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1.
J Biomed Sci ; 30(1): 41, 2023 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-37316861

RESUMEN

BACKGROUND: Flavivirus causes many serious public health problems worldwide. However, licensed DENV vaccine has restrictions on its use, and there is currently no approved ZIKV vaccine. Development of a potent and safe flavivirus vaccine is urgently needed. As a previous study revealed the epitope, RCPTQGE, located on the bc loop in the E protein domain II of DENV, in this study, we rationally designed and synthesized a series of peptides based on the sequence of JEV epitope RCPTTGE and DENV/ZIKV epitope RCPTQGE. METHODS: Immune sera were generated by immunization with the peptides which were synthesized by using five copies of RCPTTGE or RCPTQGE and named as JEV-NTE and DV/ZV-NTE. Immunogenicity and neutralizing abilities of JEV-NTE or DV/ZV-NTE-immune sera against flavivirus were evaluated by ELISA and neutralization tests, respectively. Protective efficacy in vivo were determined by passive transfer the immune sera into JEV-infected ICR or DENV- and ZIKV-challenged AG129 mice. In vitro and in vivo ADE assays were used to examine whether JEV-NTE or DV/ZV-NTE-immune sera would induce ADE. RESULTS: Passive immunization with JEV-NTE-immunized sera or DV/ZV-NTE-immunized sera could increase the survival rate or prolong the survival time in JEV-challenged ICR mice and reduce the viremia levels significantly in DENV- or ZIKV-infected AG129 mice. Furthermore, neither JEV -NTE- nor DV/ZV-NTE-immune sera induced antibody-dependent enhancement (ADE) as compared with the control mAb 4G2 both in vitro and in vivo. CONCLUSIONS: We showed for the first time that novel bc loop epitope RCPTQGE located on the amino acids 73 to 79 of DENV/ZIKV E protein could elicit cross-neutralizing antibodies and reduced the viremia level in DENV- and ZIKV-challenged AG129 mice. Our results highlighted that the bc loop epitope could be a promising target for flavivirus vaccine development.


Asunto(s)
Infección por el Virus Zika , Virus Zika , Animales , Ratones , Ratones Endogámicos ICR , Anticuerpos Neutralizantes , Viremia , Sueros Inmunes , Epítopos , Factores de Transcripción
2.
J Virol ; 96(1): e0166521, 2022 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-34643435

RESUMEN

Zinc-finger protein 36, CCCH type-like 1 (ZFP36L1), containing tandem CCCH-type zinc-finger motifs with an RNA-binding property, plays an important role in cellular RNA metabolism mainly by RNA decay pathways. Recently, we demonstrated that human ZFP36L1 has potent antiviral activity against influenza A virus infection. However, its role in the host defense response against flaviviruses has not been addressed. Here, we demonstrate that ZFP36L1 functions as a host innate defender against flaviviruses, including Japanese encephalitis virus (JEV) and dengue virus (DENV). Overexpression of ZFP36L1 reduced JEV and DENV infection, and ZFP36L1 knockdown enhanced viral replication. ZFP36L1 destabilized the JEV genome by targeting and degrading viral RNA mediated by both 5'-3' XRN1 and 3'-5' RNA-exosome RNA decay pathways. Mutation in both zinc-finger motifs of ZFP36L1 disrupted RNA-binding and antiviral activity. Furthermore, the viral RNA sequences specifically recognized by ZFP36L1 were mapped to the 3'-untranslated region of the JEV genome with the AU-rich element (AUUUA) motif. We extend the function of ZFP36L1 to host antiviral defense by directly binding and destabilizing the viral genome via recruiting cellular mRNA decay machineries. IMPORTANCE Cellular RNA-binding proteins are among the first lines of defense against various viruses, particularly RNA viruses. ZFP36L1 belongs to the CCCH-type zinc-finger protein family and has RNA-binding activity; it has been reported to bind directly to the AU-rich elements (AREs) of a subset of cellular mRNAs and then lead to mRNA decay by recruiting mRNA-degrading enzymes. However, the antiviral potential of ZFP36L1 against flaviviruses has not yet been fully demonstrated. Here, we reveal the antiviral potential of human ZFP36L1 against Japanese encephalitis virus (JEV) and dengue virus (DENV). ZFP36L1 specifically targeted the ARE motif within viral RNA and triggered the degradation of viral RNA transcripts via cellular degrading enzymes 5'-3' XRN1 and 3'-5' RNA exosome. These findings provide mechanistic insights into how human ZFP36L1 serves as a host antiviral factor to restrict flavivirus replication.


