RESUMEN
The human multiprotein bridging factor 1 (hMBF1) has been established in different cellular types to have the role of transcriptional coactivator. It is also reported to be a putative Calmodulin (CaM) target, able to bind CaM in its calcium-free state, but little is known about the structural features and the biological relevance of this interaction. We applied NMR to investigate the interaction between the two proteins in solution and compared the results with those obtained with CaM-agarose affinity chromatography. No changes in ¹H-¹5N HSQC spectrum of both apo-CaM and Ca²âº-CaM upon addition of hMBF1 prove that the two proteins do not interact in vitro. These results were confirmed by CaM-agarose affinity chromatography when operating under the same conditions. The discrepancy between present and previous experiments performed with CaM-agarose affinity chromatography depends on different experimental parameters suggesting that particular attention must be paid when CaM, or other immobilized proteins, are used to measure their affinity with putative partners. These results also imply that if an interaction between the two proteins exists in vivo, as reported for hMBF1 of endothelial cells, it might involve a posttranslational modified form of the proteins or it relies on other conditions imposed by the cellular environment.
Asunto(s)
Proteínas de Unión a Calmodulina/metabolismo , Calmodulina/metabolismo , Secuencia de Aminoácidos , Calmodulina/química , Proteínas de Unión a Calmodulina/química , Cromatografía en Agarosa , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Unión ProteicaRESUMEN
The content of essential amino acids is an important aspect for determining the nutritional value of food proteins, but their digestibility is also a key property, deeply affected by food processing. The production of Parmigiano Reggiano cheese is closely related to the nutritional quality of the final product; in particular the high digestibility of its proteins is claimed to be proportional to cheese aging. Two different kinds of Parmigiano Reggiano, young (aged 15 months) and old (aged 30 months), were separately digested using an in vitro system that simulates digestive processes in the mouth, stomach and small intestine. Samples were collected at different stages of digestion and the process of protein hydrolysis was explored and compared by low-field (LF) and high-resolution (HR) NMR, together with other biochemical methods. HR-NMR allowed to simultaneously observe the quantity of free amino acids, peptides and proteins, also giving for these latter qualitative information about their dimension. LF-NMR, instead, gave the possibility to observe digestion with no treatments whatsoever, thus representing a technique suitable for on-line measurements. The results pointed out that cheeses with different aging times, although starting from distinct initial compositions, conclude digestion in a similar way, in terms of free amino acids and small organic compounds, but evolve with different kinetics of hydrolysis and peptide formation, discriminating the young from the old cheese.