabolismo , Ligando RANK/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos C57BL , Monocitos/citología , Monocitos/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción NFATC/genética , Osteoclastos/citología , ARN Mensajero/biosíntesis , ARN Interferente PequeñoRESUMEN
N-(4-hydroxyphenyl)-retinamide (4-HPR) inhibits the dihydroceramide Δ4-desaturase 1 (DEGS1) enzymatic activity. We previously reported that 4-HPR suppresses the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) spike protein-mediated membrane fusion through a decrease in membrane fluidity in a DEGS1-independent manner. However, the precise mechanism underlying the inhibition of viral entry by 4-HPR remains unclear. In this study, we examined the role of reactive oxygen species (ROS) in the inhibition of membrane fusion by 4-HPR because 4-HPR is a well-known ROS-inducing agent. Intracellular ROS generation was found to be increased in the target cells in a cell-cell fusion assay after 4-HPR treatment, which was attenuated by the addition of the antioxidant, α-tocopherol (TCP). The reduction in membrane fusion susceptibility by 4-HPR treatment in the cell-cell fusion assay was alleviated by TCP addition. Furthermore, fluorescence recovery after photobleaching analysis showed that the lateral diffusion of glycosylphosphatidylinositol-anchored protein and SARS CoV-2 receptor was reduced by 4-HPR treatment and restored by TCP addition. These results indicate that the decrease in SARS-CoV-2 spike protein-mediated membrane fusion and membrane fluidity by 4-HPR was due to ROS generation. Taken together, these results demonstrate that ROS production is associated with the 4-HPR inhibitory effect on SARS-CoV-2 entry.
Asunto(s)
Antineoplásicos , COVID-19 , Fenretinida , Humanos , Fenretinida/farmacología , Especies Reactivas de Oxígeno/metabolismo , Antineoplásicos/farmacología , SARS-CoV-2/metabolismo , Apoptosis , OxidorreductasasRESUMEN
Coronaviruses, including SARS-CoV-2, SARS-CoV, MERS-CoV and influenza A virus, require the host proteases to mediate viral entry into cells. Rather than targeting the continuously mutating viral proteins, targeting the conserved host-based entry mechanism could offer advantages. Nafamostat and camostat were discovered as covalent inhibitors of TMPRSS2 protease involved in viral entry. To circumvent their limitations, a reversible inhibitor might be required. Considering nafamostat structure and using pentamidine as a starting point, a small set of structurally diverse rigid analogues were designed and evaluated in silico to guide selection of compounds to be prepared for biological evaluation. Based on the results of in silico study, six compounds were prepared and evaluated in vitro. At the enzyme level, compounds 10-12 triggered potential TMPRSS2 inhibition with low micromolar IC50 concentrations, but they were less effective in cellular assays. Meanwhile, compound 14 did not trigger potential TMPRSS2 inhibition at the enzyme level, but it showed potential cellular activity regarding inhibition of membrane fusion with a low micromolar IC50 value of 10.87 µM, suggesting its action could be mediated by another molecular target. Furthermore, in vitro evaluation showed that compound 14 inhibited pseudovirus entry as well as thrombin and factor Xa. Together, this study presents compound 14 as a hit compound that might serve as a starting point for developing potential viral entry inhibitors with possible application against coronaviruses.
Asunto(s)
COVID-19 , Coronavirus del Síndrome Respiratorio de Oriente Medio , Humanos , SARS-CoV-2 , Benzamidinas/farmacología , Internalización del Virus , Antivirales/farmacología , Antivirales/químicaRESUMEN
Interferon regulatory factors (IRFs) are key elements of antiviral innate responses that regulate the transcription of interferons (IFNs) and IFN-stimulated genes (ISGs). While the sensitivity of human coronaviruses to IFNs has been characterized, antiviral roles of IRFs during human coronavirus infection are not fully understood. Type I or II IFN treatment protected MRC5 cells from human coronavirus 229E infection, but not OC43. Cells infected with 229E or OC43 upregulated ISGs, indicating that antiviral transcription is not suppressed. Antiviral IRFs, IRF1, IRF3 and IRF7, were activated in cells infected with 229E, OC43 or severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2). RNAi knockdown and overexpression of IRFs demonstrated that IRF1 and IRF3 have antiviral properties against OC43, while IRF3 and IRF7 are effective in restricting 229E infection. IRF3 activation effectively promotes transcription of antiviral genes during OC43 or 229E infection. Our study suggests that IRFs may be effective antiviral regulators against human coronavirus infection.