Asunto(s)
Factor 1 de Respuesta al Butirato/metabolismo , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Infecciones por Flavivirus/metabolismo , Infecciones por Flavivirus/virología , Flavivirus/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Estabilidad del ARN , Replicación Viral , Regiones no Traducidas 3' , Secuencias de Aminoácidos , Factor 1 de Respuesta al Butirato/química , Virus del Dengue/fisiología , Virus de la Encefalitis Japonesa (Especie)/fisiología , Interacciones Huésped-Patógeno , Humanos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN Viral/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN
3.
Nucleic Acids Res ; 48(13): 7371-7384, 2020 07 27.
Artículo en Inglés | MEDLINE | ID: mdl-32556261

RESUMEN

ZFP36L1, a CCCH-type zinc finger protein, is an RNA-binding protein that participates in controlling cellular mRNA abundance and turnover by posttranscriptional regulation. Here, we demonstrated that ZFP36L1 has an important role in host defense against influenza A virus (IAV) infection. Overexpression of ZFP36L1 reduced IAV replication via translational repression of HA, M and NS RNA segment transcripts. IAV infection upregulated cellular ZFP36L1 expression, and endogenous ZFP36L1 knockdown significantly enhanced IAV replication. ZFP36L1 directly binds to IAV NS1 mRNA in the cytoplasm and blocks the expression and function of NS1 protein. Mutation of CCCH-type zinc finger domains of ZFP36L1 lost its antiviral potential and NS1 mRNA binding. Thus, ZFP36L1 can act as a host innate defense by targeting HA, M and NS mRNA transcripts to suppress viral protein translation.


Asunto(s)
Factor 1 de Respuesta al Butirato/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas no Estructurales Virales/genética , Células A549 , Animales , Sitios de Unión , Factor 1 de Respuesta al Butirato/química , Factor 1 de Respuesta al Butirato/genética , Perros , Células HEK293 , Humanos , Virus de la Influenza A/metabolismo , Virus de la Influenza A/fisiología , Células de Riñón Canino Madin Darby , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de la Matriz Viral/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación Viral
4.
Sci Rep ; 9(1): 17457, 2019 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-31745208

RESUMEN

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

5.
Artículo en Inglés | MEDLINE | ID: mdl-31636070

RESUMEN

Flaviviruses comprise several medically important viruses, including Japanese encephalitis virus, West Nile virus, dengue virus (DENV), yellow fever virus, and Zika virus (ZIKV). A large outbreak of DENV and ZIKV occurred recently, leading to many cases of illness and death. However, despite decades of effort, we have no clinically specific therapeutic drugs against DENV and ZIKV. Previous studies showed that inflammatory responses play a critical role in dengue and Zika virus pathogenesis. Thus, in this study, we examined a series of novel anti-inflammatory compounds and found that treatment with compound 2d could dose dependently reduce viral protein expression and viral progeny production in HEK-293 and Raw264.7 cells infected with four serotypes of DENV and ZIKV. In addition, considering medication safety, compound 2d could not suppress cyclooxygenase-1 (COX-1) enzymatic activities and thus could prevent the side effect of bleeding. Moreover, compound 2d significantly inhibited COX-2 enzymatic activities and prostaglandin E2 levels, associated with viral replication, compared to results with a selective COX-2 inhibitor, celecoxib. Furthermore, administering 5 mg/kg compound 2d to DENV-2-infected AG129 mice prolonged survival and reduced viremia and serum cytokine levels. Overall, compound 2d showed therapeutic safety and efficacy in vitro and in vivo and could be further developed as a potential therapeutic agent for flavivirus infection.


Asunto(s)
Antiinflamatorios/farmacología , Dengue/tratamiento farmacológico , Infección por el Virus Zika/tratamiento farmacológico , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/química , Antivirales/administración & dosificación , Antivirales/química , Antivirales/farmacología , Celecoxib/farmacología , Ciclooxigenasa 1/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Dengue/enzimología , Dengue/virología , Virus del Dengue/clasificación , Virus del Dengue/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Ratones , Ratones de la Cepa 129 , Células RAW 264.7 , Seguridad , Serogrupo , Resultado del Tratamiento , Replicación Viral/efectos de los fármacos , Virus Zika/efectos de los fármacos , Infección por el Virus Zika/enzimología , Infección por el Virus Zika/virología
6.
J Biomed Sci ; 26(1): 55, 2019 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-31366399

RESUMEN

BACKGROUND: Mutations in the PB1 subunit of RNA-dependent RNA polymerase (RdRp) of influenza A virus can affect replication fidelity. Before the influenza A/H1N1 pandemic in 2009, most human influenza A/H1N1 viruses contained the avian-associated residue, serine, at position 216 in PB1. However, near the onset of the 2009 pandemic, human viruses began to acquire the mammalian-associated residue, glycine, at PB1-216, and PB1-216G became predominant in human viruses thereafter. METHODS: Using entropy-based analysis algorithm, we have previously identified several host-specific amino-acid signatures that separated avian and swine viruses from human influenza viruses. The presence of these host-specific signatures in human influenza A/H1N1 viruses suggested that these mutations were the result of adaptive genetic evolution that enabled these influenza viruses to circumvent host barriers, which resulted in cross-species transmission. We investigated the biological impact of this natural avian-to-mammalian signature substitution at PB1-216 in human influenza A/H1N1 viruses. RESULTS: We found that PB1-216G viruses had greater mutation potential, and were more sensitive to ribavirin than PB1-216S viruses. In oseltamivir-treated HEK293 cells, PB1-216G viruses generated mutations in viral neuraminidase at a higher rate than PB1-216S viruses. By contrast, PB1-216S viruses were more virulent in mice than PB1-216G viruses. These results suggest that the PB1-S216G substitution enhances viral epidemiological fitness by increasing the frequency of adaptive mutations in human influenza A/H1N1 viruses. CONCLUSIONS: Our results thus suggest that the increased adaptability and epidemiological fitness of naturally arising human PB1-216G viruses, which have a canonical low-fidelity replicase, were the biological mechanisms underlying the replacement of PB1-216S viruses with a high-fidelity replicase following the emergence of pdmH1N1. We think that continued surveillance of such naturally occurring PB1-216 variants among others is warranted to assess the potential impact of changes in RdRp fidelity on the adaptability and epidemiological fitness of human A/H1N1 influenza viruses.


