RESUMEN
Treatment of malignant neoplasms often requires the use of combinations of chemotherapeutic agents. However, in order to select combinations that are effective against specific tumor cells, it is necessary to understand the mechanisms of action of the drugs that make up the combination. Bacillus pumilus ribonuclease (binase) is considered as an adjuvant antitumor agent, and the sensitivity of malignant cells to the apoptogenic effect of binase depends on the presence of certain oncogenes. In the acute myelogenous leukemia cell line Kasumi-1, binase blocks the proliferation pathway mediated by the mutant tyrosine kinase KIT, which, as shown in our work, activates an alternative proliferation pathway through AKT kinase. In Kasumi-1 cells, binase in combination with an Akt1/2 inhibitor induces apoptosis, and their toxic effects add up: the Akt1/2 inhibitor blocks the binase-induced pathway after suppression of the KIT-dependent pathway. Thus, a combination of binase and AKT kinase inhibitors can effectively block various pathways of tumor cell proliferation and be used for their elimination.
Asunto(s)
Antineoplásicos , Proteínas Proto-Oncogénicas c-akt , Antineoplásicos/farmacología , Apoptosis , Endorribonucleasas/metabolismo , Inhibidores de Proteínas Quinasas , Proteínas Tirosina Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Ribonucleasas/genética , Ribonucleasas/farmacologíaRESUMEN
Neuronal dysfunction and loss associated with the accumulation of amyloid-ß (Aß) in the form of extracellular amyloid plaques and hyperphosphorylated tau in the form of intraneuronal neurofibrillary tangles represent key features of Alzheimer's disease (AD). Amyloid plaques found in the brains of AD patients are predominantly composed of Aß42 and its multiple chemically or structurally modified isoforms. Recently, we demonstrated that Aß42 with isomerised Asp7 (isoAß42) which is one of the most abundant Aß isoform in plaques, exhibited high neurotoxicity in human neuronal cells. Here, we show that, in SH-SY5Y neuroblastoma cells, the administration of synthetic isoAß42 rather than intact Aß42 resulted in a significantly higher level of protein phosphorylation, especially the phosphorylation of tau, tubulins, and matrin 3. IsoAß42 induced a drastic reduction of tau protein levels. Our data demonstrate, for the first time, that isoAß42, being to date the only known synthetic Aß species to cause AD-like amyloidogenesis in an animal AD model, induced cell death by disabling structural proteins in a manner characteristic of that observed in the neurons of AD patients. The data emphasize an important role of isoAß42 in AD progression and provide possible neurotoxicity paths for this particular isoform.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Ácido Aspártico/metabolismo , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/toxicidad , Fosfoserina/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Electroforesis en Gel Bidimensional , Humanos , Modelos Biológicos , Neuronas/metabolismo , Neuronas/patología , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Fosforilación , Isoformas de Proteínas/toxicidad , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas tau/genéticaRESUMEN
Monitoring of peptides offers a promising approach for the discovery of novel biomarkers, which might be valuable for detection, treatment and prevention of large variety of diseases. Development of highly effective methods for plasma peptide extraction remains an important task. In the current study, we applied different types of plasma peptide extraction approaches to reveal efficient methods which would provide the highest peptide yield. We used different combinations of plasma treatment with acetonitrile and/or urea/guanidine, protein precipitation by acetone, gel-filtration, ultrafiltration, and two types of solid phase extraction. The extracted peptides were analyzed by LC-MS/MS. The obtained results suggest that several methods, including differential solubilization, organic precipitation, as well as some variants of ultrafiltration and solid phase extraction, provide effective plasma peptide enrichment convenient for further LC-MS/MS analysis. Alas, most of the identified peptides were extracted by only one of the applied methods. Hence, it seems reasonable to consider several methods to increase the possibility of novel biomarker discovery.
Asunto(s)
Péptidos/sangre , Péptidos/aislamiento & purificación , Espectrometría de Masas en Tándem/métodos , Precipitación Química , Cromatografía Liquida/métodos , Proteoma/análisis , Proteoma/aislamiento & purificación , Extracción en Fase Sólida , UltrafiltraciónRESUMEN
Interaction of Zn(2+) with the metal-binding domain of the English (H6R) amyloid-ß mutant results in the formation of peptide dimers. The mutation causes the exclusion of His6 from the zinc chelation pattern observed in the intact domain and triggers the assembly of the dimers via zinc ions coordinated by (11)EVHH(14) fragments.