Comparative analysis of brain proteins from p53-deficient mice by two-dimensional electrophoresis

Abstract

p53 is a tumor suppressor protein that regulates many cellular processes including the cell cycle, DNA repair, and apoptosis. It also serves as a critical regulator of neuronal apoptosis in the central nervous system (CNS). To elucidate the role of p53 in the CNS, brain proteins of p53 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-week-old 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 2-D gel analysis showed that, on average, 7 of 886 spots from brain tissue were p53-/- specific, whereas 12 of 1008 spots from lung tissue were p53-/- specific. N-terminal amino acid sequence was determined for p53-/- specific proteins. In all brain tissues from p53-/- mice, a newly identified mouse mitochondrial NADH-ubiquinone oxidoreductase 24 kDa subunit showed decreased expression, and apolipoprotein A1 acidic forms showed increased expression. In addition, brain-type creatine kinase B chain and tubulin beta-5 N-terminal fragment were increased in the p53-/- cerebellum, and a new protein in mouse, hydroxyacylglutathione hydrolase (glyoxalase II) was decreased in the temporal cortex of p53-/- mice. The alterations in protein expression identified in this study may imply a p53-related brain function. This is the first proteomic analysis on the p53-/- mouse brain, and further information based on this study will provide new insights into the p53 function in the CNS.

Buffer optimization for high resolution of human lung cancer tissue proteins by two-dimensional gel electrophoresis

“…Human lung tissue, 30-50 mg, was ground to powder in liquid N 2 and lysed in lysis buffer (8 M urea, 2 M thiourea, 100 mM DTT, 4% (w/v) CHAPS, 25 mM Tris/HCl pH 7.1, 50 mM KCl, 0.2% (v/v) carrier ampholyte pH 3-10 NL, and a protease inhibitor cocktail (Roche). The lysates were vortexed followed by incubation at 37°C for 1 h and the lysates were centrifuged at 15,000 9 g for 30 min at 4°C (Araki et al 2000). Supernatants were collected and protein concentrations in samples were determined with the 2-D Quant protein assay kit (Amersham Biosciences).…”

Mitochondrial Liaisons of p53

“…Similarly, p53 can transactivate the gene encoding hexokinase II (147), which catalyzes the rate-limiting step of glycolysis (i.e., the conversion of glucose into G6P) while interacting with the PTPC, favoring the Warburg effect and exerting consistent antiapoptotic effects (67,148,183). p53 also induces the transcription of the genes coding for the muscular and brain isoforms of creatine kinase (CKM and CKB, respectively), which exert prosurvival functions by maintaining ATP levels at the expenses of phosphocreatine (7,249). These apparently counterintuitive prosurvival activities of the p53 system presumably reflect a high degree of complexity that has not yet been entirely disentangled (244).…”

Homeostatic functions of the p53 tumor suppressor: Regulation of energy metabolism and antioxidant defense

“…Functional p53 responsive element is also contained in the gene encoding the brain isoform of creatine kinase [102]. Creatine kinase restores ATP level by phosphorylating ADP and by consuming phosphocreatine, which serves as energy reservoir in tissues that are actively spending ATP, such as skeletal muscle, brain and smooth muscles.…”