Asunto(s)
Virus de la Influenza A/fisiología , Proteínas Virales/genética , Replicación Viral/genética , Adaptación Fisiológica/genética , Animales , Perros , Células HEK293 , Humanos , Virus de la Influenza A/genética , Células de Riñón Canino Madin Darby , Mutación/genética , Proteínas Virales/metabolismo , Virulencia/genética
7.
PLoS Pathog ; 14(7): e1007166, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-30016363

RESUMEN

CCCH-type zinc-finger antiviral protein (ZAP) is a host factor that restricts the infection of many viruses mainly through RNA degradation, translation inhibition and innate immune responses. So far, only one flavivirus, yellow fever virus, has been reported to be ZAP-resistant. Here, we investigated the antiviral potential of human ZAP (isoform ZAP-L and ZAP-S) against three flaviviruses, Japanese encephalitis virus (JEV), dengue virus (DENV) and Zika virus (ZIKV). Infection of JEV but not DENV or ZIKV was blocked by ZAP overexpression, and depletion of endogenous ZAP enhanced JEV replication. ZAP hampered JEV translation and targeted viral RNA for 3'-5' RNA exosome-mediated degradation. The zinc-finger motifs of ZAP were essential for RNA targeting and anti-JEV activity. JEV 3'-UTR, especially in the region with dumbbell structures and high content of CG dinucleotide, was mapped to bind ZAP and confer sensitivity to ZAP. In summary, we identified JEV as the first ZAP-sensitive flavivirus. ZAP may act as an intrinsic antiviral factor through specific RNA binding to fight against JEV infection.


Asunto(s)
Virus de la Encefalitis Japonesa (Especie)/inmunología , Encefalitis Japonesa/inmunología , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata/inmunología , Proteínas de Unión al ARN/inmunología , Humanos
8.
Int J Mol Sci ; 19(5)2018 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-29751537

RESUMEN

Monocyte chemotactic protein induced protein 3 (MCPIP3) belongs to the Cys⁻Cys⁻Cys⁻His (CCCH)-zinc finger protein family and contains a highly conserved CCCH-zinc finger domain and a Nedd4-BP1 YacP nuclease (NYN) domain. Previous studies showed that MCPIP3 inhibits the expression of proinflammatory genes, such as vascular cell adhesion molecule (VCAM)-1, in human endothelial cells, but the roles and functions of MCPIP3 in cancer cells are still unknown. In human colorectal cancer specimens, we found that the messenger RNA expression of MCPIP3 was significantly downregulated in cancer tissues compared to adjacent normal tissues (18/25; average fold change of 8.18). Two cell models were used to demonstrate the anti-migration activity of MCPIP3. First, Tet-on T-REx-293/HA-MCPIP3 cells were used to examine whether MCPIP3 can change epithelial⁻mesenchymal transition (EMT)-related gene expressions. Second, we used two human colorectal cancer cell lines, SW620 and HCT116, to prove the role of MCPIP3 in regulating EMT-related gene expressions. We found that overexpression of MCPIP3 inhibited cell migration according to a wound-healing assay and Transwell invasion assay and vimentin expression, and increased E-cadherin expression in these two cell lines. These results suggest that MCPIP3 might play a negative role in cell migration of human colorectal cancer cells.


Asunto(s)
Neoplasias Colorrectales/metabolismo , Ribonucleasas/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Movimiento Celular/genética , Movimiento Celular/fisiología , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Células HCT116 , Humanos , Ribonucleasas/genética , Vimentina/genética , Vimentina/metabolismo , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiología
9.
Sci Rep ; 8(1): 2742, 2018 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-29426877

