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アクティブボード・２００１年１１月
研究発表を行った学会；The International Proteomics
Conference 2001；ComBio2001;
２００１年９月３０日〜１０月４日
（CANBERRA AUSTRALIABR）
タイトル；Functional Proteomic Analysis of
Tumor Suppressor Related Proteins in Brain
発表者；荒木 令江
（医学部・腫瘍医学講座）
Abstract；
Tumor suppressor proteins regulate many cellular processes including cell cycle, DNA repair, and apoptosis. P53, NF1, NF2, and NE-dlg gene products have been known as tumor suppressor and functional regulator of the neuronal cells in the central nervous system (CNS).
To elucidate the role of their functions in the CNS, brain proteins that contribute to control their
activation were analyzed with two proteomic strategies.
First, brain tissue proteins of p53 gene knock-out mice (p53-/-) were analyzed by two-dimensional gel electrophoresis (2-DE) and compared with those from p53 wild type (p53+/+) mice. Six types of brain tissue (temporal cortex, cerebellum, hippocampus, striatum, olfactory bulb, and cervical spinal cord)
and other control tissues (lung and blood) from 18 weeks old of non-stress induced mice were analyzed. The morphology of brains from p53-/- mice appeared to be normal and identical to that of p53+/+ mice, although lungs showed diffuse tumors that may have been caused by p53 deficiency.
Comparative 2D-gel analysis showed that, on average, 7 of 886 spots from brain tissue were p53-/- specific. Identification of those proteins by the N-terminal and internal amino acid analysis revealed that p53-/- brain contained several specific proteins that are related to the apoptosis, cellular damages, cell cycle, and tumor generation.
Secondly, the specific binding proteins that interact with NF1, NF2, NE-dlg gene products were determined from the normal brain tissue and brain cell lysates, using micro affinity beads immobilized each target gene products.
Eight proteins for NF1, five proteins for NF2, and one novel protein for NE-dlg were identified. These cellular binding proteins appeared to belong in the DNA-repair enzymes, growth and apoptosis regulators.
The evidences of those intracellular associations and colocalizations were further confirmed by the immunoprecipitation assay, and confocal microscopic analysis. The precise binding sites of each molecule, biochemical alterations and the cellular phenotypic changes after the interaction were also analyzed. The lack of these interactions caused by mutations of the tumor suppressor genes affected the cellular signals and phenotypes.
This is the first functional proteomic analysis of the brain, and further information based on this study will provide new sights into the function of tumor suppressor protein in CNS.
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