RESUMEN
Characterization of Human Respiratory Syncytial Virus (HRSV) protein interactions with host cell components is crucial to devise antiviral strategies. Viral nucleoprotein, phosphoprotein and matrix protein genes were optimized for human codon usage and cloned into expression vectors. HEK-293T cells were transfected with these vectors, viral proteins were immunoprecipitated, and co-immunoprecipitated cellular proteins were identified through mass spectrometry. Cell proteins identified with higher confidence scores were probed in the immunoprecipitation using specific antibodies. The results indicate that nucleoprotein interacts with arginine methyl-transferase, methylosome protein and Hsp70. Phosphoprotein interacts with Hsp70 and tropomysin, and matrix with tropomysin and nucleophosmin. Additionally, we performed immunoprecipitation of these cellular proteins in cells infected with HRSV, followed by detection of co-immunoprecipitated viral proteins. The results indicate that these interactions also occur in the context of viral infection, and their potential contribution for a HRSV replication model is discussed.
Asunto(s)
Nucleoproteínas/metabolismo , Fosfoproteínas/metabolismo , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/metabolismo , Proteínas de la Matriz Viral/metabolismo , Células HEK293 , Proteínas del Choque Térmico HSP72/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Nucleofosmina , Nucleoproteínas/genética , Fosfoproteínas/genética , Unión Proteica , Proteína-Arginina N-Metiltransferasas/metabolismo , Virus Sincitial Respiratorio Humano/genética , Proteínas de la Matriz Viral/genética , Proteínas Estructurales ViralesRESUMEN
Human Respiratory Syncytial Virus P protein plus the viral RNA, N and L viral proteins, constitute the viral replication complex. In this report we describe that HRSV P protein has putative intrinsically disordered domains predicted by in silico methods. These two domains, located at the amino and caboxi terminus, were identified by mass spectrometry analysis of peptides obtained from degradation fragments observed in purified P protein expressed in bacteria. The degradation is not occurring at the central oligomerization domain, since we also demonstrate that the purified fragments are able to oligomerize, similarly to the protein expressed in cells infected by HRSV. Disordered domains can play a role in protein interaction, and the present data contribute to the comprehension of HRSV P protein interactions in the viral replication complex.
Asunto(s)
Humanos , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Fragmentos de Péptidos/análisis , Técnicas In Vitro , ARN Viral , Replicación Viral , Virus Sincitial Respiratorio Humano/aislamiento & purificación , Técnicas y Procedimientos Diagnósticos , MétodosRESUMEN
Human Respiratory Syncytial Virus P protein plus the viral RNA, N and L viral proteins, constitute the viral replication complex. In this report we describe that HRSV P protein has putative intrinsically disordered domains predicted by in silico methods. These two domains, located at the amino and caboxi terminus, were identified by mass spectrometry analysis of peptides obtained from degradation fragments observed in purified P protein expressed in bacteria. The degradation is not occurring at the central oligomerization domain, since we also demonstrate that the purified fragments are able to oligomerize, similarly to the protein expressed in cells infected by HRSV. Disordered domains can play a role in protein interaction, and the present data contribute to the comprehension of HRSV P protein interactions in the viral replication complex.
RESUMEN
In this study, we have identified the E-NTPDase family members and ecto-5'-nucleotidase/CD73 in rat heart left ventricle. Moreover, we characterize the biochemical properties and enzyme activities from synaptosomes of the nerve terminal endings of heart left ventricle. We observe divalent cation-dependent enzymes that presented optimum pH of 8.0 for ATP and ADP hydrolysis, and 9.5 for AMP hydrolysis. The apparent K(M) values are 40 microM, 90 microM and 39 microM and apparent V(max) values are 537, 219 and 111 nmol Pi released/min/mg of protein for ATP, ADP and AMP hydrolysis, respectively. Ouabain, orthovanadate, NEM, lanthanum and levamisole do not affect ATP and ADP hydrolysis in rat cardiac synaptosomes. Oligomycin (2 microg/mL) and sodium azide (0.1 mM), both mitochondrial ATPase inhibitors, inhibit only the ATP hydrolysis. High concentrations of sodium azide and gadolinium chloride show an inhibition on both, ATP and ADP hydrolysis. Suramin inhibit more strongly ATP hydrolysis than ADP hydrolysis whereas Evans blue almost abolish both hydrolysis. AMP hydrolysis is not affected by levamisole and tetramisole, whereas 0.1 mM ammonium molybdate practically abolish the ecto-5'-nucleotidase activity. RT-PCR analysis from left ventricle tissue demonstrate different levels of expression of Entpd1 (Cd39), Entpd2 (Cd39L1), Entpd3 (Cd39L3), Entpd5 (Cd39L4) Entpd6, (Cd39L2) and 5'-NT/CD73. By quantitative real-time PCR we identify the Entpd2 as the enzyme with the highest expression in rat left ventricle. Our results contribute to the understanding about the control of the extracellular nucleotide levels in and cardiac system.