RESUMEN

ZFP36 family members include ZFP36, ZFP36L1, and ZFP36L2, which belong to CCCH-type zinc finger proteins with two tandem zinc finger (TZF) regions. Whether ZFP36L1 and ZFP36L2 have antiproliferative activities similar to that of ZFP36 is unclear. In this study, when ZFP36L1 or ZFP36L2 was overexpressed in T-REx-293 cells, cell proliferation was dramatically inhibited and the cell cycle was arrested at the G1 phase. The levels of cell-cycle-related proteins, including cyclin B, cyclin D, cyclin A, and p21, decreased; however, p53 increased in ZFP36L1-or ZFP36L2-overexpressing T-REx-293 cells. Forced expression of ZFP36L1 or ZFP36L2 also inhibited cell proliferation and cyclin D gene expression in three human colorectal cancer cell lines: HCT116 p53+/+, HCT116 p53-/-, and SW620 (mutated p53) cells. However, it increased p53 and p21 expression only in HCT116 p53+/+ cells. Knockdown of ZFP36L1 or ZFP36L2 increased cell proliferation and cyclin D expression; furthermore, the mutation of the TZF of ZFP36L1 or ZFP36L2 caused them to lose their antiproliferative ability, to the extent that they could not inhibit cyclin D expression in these three cell lines. The results indicated that ZFP36L1 and ZFP36L2 play a negative role in cell proliferation; the underlying mechanisms might be mediated through a cyclin D-dependent and p53-independent pathway.


Asunto(s)
Factor 1 de Respuesta al Butirato/fisiología , Proliferación Celular , Ciclina D/metabolismo , Factores de Transcripción/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Ciclina A/metabolismo , Ciclina B/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Transducción de Señal
10.
Oncotarget ; 6(28): 25988-6001, 2015 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-26317903

RESUMEN

Bortezomib (Velcal) was the first proteasome inhibitor to be approved by the US Food and Drug Administration to treat patients with relapsed/refractory multiple myelomas. Previous studies have demonstrated that bortezomib inhibits tumor cell proliferation and induces apoptosis by blocking the nuclear factor (NF)-κB pathway. However, the exact mechanism by which bortezomib induces cancer cell apoptosis is still not well understood. In this study, we found that bortezomib significantly inhibited cell proliferation in both human Burkitt's lymphoma CA46 and Daudi cells. Through proteomic analysis, we found that bortezomib treatment changed the expression of various proteins in distinct functional categories including unfolding protein response (UPS), RNA processing, protein targeting and biosynthesis, apoptosis, and signal transduction. Among the proteins with altered expression, hnRNP K, hnRNP H, Hsp90α, Grp78, and Hsp7C were common to both Daudi and CA46 cells. Interestingly, bortezomib treatment downregulated the expression of high-molecular-weight (HMw) hnRNP K and c-Myc but upregulated the expression of low-molecular-weight (LMw) hnRNP K. Moreover, cell proliferation was significantly correlated with high expression of HMw hnRNP K and c-Myc. HMw and LMw hnRNP K were identified as sumoylated and desumoylated hnRNP K, respectively. Using transient transfection, we found that sumoylated hnRNP K increased c-Myc expression at the translational level and contributed to cell proliferation, and that Lys422 of hnRNP K is the candidate sumoylated residue. Our results suggest that besides inhibiting the ubiquitin-proteasome pathway, bortezomib may inhibit cell proliferation by downregulating sumoylated hnRNP K and c-Myc expression in Burkitt's lymphoma cells.


Asunto(s)
Bortezomib/farmacología , Proliferación Celular/efectos de los fármacos , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Antineoplásicos/farmacología , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Línea Celular Tumoral , Proliferación Celular/genética , Electroforesis en Gel Bidimensional , Chaperón BiP del Retículo Endoplásmico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Humanos , Immunoblotting , Lisina/genética , Lisina/metabolismo , Mutación , Proteómica/métodos , Proteínas Proto-Oncogénicas c-myc/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Sumoilación
11.
J Infect Dis ; 212(12): 2011-20, 2015 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-26063222

RESUMEN

Dengue is a mosquito-borne viral disease that afflicts millions of individuals worldwide every year. Infection by any of the 4 dengue virus (DENV) serotypes can result in a spectrum of disease severity. We investigated the impact of variants of interferon-regulated innate immunity genes with a potent antiviral effect on the outcome of DENV infection. We compared the effect of OAS gene family variants on 2 DENV serotypes in cell culture. While both OAS1-p42 and p46 showed antiviral activity against DENV-2, only OAS1-p42 presented anti-DENV-1 activity. Conversely, whereas both OAS3_S381 and R381 variants were able to block DENV-1 infection, the anti-DENV-2 activity observed for OAS3_S381 was largely lost for the R381 variant. By means of an allelic association study of a cohort of 740 patients with dengue, we found a protective effect of OAS3_R381 against shock (odds ratio [OR], 0.37; P < .001). This effect was due to DENV-2 infections (OR, 0.13; P = .007) but was absent for DENV-1, in accordance with the serotype-dependent OAS3 activity found in the functional study. Severe dengue has long been associated with a cytokine storm of unclear origin. This work identifies an early innate immunity process that could lead to the immune overreaction observed in severe dengue and could be triggered by a specific host genotype-pathogen genotype interaction.


