TAILIEUCHUNG - Báo cáo khoa học: Identification of intracellular target proteins of the calcium-signaling protein S100A12

In this report, we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca 2+ -dependent manner. Using S100A12 affinity chroma-tography, we have identified cytosolic NADP + -dependent isocitrate dehydrogenase (IDH), fructose-1,6-bisphosphate aldolase A (aldolase), glyceraldehyde-3-phosphate dehy-drogenese (GAPDH), annexin V, S100A9, and S100A12 itself as S100A12-binding proteins. Immunoprecipitation experiments indicated the formation of stable complexes between S100A12 and IDH, aldolase, GAPDH, annexin V and S100A9in vivo | Eur. J. Biochem. 271 3765-3775 2004 FEBS 2004 doi Identification of intracellular target proteins of the calcium-signaling protein S100A12 Takashi Hatakeyama1 Miki Okada1 Seiko Shimamoto1 Yasuo Kubota2 and Ryoji Kobayashi1 Departments of 1 Signal Transduction Sciences and 2Dermatology Kagawa University Faculty of Medicine Japan In this report we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca2 -dependent manner. Using S100A12 affinity chromatography we have identified cytosolic NADP -dependent isocitrate dehydrogenase IDH fructose-1 6-bisphosphate aldolase A aldolase glyceraldehyde-3-phosphate dehy-drogenese GAPDH annexin V S100A9 and S100A12 itself as S100A12-binding proteins. Immunoprecipitation experiments indicated the formation of stable complexes between S100A12 and IDH aldolase GAPDH annexin V and S100A9 in vivo. Surface plasmon resonance analysis showed that the binding to S100A12 of S100A12 S100A9 and annexin V was strictly Ca2 -dependent whereas that of GAPDH and IDH was only weakly Ca2 -dependent. To localize the site of S100A12 interaction we examined the binding of a series of C-terminal truncation mutants to the S100A12-immobilized sensor chip. The results indicated that the S100A12-binding site on S100A12 itself is located at the C-terminus residues 87-92 . However cross-linking experiments with the truncation mutants indicated that residues 87-92 were not essential for S100A12 dimerization. Thus the interaction between S100A12 and S100A9 or immobilized S100A12 should not be viewed as a typical S100 homo- or heterodimerization model. Ca2 -dependent affinity chromatography revealed that C-terminal residues 75-92 are not necessary for the interaction of S100A12 with IDH aldolase GAPDH and annexin V. To analyze the functional properties of S100A12 we studied its action in protein folding reactions in vitro. The thermal aggregation of IDH or GAPDH was facilitated by .

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