RESUMO
BACKGROUND: RBBP6 is a 250 kDa splicing-associated protein that has been identified as an E3 ligase due to the presence of a RING finger domain. In humans and mice it interacts with both p53 and Rb, and plays a role in the induction of apoptosis and regulation of the cell cycle. RBBP6 has recently been shown to be highly up-regulated in oesophageal cancer, and to be a promising target for immunotherapy against the disease. RESULTS: We show here using heteronuclear NMR that the N-terminal 81 amino acids of RBBP6 constitute a novel ubiquitin-like domain, which we have called the DWNN domain. The domain lacks conserved equivalents of K48 and K63, although the equivalents of K6 and K29 are highly, although not absolutely, conserved. The di-glycine motif that is characteristic of proteins involved in ubiquitination is found in the human and mouse form of the domain, although it is not present in all organisms. It forms part of a three-domain form of RBBP6 containing the DWNN domain, a zinc knuckle and a RING finger domain, which is found in all eukaryotic genomes so far examined, in the majority of cases at single copy number. The domain is also independently expressed in vertebrates as a single domain protein. CONCLUSION: DWNN is a novel ubiquitin-like domain found only at the N-terminus of the RBBP6 family of splicing-associated proteins. The ubiquitin-like structure of the domain greatly increases the likelihood that RBBP6 functions through some form of ubiquitin-like modification. Furthermore, the fact that the DWNN domain is independently expressed in higher vertebrates leads us to propose that the domain may itself function as a novel ubiquitin-like modifier of other proteins.
Assuntos
Proteínas de Transporte/química , Proteínas de Ligação a DNA/química , Processamento Alternativo , Sequência de Aminoácidos , Proteínas de Transporte/fisiologia , Biologia Computacional , DNA/química , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/fisiologia , Neoplasias Esofágicas/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Modelos Estatísticos , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Ubiquitina/química , Ubiquitina-Proteína Ligases , Regulação para Cima , Dedos de ZincoRESUMO
The application of HPLC-NMR for the analysis of a mixture of fatty alcohol ethoxylates (FAEs) is described. The use of the new generation, cryogenically cooled probes is investigated in respect of the sensitivity advantage that they provide. The FAE mixture is separated using liquid chromatography at the critical point of adsorption. The ability of the method to differentiate between the different end groups and the degree of polymerization of the mixture constituents is investigated. Both on-flow and stop-flow HPLC-NMR techniques were used together with two-dimensional NMR spectroscopy. The results are compared with those obtained by using an evaporative light scattering detector for the HPLC.