Asunto(s)
2',5'-Oligoadenilato Sintetasa/genética , Virus del Dengue/inmunología , Dengue/patología , Predisposición Genética a la Enfermedad , 2',5'-Oligoadenilato Sintetasa/metabolismo , Adolescente , Adulto , Células Cultivadas , Niño , Preescolar , Dengue/genética , Dengue/inmunología , Femenino , Estudios de Asociación Genética , Humanos , Lactante , Masculino , Adulto Joven
12.
PLoS Pathog ; 11(3): e1004750, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25816318

RESUMEN

Infection with Japanese encephalitis virus (JEV) can induce the expression of pro-inflammatory cytokines and cause acute encephalitis in humans. ß-oxidation breaks down fatty acids for ATP production in mitochondria, and impaired ß-oxidation can induce pro-inflammatory cytokine expression. To address the role of fatty-acid ß-oxidation in JEV infection, we measured the oxygen consumption rate of mock- and JEV-infected cells cultured with or without long chain fatty acid (LCFA) palmitate. Cells with JEV infection showed impaired LCFA ß-oxidation and increased interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) expression. JEV nonstructural protein 5 (NS5) interacted with hydroxyacyl-CoA dehydrogenase α and ß subunits, two components of the mitochondrial trifunctional protein (MTP) involved in LCFA ß-oxidation, and NS5 proteins were detected in mitochondria and co-localized with MTP. LCFA ß-oxidation was impaired and higher cytokines were induced in cells overexpressing NS5 protein as compared with control cells. Deletion and mutation studies showed that the N-terminus of NS5 was involved in the MTP association, and a single point mutation of NS5 residue 19 from methionine to alanine (NS5-M19A) reduced its binding ability with MTP. The recombinant JEV with NS5-M19A mutation (JEV-NS5-M19A) was less able to block LCFA ß-oxidation and induced lower levels of IL-6 and TNF-α than wild-type JEV. Moreover, mice challenged with JEV-NS5-M19A showed less neurovirulence and neuroinvasiveness. We identified a novel function of JEV NS5 in viral pathogenesis by impairing LCFA ß-oxidation and inducing cytokine expression by association with MTP.


Asunto(s)
Virus de la Encefalitis Japonesa (Especie)/metabolismo , Encefalitis Japonesa/metabolismo , Ácidos Grasos/metabolismo , Subunidad alfa de la Proteína Trifuncional Mitocondrial/metabolismo , Subunidad beta de la Proteína Trifuncional Mitocondrial/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Virus de la Encefalitis Japonesa (Especie)/genética , Encefalitis Japonesa/genética , Ácidos Grasos/genética , Células HEK293 , Humanos , Ratones , Subunidad alfa de la Proteína Trifuncional Mitocondrial/genética , Subunidad beta de la Proteína Trifuncional Mitocondrial/genética , Oxidación-Reducción , Mutación Puntual , Proteínas no Estructurales Virales/genética
13.
Molecules ; 20(3): 4516-29, 2015 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-25768846

RESUMEN

Our previous studies found that osthole markedly reduced blood glucose levels in both db/db and ob/ob mice. To improve the antidiabetic activity of osthole, a series of N-hydroxycinnamide derivatives of osthole were synthesized, and their hypoglycemia activities were examined in vitro and in vivo. Both N-hydroxycinnamide derivatives of osthole, OHC-4p and OHC-2m, had the greatest potential for activating AMPK and increasing glucose uptake by L6 skeletal muscle cells. In addition, OHC-4p and OHC-2m time- and dose-dependently increased phosphorylation levels of AMPK and p38 MAPK. The AMPK inhibitor, compound C, and the p38 MAPK inhibitor, SB203580, significantly reversed activation of AMPK and p38 MAPK, respectively, in OHC-4p- and OHC-2m-treated cells. Compound C and SB203580 also inhibited glucose uptake induced by OHC-4p and OHC-2m. Next, we found that OHC-4p and OHC-2m significantly increased glucose transporter 4 (GLUT4) translocation to plasma membranes and counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that activation of AMPK and p38 MAPK by OHC-4p and OHC-2m is associated with increased glucose uptake and GLUT4 translocation and subsequently led to amelioration of hyperglycemia. Therefore, OHC-4p and OHC-2m might have potential as antidiabetic agents for treating type 2 diabetes. Our previous studies found that osthole markedly reduced blood glucose levels in both db/db and ob/ob mice. To improve the antidiabetic activity of osthole, a series of N-hydroxycinnamide derivatives of osthole were synthesized, and their hypoglycemia activities were examined in vitro and in vivo. Both N-hydroxycinnamide derivatives of osthole, OHC-4p and OHC-2m, had the greatest potential for activating AMPK and increasing glucose uptake by L6 skeletal muscle cells. In addition, OHC-4p and OHC-2m time- and dose-dependently increased phosphorylation levels of AMPK and p38 MAPK. The AMPK inhibitor, compound C, and the p38 MAPK inhibitor, SB203580, significantly reversed activation of AMPK and p38 MAPK, respectively, in OHC-4p- and OHC-2m-treated cells. Compound C and SB203580 also inhibited glucose uptake induced by OHC-4p and OHC-2m. Next, we found that OHC-4p and OHC-2m significantly increased glucose transporter 4 (GLUT4) translocation to plasma membranes and counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that activation of AMPK and p38 MAPK by OHC-4p and OHC-2m is associated with increased glucose uptake and GLUT4 translocation and subsequently led to amelioration of hyperglycemia. Therefore, OHC-4p and OHC-2m might have potential as antidiabetic agents for treating type 2 diabetes.


