RESUMEN
The MYC family of oncogenes encodes a set of three related transcription factors that are overexpressed in many human tumors and contribute to the cancer-related deaths of more than 70,000 Americans every year. MYC proteins drive tumorigenesis by interacting with co-factors that enable them to regulate the expression of thousands of genes linked to cell growth, proliferation, metabolism and genome stability. One effective way to identify critical co-factors required for MYC function has been to focus on sequence motifs within MYC that are conserved throughout evolution, on the assumption that their conservation is driven by protein-protein interactions that are vital for MYC activity. In addition to their DNA-binding domains, MYC proteins carry five regions of high sequence conservation known as Myc boxes (Mb). To date, four of the Mb motifs (MbI, MbII, MbIIIa and MbIIIb) have had a molecular function assigned to them, but the precise role of the remaining Mb, MbIV, and the reason for its preservation in vertebrate Myc proteins, is unknown. Here, we show that MbIV is required for the association of MYC with the abundant transcriptional coregulator host cell factor-1 (HCF-1). We show that the invariant core of MbIV resembles the tetrapeptide HCF-binding motif (HBM) found in many HCF-interaction partners, and demonstrate that MYC interacts with HCF-1 in a manner indistinguishable from the prototypical HBM-containing protein VP16. Finally, we show that rationalized point mutations in MYC that disrupt interaction with HCF-1 attenuate the ability of MYC to drive tumorigenesis in mice. Together, these data expose a molecular function for MbIV and indicate that HCF-1 is an important co-factor for MYC.
Asunto(s)
Secuencias de Aminoácidos/genética , Transformación Celular Neoplásica/genética , Factor C1 de la Célula Huésped/genética , Mutación , Proteínas Proto-Oncogénicas c-myc/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Secuencia Conservada/genética , Evolución Molecular , Células HEK293 , Factor C1 de la Célula Huésped/metabolismo , Humanos , Inmunoprecipitación , Ratones , Células 3T3 NIH , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/metabolismo , Homología de Secuencia de AminoácidoAsunto(s)
Antineoplásicos/química , Hidrocarburos Aromáticos con Puentes/química , Paclitaxel/análogos & derivados , Paclitaxel/química , Taxoides , Alcaloides/síntesis química , Alcaloides/química , Alcaloides/toxicidad , Animales , Antineoplásicos/síntesis química , Antineoplásicos/toxicidad , Hidrocarburos Aromáticos con Puentes/síntesis química , Cristalografía por Rayos X , Femenino , Humanos , Melanoma Experimental/tratamiento farmacológico , Ratones , Ratones Endogámicos , Conformación Molecular , Estructura Molecular , Neoplasias Ováricas , Paclitaxel/síntesis química , Paclitaxel/toxicidad , Células Tumorales CultivadasRESUMEN
A tandem mass spectrometric (ms/ms) method using desorption chemical ionization is described for the quantitation of taxol [1], cephalomannine [2], and baccatin III [3] found in Taxus brevifolia bark and needle extracts. A parent ion scan was used to simultaneously determine the weight percentages of 1-3 in bark and needle samples by the method of standard addition. In an alternative experiment, the concentration of 1 in the same samples was determined by ms/ms using trideuterated 10-acetyltaxol [7a] as an internal standard. High-performance liquid chromatography (hplc) was also used to determine the weight percentages of 1-3 in the same T. brevifolia bark and needle extracts with an external standard. The ms/ms method of quantitation by internal standard is the best overall method of analysis examined. With this method, 1 was quantitated in the T. brevifolia extracts at the low picomole level with a relative standard deviation of 17% or better for all samples analyzed with an analysis time of less than five min per sample. The precision, level of quantitation, and speed of analysis of the three methods of taxane quantitation are compared.