Asunto(s)
COVID-19 , Coronavirus Humano 229E , Humanos , Factor 3 Regulador del Interferón , SARS-CoV-2/metabolismo , Interferones/metabolismo , Antivirales/farmacología , Factores Reguladores del InterferónRESUMEN
Pathological bone resorption by osteoclasts is primarily treated with bisphosphonates. Because the administration of bisphosphonates is associated with a risk for multiple adverse symptoms, a precise understanding of the mechanisms underlying osteoclastogenesis is required to develop drugs with minimal side-effects. Osteoclastogenesis depends on receptor activator of nuclear factor kappa B (RANK) signaling mediated by TRAF6. We previously identified a highly conserved domain in the cytoplasmic tail of RANK (HCR), which did not share any significant homology with other proteins and was essential for osteoclastogenesis. HCR acts as a platform for the formation of Gab2- and Vav3-containing signal complexes, and ectopic expression of the HCR peptide inhibits osteoclastogenesis. Here, we uncover the mechanisms of HCR peptide-mediated inhibition of osteoclastogenesis. Expression of either the amino- or carboxyl-terminal half of the HCR peptide (N- or C-peptide) independently inhibited RANK signaling prior to cell-cell fusion. In contrast, expression of the GY-peptide, which is a part of the C-peptide, did not significantly affect prefusion RANK signaling, but did inhibit cell-cell fusion to prevent formation of multinucleated mature osteoclasts. Moreover, Gab2, which is involved in RANK signaling by binding TRAF6, bound the C-peptide but not the N-peptide, suggesting that the C- and the N-peptides sequester TRAF6 in a Gab2-dependent and Gab2-independent manner, respectively. In contrast, the GY-peptide did not bind Gab2 but could bind Vav3, which mediates signaling for cell-cell fusion. Collectively, we propose that the HCR peptide inhibits osteoclastogenesis through two modes of action-inhibition of (1) prefusion RANK signaling and (2) cell-cell fusion by blocking TRAF6- and Vav3-mediated signaling, respectively.
Asunto(s)
Citoplasma/metabolismo , Osteoclastos/metabolismo , Péptidos/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Células de la Médula Ósea/metabolismo , Péptido C/genética , Péptido C/metabolismo , Fusión Celular , Citoplasma/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Péptidos/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas Proto-Oncogénicas c-vav/metabolismo , Receptor Activador del Factor Nuclear kappa-B/genética , Transducción de Señal/genética , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismoRESUMEN
The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently, virus variants that can evade the immunity in a host achieved through vaccination have emerged. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread, are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner and is TMPRSS2 independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific small interfering RNAS (siRNAs) revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytium formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosomal pathways could be an effective strategy by which to cure COVID-19 in the future. IMPORTANCE To develop effective therapeutics against COVID-19, it is necessary to elucidate in detail the infection mechanism of the causative agent, SARS-CoV-2. SARS-CoV-2 binds to the cell surface receptor ACE2 via the spike protein, and then the spike protein is cleaved by host proteases to enable entry. Here, we found that the metalloproteinase-mediated pathway is important for SARS-CoV-2 infection in addition to the TMPRSS2-mediated pathway and the endosomal pathway. The metalloproteinase-mediated pathway requires both the prior cleavage of spike into two domains and a specific sequence in the second domain, S2, conditions met by SARS-CoV-2 but lacking in the related human coronavirus SARS-CoV. Besides the contribution of metalloproteinases to SARS-CoV-2 infection, inhibition of metalloproteinases was important in preventing cell death, which may cause organ damage. Our study provides new insights into the complex pathogenesis unique to COVID-19 and relevant to the development of effective therapies.