Asunto(s)
Ácidos Cumáricos/administración & dosificación , Ácidos Cumáricos/síntesis química , Cumarinas/administración & dosificación , Cumarinas/química , Cumarinas/síntesis química , Diabetes Mellitus Experimental/tratamiento farmacológico , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/síntesis química , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Ácidos Cumáricos/farmacología , Cumarinas/farmacología , Diabetes Mellitus Experimental/enzimología , Transportador de Glucosa de Tipo 4/metabolismo , Hipoglucemiantes/farmacología , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Ratas , Estreptozocina , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
14.
J Immunol ; 193(8): 4159-68, 2014 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-25225661

RESUMEN

Human MCP-1-induced protein 1 (MCPIP1, also known as ZC3H12A and Regnase-1) plays important roles in negatively regulating the cellular inflammatory response. Recently, we found that as an RNase, MCPIP1 has broad-spectrum antiviral effects by targeting viral RNA. In this study, we demonstrated that MCPIP1 expression was induced by hepatitis C virus (HCV) infection in Huh7.5 hepatoma cells. MCPIP1 expression was higher in liver tissue from patients with chronic HCV infection compared with those without chronic HCV infection. Knockdown of MCPIP1 increased HCV replication and HCV-mediated expression of proinflammatory cytokines, such as TNF-α, IL-6, and MCP-1. However, overexpression of MCPIP1 significantly inhibited HCV replication and HCV-mediated expression of proinflammatory cytokines. Various mutants of functional domains of MCPIP1 showed disruption of the RNA binding and oligomerization abilities, as well as RNase activity, but not deubiquitinase activity, which impaired the inhibitory activity against HCV replication. On immunocytochemistry, MCPIP1 colocalized with HCV RNA. Use of a replication-defective HCV John Cunningham 1/AAG mutant and in vitro RNA cleavage assay demonstrated that MCPIP1 could directly degrade HCV RNA. MCPIP1 may suppress HCV replication and HCV-mediated proinflammatory responses with infection, which might contribute to the regulation of host defense against the infection and virus-induced inflammation.


Asunto(s)
Hepacivirus/fisiología , Hepatitis C Crónica/inmunología , Factores de Transcripción/fisiología , Replicación Viral , Línea Celular Tumoral , Quimiocina CCL2/biosíntesis , Células HEK293 , Hepacivirus/genética , Hepatitis C Crónica/patología , Hepatitis C Crónica/virología , Humanos , Interleucina-6/biosíntesis , Hígado/inmunología , Hígado/patología , Hígado/virología , Mutación , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleasas , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/biosíntesis , Proteasas Ubiquitina-Específicas
15.
Chem Biol Interact ; 217: 1-8, 2014 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-24727557

RESUMEN

WJ1376-1 and WJ1398-1 are new synthetic compounds developed based on the structure of the Chinese herbal medicine osthole. Previously, we reported that WJ1376-1 and WJ1398-1 can induce cell-cycle arrest by activating ATR kinase (ataxia telangiectasia and rad3 related kinase) and inhibiting the phosphorylation of Aurora A kinase. In this study, we determined that WJ1376-1 and WJ1398-1 strongly inhibited the migration and invasion in human colorectal cancer cells at concentrations as low as 1µM. In the transforming growth factor (TGF)-ß-induced epithelial-mesenchymal transition model, WJ1376-1 and WJ1398-1 potently downregulated the transcription factor Snail1, the mesenchymal protein vimentin, and matrix metalloprotease-9, but upregulated the epithelial protein E-cadherin. WJ1376-1 and WJ1398-1 also inhibited the TGF-ß-induced phosphorylation of Smad2 and of Akt at Ser 473, and the nuclear translocation of Smad2 was substantially lower in WJ1376-1- and WJ1398-1-treated cells than it was in control cells. In transient transfection experiments, we observed that WJ1376-1 and WJ1398-1 strongly inhibited TGF-ß-stimulated activity of a Smad reporter. Finally, WJ1376-1 and WJ1398-1 blocked TGF-ß-induced phosphorylation of the TGF-ß Type I receptor (TGF-ßRI). These results suggest that WJ1376-1 and WJ1398-1 inhibit cell migration and invasion by suppressing TGF-ßRI phosphorylation and subsequently hindering both Smad2 and phosphatidylinositol 3-kinase/Akt signaling pathways.


Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Movimiento Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Cumarinas/farmacología , Ácidos Hidroxámicos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Smad2/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Células HCT116 , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , ARN/química , ARN/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Smad2/genética , Factor de Crecimiento Transformador beta/metabolismo
16.
J Virol ; 88(12): 6793-804, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24696471

RESUMEN

UNLABELLED: Positive-sense RNA viruses, such as dengue virus (DENV), hijack the intracellular membrane machinery for their own replication. The Rab18 protein, a member of the Rab GTPase family, key regulators of membrane trafficking, is located on the organelles involved in DENV infection, such as the endoplasmic reticulum (ER) and lipid droplets (LDs). In this study, we addressed the potential involvement of Rab18 in DENV infection by using cells overexpressing the wild-type, GTP-bound active form, or GDP-bound inactive form of Rab18 and cells with Rab18 knockdown. DENV replication, measured by viral protein, viral RNA, and viral progeny production, as well as LD induction, was reduced in cells with inactive Rab18 and in cells deprived of Rab18 expression, suggesting a positive role of Rab18 in the DENV life cycle. Interestingly, the interaction of fatty acid synthase (FASN), a key lipogenic enzyme in lipid biosynthesis, with DENV NS3 protein relied on the conversion of the GDP-bound to the GTP-bound form of Rab18. Furthermore, the targeting of FASN to sites participating in DENV infection, such as the ER and LDs, depends on functional Rab18. Thus, Rab18-mediated membrane trafficking of FASN and NS3 facilitates DENV replication, probably by ensuring a sufficient and coordinated lipid supply for membrane proliferation and arrangement. IMPORTANCE: Infection by dengue virus (DENV), an important mosquito-borne virus threatening ∼40% of the world's population, can cause mild dengue fever or severe dengue hemorrhagic fever and dengue shock syndrome. The pathogenesis mechanisms of DENV-related diseases are not clear, but high viral replication is believed to be a risk factor for the severe form of DENV infection. Thus, understanding the detailed mechanism of DENV replication might help address this devastating virus. Here, we found that Rab18, a small GTPase involved in vesicle trafficking and located in the endoplasmic reticulum network and on the surfaces of lipid droplets, positively regulates DENV replication. The functional machinery of Rab18 is required to recruit the enzyme fatty acid synthase to sites of DENV replication and to interact with DENV NS3 protein to promote fatty acid biosynthesis. Thus, DENV usurps Rab18 to facilitate its own replication.


Asunto(s)
Virus del Dengue/fisiología , Dengue/enzimología , Ácido Graso Sintasas/metabolismo , Replicación Viral , Proteínas de Unión al GTP rab/metabolismo , Dengue/metabolismo , Dengue/virología , Virus del Dengue/genética , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/virología , Ácido Graso Sintasas/genética , Humanos , Unión Proteica , Transporte de Proteínas , ARN Helicasas/genética , ARN Helicasas/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas de Unión al GTP rab/genética
17.
Chem Res Toxicol ; 26(11): 1683-91, 2013 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-24127835

RESUMEN

Osthole is extracted from the Chinese herbs Cnidium monnieri and Angelica pubescens, and it was found to have antitumor activity in vitro and in vivo. A series of osthole derivatives have been synthesized, and the N-hydroxycinnamide derivatives of osthole, WJ1376-1 and WJ1398-1 were found to have the greatest potential against human colon adenocarcinoma cells. In contrast to the parental osthole, both WJ1376-1 and WJ1398-1 were found to induce multinucleation and polyploidy by microscopic observation and flow cytometry. WJ1376-1 and WJ1398-1 significantly activated ataxia telangiectasia and rad3 related (ATR) kinase, which triggered activation of the checkpoint kinase 2 (Chk2) signaling pathway and then down regulated Cdc25 phosphatase and Cdc2/cyclin B kinase activities. WJ1376-1 and WJ1398-1 also inhibited the phosphorylation of Aurora A kinase, which is associated with important processes during mitosis. The presence of a "comet" DNA fragment and phosphorylation of p53 at Ser 15 clearly indicated that DNA damage occurred with WJ1376-1 and WJ1398-1 treatment. WJ1376-1 and WJ1398-1 ultimately induced apoptosis as evidenced by the upregulation of Bad and activation of caspases-3, -7, and -9. Furthermore, WJ1376-1 and WJ1398-1 also showed a great effect in attenuating tumor growth without affecting the body weight of xenograft nude mice. Taken together, these results suggest that the toxic activities of WJ1376-1 and WJ1398-1 were dissimilar to that of the parental osthole, which can induce cell polyploidy and G2/M cell cycle arrest in colon adenocarcinoma cells and may provide a potential therapeutic target for colon cancer treatment in the future.