Asunto(s)
5'-Nucleotidasa/genética , Adenosina Trifosfatasas/genética , Perfilación de la Expresión Génica , Miocardio/enzimología , Terminaciones Nerviosas/metabolismo , Nucleótidos/metabolismo , 5'-Nucleotidasa/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Espacio Extracelular/metabolismo , Ventrículos Cardíacos , Concentración de Iones de Hidrógeno , Hidrólisis/efectos de los fármacos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Masculino , Ouabaína/farmacología , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Azida Sódica/farmacología , Suramina/farmacología , Sinaptosomas/metabolismoRESUMEN
In the present study we investigate the biochemical properties of the members of NPP family in synaptosomes prepared from rat heart left ventricles. Using p-nitrophenyl-5'-thymidine monophosphate (p-Nph-5'-TMP) as substrate for E-NPPs in rat cardiac synaptosomes, we observed an alkaline pH dependence, divalent cation dependence and the K ( M ) value corresponded to 91.42 +/- 13.97 microM and the maximal velocity (V ( max )) value calculated was 63.79 +/- 3.59 nmol p-nitrophenol released/min/mg of protein (mean +/- SD, n = 4). Levamisole (1 mM), was ineffective as inhibitor of p-Nph-5'-TMP hydrolysis in pH 8.9 (optimum pH for the enzyme characterized). Suramin (0.25 mM) strongly reduced the hydrolysis of p-Nph-5'-TMP by about 46%. Sodium azide (10 and 20 mM) and gadolinium chloride (0.3 and 0.5 mM), E-NTPases inhibitors, had no effects on p-Nph-5'-TMP hydrolysis. RT-PCR analysis of left ventricle demonstrated the expression of NPP2 and NPP3 enzymes, but excluded the presence of NPP1 member. By quantitative real-time PCR we identified the NPP3 as the enzyme with the highest expression in rat left ventricle. The demonstration of the presence of the E-NPP family in cardiac system, suggest that these enzymes could contribute with the fine-tuning control of the nucleotide levels at the nerve terminal endings of left ventricles that are involved in several cardiac pathologies.
Asunto(s)
Ventrículos Cardíacos/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/metabolismo , Sinaptosomas/enzimología , Animales , Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Hidrólisis , Masculino , Nitrofenoles/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Pirofosfatasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Azida Sódica/farmacología , Suramina/farmacología , Timidina Monofosfato/metabolismoRESUMEN
Nucleotide excision repair (NER) is one of the most versatile DNA repair mechanisms, ensuring the proper functioning and trustworthy transmission of genetic information in all living cells. The phenotypic consequences caused by NER defects in humans are autosomal recessive diseases such as xeroderma pigmentosum (XP). This syndrome is the most sun-sensitive disorder leading to a high frequency of skin cancer. The majority of patients with XP carry mutations in the XPA or XPC genes that encode proteins involved in recognition of DNA damage induced by UV light at the beginning of the NER process. Cells cultured from XPA and XPC patients are hypersensitive to UV light, as a result of malfunctioning DNA repair. So far there is no effective long-term treatment for these patients. Skin cancer prevention can only be achieved by strict avoidance of sunlight exposure or by the use of sunscreen agents. We have constructed recombinant adenoviruses carrying the XPA and XPC genes that were used to infect XP-A and XP-C immortalized and primary fibroblast cell lines. UV survival curves and unscheduled DNA synthesis confirmed complete phenotypic reversion in XP DNA repair deficient cells with no trace of cytotoxicity. Moreover, transgene expression is stable for at least 60 days after infection. This efficient adenovirus gene delivery approach may be an important tool to better understand XP deficiency and the causes of DNA damage induced skin cancer.