Asunto(s)
COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Internalización del Virus , Humanos , Metaloproteasas/genética , SARS-CoV-2/metabolismo , Serina Endopeptidasas/genética , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Various chimeric receptors have been developed and used for biological experiments. In the present study, we constructed three types of chimeric receptor activator of nuclear factor-kappa B (RANK) with the glutathione S-transferase (GST) protein in the extracellular domain, and stimulated them using newly synthesized chemical trimerizers with three glutathiones. Although this stimulation did not activate these proteins, we unexpectedly found that the chimera named RANK-GST-SC, in which GST replaced a major part of the RANK extracellular domain, activated nuclear factor-kappa B (NF-κB) signaling approximately sixfold more strongly than wild-type RANK without the ligand. The dimerization of extracellular GST is considered to function as a switch outside the cell, and signal transduction then occurs. GST has been widely employed as a tag for protein purification; GST-fusion protein can be conveniently captured by glutathione-conjugated beads and easily purified from impurity. The present study is a pioneering example of the novel utility of GST and provides information for the development of new chemical biology systems.
Asunto(s)
FN-kappa B , Ligando RANK , Quimera/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , FN-kappa B/metabolismo , Osteoclastos/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismoRESUMEN
Although the importance of virus-specific cytotoxic T lymphocytes (CTL) in virus clearance is evident in COVID-19, the characteristics of virus-specific CTLs related to disease severity have not been fully explored. Here we show that the phenotype of virus-specific CTLs against immunoprevalent epitopes in COVID-19 convalescents might differ according to the course of the disease. We establish a cellular screening method that uses artificial antigen presenting cells, expressing HLA-A*24:02, the costimulatory molecule 4-1BBL, SARS-CoV-2 structural proteins S, M, and N and non-structural proteins ORF3a and nsp6/ORF1a. The screen implicates SARS-CoV-2 M protein as a frequent target of IFNγ secreting CD8+ T cells, and identifies M198-206 as an immunoprevalent epitope in our cohort of HLA-A*24:02 positive convalescent COVID-19 patients recovering from mild, moderate and severe disease. Further exploration of M198-206-specific CD8+ T cells with single cell RNA sequencing reveals public TCRs in virus-specific CD8+ T cells, and shows an exhausted phenotype with less differentiated status in cells from the severe group compared to cells from the moderate group. In summary, this study describes a method to identify T cell epitopes, indicate that dysfunction of virus-specific CTLs might be an important determinant of clinical outcomes.
Asunto(s)
Linfocitos T CD8-positivos , COVID-19 , Humanos , SARS-CoV-2 , Linfocitos T Citotóxicos , Epítopos de Linfocito T , Antígenos HLA-ARESUMEN
Nuclear factor-κB (NF-κB) is an inducible transcription factor activated by a variety of cytokines, and promotes the transcription of genes involved in cancer, inflammation, autoimmune disease, and viral infection, among others. Because of its involvement in numerous disease processes, considerable research has focused on NF-κB as a potential drug target. We previously reported that cupric ion (Cu(2+)) blocks NF-κB activation. However, Cu(2+) is unsuitable for drug applications. The copper complex of an artificial peptide HPH-Pep (HPH-Pep-Cu(2+)) was a promising alternative, but it did not easily cross the cell membrane. We report the development of a NF-κB inhibiting Cu(2+) complex with improved cell-penetrating activity arising from the coupling of a Tat peptide to HPH-Pep-Cu(2+).