Asunto(s)
Apoptosis/efectos de los fármacos , Cumarinas/toxicidad , Daño del ADN/efectos de los fármacos , Ácidos Hidroxámicos/toxicidad , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Cumarinas/química , Cumarinas/uso terapéutico , Regulación hacia Abajo/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/uso terapéutico , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos
18.
Anticancer Res ; 33(8): 3225-31, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23898083

RESUMEN

15,16-Dihydrotanshinone I (DHTS) is a component of the traditional Chinese medicinal plant Salvia miltiorrhiza Bunge. In this study, DHTS at as low as 2.5 µg/ml concentration significantly inhibited proliferation of human benign (SW480) and malignant (SW620) colorectal cancer cells, as shown by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide (MTT) and flow cytometric analysis. Activating transcription factor (ATF)-3, a basic leucine zipper-type transcription factor, was found to be predominantly up-regulated in DHTS-treated SW480 and SW620 cells. The up-regulation of ATF3 was blocked by a c-JUN N-terminal kinase (JNK) or p38 inhibitor. Overexpression of ATF3 resulted in a significant augmentation of DHTS-induced apoptosis of SW480 cells, but resistance to DHTS-induced apoptosis of SW620 cells. These results suggest that DHTS has a strong therapeutic or preventive potential against cancer. In addition, ATF3 has a dual role in DHTS-induced apoptosis, depending on the degree of malignancy of colorectal cancer.


Asunto(s)
Factor de Transcripción Activador 3/metabolismo , Apoptosis/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Fenantrenos/farmacología , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/enzimología , Ensayos de Selección de Medicamentos Antitumorales , Furanos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Metástasis de la Neoplasia , Fosforilación/efectos de los fármacos , Quinonas
19.
Nucleic Acids Res ; 41(5): 3314-26, 2013 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-23355615

RESUMEN

Monocyte chemoattractant protein 1-induced protein 1 (MCPIP1), belonging to the MCPIP family with highly conserved CCCH-type zinc finger and Nedd4-BP1, YacP Nuclease domains, has been implicated in negative regulation of the cellular inflammatory responses. In this report, we demonstrate for the first time that this RNA-binding nuclease also targets viral RNA and possesses potent antiviral activities. Overexpression of the human MCPIP1, but not MCPIP2, MCPIP3 or MCPIP4, inhibited Japanese encephalitis virus (JEV) and dengue virus (DEN) replication. The functional analysis of MCPIP1 revealed that the activities of RNase, RNA binding and oligomerization, but not deubiqutinase, are required for its antiviral potential. Furthermore, infection of other positive-sense RNA viruses, such as sindbis virus and encephalomyocarditis virus, and negative-sense RNA virus, such as influenza virus, as well as DNA virus, such as adenovirus, can also be blocked by MCPIP1. Moreover, the endogenous MCPIP1 gene expression was induced by JEV and DEN infection, and knockdown of MCPIP1 expression enhanced the replication of JEV and DEN in human cells. Thus, MCPIP1 can act as a host innate defense via RNase activity for targeting and degrading viral RNA.


Asunto(s)
Virus del Dengue/fisiología , Virus de la Encefalitis Japonesa (Especie)/fisiología , Estabilidad del ARN , ARN Viral/metabolismo , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Secuencia Conservada , Virus del Dengue/genética , Virus del Dengue/inmunología , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis Japonesa (Especie)/inmunología , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Ribonucleasas , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Replicación Viral , Dedos de Zinc
20.
Drug Chem Toxicol ; 36(3): 313-9, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23030068

RESUMEN

Vitis thunbergii var. taiwaniana (VTT) is an indigenous Taiwanese wild grape and is used as a folk medicine in Taiwan. VTT is rich in polyphenols, especially quercetin and resveratrol derivatives, which were demonstrated to exhibit inhibitory activities against carcinogenesis and prevent some neurodegenerative diseases. (-)-Vitisin B is one of the resveratrol tetramers extracted from VTT. In this study, we investigated the mechanisms of (-)-vitisin B on the induction of apoptosis in human HL-60 promyelocytic leukemia cells. First, (-)-vitisin B significantly inhibited cell proliferation through inducing cell apoptosis. This effect appeared to occur in a time- and dose-dependent manner. Cell-cycle distribution was also examined, and we found that (-)-vitisin B significantly induced a sub-G1 population in a dose-dependent manner. In addition, (-)-vitisin B exhibited stronger inhibitory effects on cell proliferation than resveratrol. Second, (-)-vitisin B dose dependently induced apoptosis-related protein expressions, such as the cleavage form of caspase-3, caspase-8, caspase-9, poly(ADP ribose) polymerase, and the proapoptotic Bax protein. Third, (-)-vitisin B treatment also resulted in increases in c-Jun N-terminal kinase (JNK) phosphorylation and Fas ligand (FasL) expression. Moreover, the (-)-vitisin B-induced FasL expression and caspase-3 activation could be reversed by a JNK inhibitor. These results suggest that (-)-vitisin B-induced apoptosis of leukemia cells might be mediated through activation of JNK and Fas death-signal transduction.


Asunto(s)
Antineoplásicos/farmacología , Benzofuranos/farmacología , Leucemia Promielocítica Aguda/tratamiento farmacológico , Fenoles/farmacología , Vitis/química , Antineoplásicos/análisis , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Benzofuranos/análisis , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/patología , Medicina Tradicional de Asia Oriental , Fenoles/análisis , Extractos Vegetales/análisis , Extractos Vegetales/farmacología , Taiwán
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