[Show abstract][Hide abstract] ABSTRACT: Proteins tightly bound to DNA (TBP) comprise a group of proteins that remain bound to DNA even after harsh deproteinization procedures. The amount of these proteins is 20-100 μg for mg of DNA depending on eukaryotic source. This experimental paper examines the possibility to use some TBP for clinical biomarker discovery, e.g. for identification of prognostic and diagnostic cancer markers. The main aim of this study was to designate differences between tightly DNA binding protein patterns extracted from rat liver and rat experimental hepatomas (Zajdela ascites hepatoma and hepatoma G-27) and to evaluate possibility that some of these proteins may be used as biomarkers for cell cancer transformation. Methods: We used proteomics aproach as a tool for comparison of pattern of TBP from rat experimental hepatomas and normal liver cells. Combination of 2DE fractionation with mass spectrometry (MALDI TOF-MS) suitable for parallel profiling of complex TBP mixtures. Results: Intriguingly 2DE protein maps of TBP from rat liver and rat experimental hepatomas (Zajdela acites hepatoma and hepatoma G-27) were quite different. We identified 9 proteins, some of them shared in all TBP patterns. Among identified tightly bound to DNA proteins there were three proteins considered as nuclear matrix proteins (lamin B1, scaffold attachment factor B1, heterogeneous nuclear ribonucleoprotein). Also we identified DNA repair protein RAD50, coiled-coil domain-containing protein 41, structural maintenance of chromosomes protein1A and some ATP -dependent RNA helicases indicating that TBP are of interest with respect to their potential involvement in the topological organization and/ or function of genomic DNA. Conclusions: We suppose that proteomic approach for TBP identification may be promising in development of biomarkers, also obtained results may be valuable for further understanding TBP functions in genome.
[Show abstract][Hide abstract] ABSTRACT: Tightly bound to DNA proteins (TBP) are a protein group that remain attached to DNA with covalent or non-covalent bonds after its deproteinisation. The distribution of TBP in genes reflects the type of cell differentiation. It has been hypothesised that TBP binding is involved in regulation of gene expression. Early studies reported uniformity of the TBPs from different sources. Later it was shown that TBPs obtained from DNA, isolated in mild conditions from evolutionary distant species, are different. Application of chloroform DNA extraction without use of externally added enzymes enabled us to reveal differences in the TBP spectrum in plant organs and changes of this spectrum in the course of plant development. The goal of this work was to study the electrophoretic pattern of the polypeptide component in the tight DNA-protein complexes in organs of animals: a mammal (rat) and a bird (chicken). Rat thymus TBPs were represented by 70 and 60 kDa proteins, and the same polypeptides were observed also in brain and skeletal muscles. Kidney TBPs were represented by 85, 70, 65, 60 and 37 kDa polypeptides; 85, 77, 70, 60, 50 and 37 kDa TBPs were characteristic of liver. Numerous minor peptides were observed in all samples studied. The spectrum of chicken liver and blood TBPs differed in distribution of proteins of 25-35 kDa. Mass-spectrometry of 14 bands from rat liver TBP gel revealed 43 different proteins. Chromatin modifying proteins and repair enzymes, transcription factors, serpins, ATPase, kinases and enzymes of ubiquitin-proteasome pathway were found among the TBPs. Thus, TBPs appear to be a vast protein group involved in several intranuclear processes. It is hypothesised that numerous functions ascribed to the nuclear matrix are performed in the TBP complexes.
Preview · Article · Jan 2010 · Proceedings of the Latvian Academy of Sciences Section B Natural Exact and Applied Sciences