Asunto(s)
Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacología , Cobre/química , Cobre/farmacología , Histamina/análogos & derivados , FN-kappa B/antagonistas & inhibidores , Piridinas/química , Piridinas/farmacología , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Células HeLa , Histamina/química , Histamina/farmacología , Humanos , FN-kappa B/inmunología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacologíaRESUMEN
Middle East respiratory syndrome coronavirus (MERS-CoV), capable of zoonotic transmission, has been associated with emerging viral pneumonia in humans. In this study, a set of highly potent peptides were designed to prevent MERS-CoV fusion through competition with heptad repeat domain 2 (HR2) at its HR1 binding site. We designed eleven peptides with stronger estimated HR1 binding affinities than the wild-type peptide to prevent viral fusion with the cell membrane. Eight peptides showed strong inhibition of spike-mediated MERS-CoV cell-cell fusion with IC50 values in the nanomolar range (0.25-2.3 µM). Peptides #4-6 inhibited 95-98.3% of MERS-CoV plaque formation. Notably, peptide four showed strong inhibition of MERS-CoV plaques formation with EC50 = 0.302 µM. All peptides demonstrated safe profiles without cytotoxicity up to a concentration of 10 µM, and this cellular safety, combined with their anti-MERS-CoV antiviral activity, indicate all peptides can be regarded as potential promising antiviral agents.
RESUMEN
A novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), caused a worldwide pandemic. Our aim in this study is to produce new fusion inhibitors against SARS-CoV-2, which can be the basis for developing new antiviral drugs. The fusion core comprising the heptad repeat domains (HR1 and HR2) of SARS-CoV-2 spike (S) were used to design the peptides. A total of twelve peptides were generated, comprising a short or truncated 24-mer (peptide #1), a long 36-mer peptide (peptide #2), and ten peptide #2 analogs. In contrast to SARS-CoV, SARS-CoV-2 S-mediated cell-cell fusion cannot be inhibited with a minimal length, 24-mer peptide. Peptide #2 demonstrated potent inhibition of SARS-CoV-2 S-mediated cell-cell fusion at 1 µM concentration. Three peptide #2 analogs showed IC50 values in the low micromolar range (4.7-9.8 µM). Peptide #2 inhibited the SARS-CoV-2 pseudovirus assay at IC50=1.49 µM. Given their potent inhibition of viral activity and safety and lack of cytotoxicity, these peptides provide an attractive avenue for the development of new prophylactic and therapeutic agents against SARS-CoV-2.
RESUMEN
TRAF6 is essential for osteoclastogenesis and for both RANK- and CD40-mediated activation of IKK and MAPKs. RANK, but not CD40, can promote osteoclastogenesis because only RANK induces NFATc1 activation through PLCgamma2-induced Ca(2+) oscillations together with the co-stimulatory signals emanating from immune receptors linked to ITAM-containing adaptors. These previous data suggest that RANK harbors a unique domain that functions in concert with the TRAF6-binding site in osteoclastogenesis. Here we identify such a domain, highly conserved domain in RANK (HCR), which is dispensable for the early phase of RANK and ITAM signaling but is essential for their late-phase signaling, including sustained activation of NF-kappaB and PLCgamma2 leading to NFATc1 activation. HCR recruits an adaptor protein, Gab2, which further associates with PLCgamma2 in the late phase. Formation of the HCR-mediated signaling complex could account for the sustained activation of NF-kappaB and PLCgamma2. The present study identifies HCR as a unique domain that plays a critical role in the long-term linkage between RANK and ITAM signals, providing a molecular basis for therapeutic strategies.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Diferenciación Celular , Osteoclastos/citología , Fosfolipasa C gamma/metabolismo , Receptor Activador del Factor Nuclear kappa-B/genética , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Antígenos CD40/metabolismo , Secuencia Conservada , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Datos de Secuencia Molecular , Factores de Transcripción NFATC/metabolismo , Estructura Terciaria de Proteína , Alineación de Secuencia , Activación TranscripcionalRESUMEN
Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 mM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19.