Post-translational modifications determination

Histones are basic proteins that are made up by a globular domain and an N-terminal tail that protrudes from the nucleosome. Nucleosomes form the basic unit of chromatin and are made up by a complex of DNA wrapped around an octamer of histones formed by pairs of the histones H2A, H2B, H3, and H4 (45,46) (Fig. 1). Post-translational modification of the core histone tails by methylation, acetylation, phosphorylation, ubiquitina-tion, or sumoylation can alter the structure of the nucleosomes and thus alter gene expression. These post-translational modifications determine the structure and pattern of chromatin condensation and determine the histone code that drives gene transcriptional regulation (47,48). Below are briefly described the factors determining the histone acetylation and methylation. 

The Edman reaction enabled amino acid sequence analysis to be automated. Mass spectrometry provides a sensitive and versatile tool for determining primary strucmre and for the identification of post-translational modifications. 

Proteomics, the measurement of the global changes in proteins produced as a result of gene expression, bridges the gap between genome sequence and cellular behaviour and takes into account the post-translational modifications that often result in the functional effect. It has the potential to determine the role of protein-protein complexes in the complex signalling cascades that... 

One of the most important considerations for the improvement of protein yields is subcellular protein targeting, because the compartment in which a recombinant protein accumulates strongly influences the interrelated processes of folding, assembly and post-translational modification. All of these contribute to protein stability and hence help to determine the final yield [88]. 

Nucleolin lacks a characteristic DNA binding domain (Ginisty et al, 1999). Its non-specific affinity for DNA is conferred by two different domains its four RNA binding domains, particularly the 3rd and the 4th ones, and its C-terminal GAR domain (Hanakahi et al, 1999 Sapp et al, 1989). Of importance, these properties were determined in vitro with the native protein purified from cell extracts or recombinant truncated proteins they are likely to be altered in vivo by interaction with other DNA binding factors (Dempsey et al, 1998) and/or by post-translational modifications. 

Expression analysis using DNA microarrays analyzes only the transcriptome it should be mentioned that mRNA abundance in a cell often correlates poorly with the amount of protein synthesized (27). Important regulation takes place at the levels of translation and enzymatic activities. The only effect of a signal transduction pathway that is observed in a gene expression experiment is that at the endpoint of a given pathway. DNA microarrays currently have little value in determining post-translational modifications, which influence the diversity, affinity, function, cellular abundance, and transport of proteins. 

Several steps were needed to determine the structure of the core particle to higher resolution (Fig. Id). The X-ray phases of the low-resolution models were insufficient to extend the structure to higher resolution, since the resolution of the early models of the NCP was severely limited by disorder in the crystals. The disorder was presumed to derive from both the random sequences of the DNA and from heterogeneity of the histone proteins caused by variability in post-translational modification of the native proteins. One strategy for developing an atomic position model of the NCP was to develop a high-resolution structure of the histone core. This structure could then be used with molecular replacement techniques to determine the histone core within the NCP and subsequently identify the DNA in difference Fourier electron density maps. 

Compared to the genome, the proteome (the entire diverse protein content of a cell) is a far more dynamic system. Proteins imdergo post-translational modifications such as phosphorylation, glycosylation and sulphation, as well as cleavage for specific proteins. These alterations determine protein activity, localisation and turnover. All are subject to change following a toxic insult and, in some ways, the study of proteins holds more promise than the study of gene expression as the former is nearer to key activities in the cell. 

Zhang, L., Freitas, MA., Wickham, J., Parthun, M.R., Klisovic, M.I., Marcucd, G. and Byrd, J.C. (2004) Differential expression of histone post-translational modifications in acute myeloid and chronic lymphocytic leukemia determined by high-pressure liquid chromatography and mass spectrometry. Journal of the American Society for Mass Spectrometry, 15, 77-86. 

Unlike the genome the proteome is not a static but a dynamic and constantly changing entity that is cell- and tissue-specific and dependent on the environment. Because of the dynamic nature of protein expression and fimction, these properties need to be determined quantitatively in a time-dependent manner. Proteomics, the study of the proteome, involves the analysis of the complete pattern of the expressed proteins and their post-translational modifications in a cell, tissue, or body fluid. An integrated view of any living system hence requires an analysis that takes into account the spatial as well as temporal distribution of all the proteins in a cell or tissue. The analytical effort that is necessary to deliver such an integrated view is by several orders of magnitude more complicated than that of the recently finished human genome (Lander ef al. 2001 Venter et al. 2001). 

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