サルカンゾウミクロソームノチュウセイプロティナーゼ : ソノカヨウカブブンセイセイオヨビセイシツ

京都大学0048新制・課程博士理学博士甲第2164号理博第565号新制||理||300(附属図書館)5997UT51-54-H14京都大学大学院理学研究科動物学専攻(主査)教授 高橋 健治, 教授 大沢 済, 教授 日高 敏隆学位規則第5条第1項該当Kyoto UniversityDA

Altered DNA binding specificity of Arnt by selection of partner bHLH–PAS proteins

The Ah receptor (AhR) and HLF are transcription factors involved in xenobiotic metabolism and hypoxic response, respectively. AhR and HLF heterodimerize with Arnt as the common partner, and bind to asymmetric E-boxes termed XRE and HRE, respectively. In order to investigate nucleotide preference of the heterodimers, reporter plasmids with oligonucleotides for XREs or HREs with systematic mutations were constructed and their activity was determined. Comparison of the activity revealed that DNA length and nucleotide preference recognized by Arnt subunit in the two heterodimers were largely different between XRE and HRE. We expressed AhR–Arnt and HLF–Arnt in Escherichia coli and used them for DNA binding. The dissociation constant of HLF–Arnt–HRE was 10.4 ± 1.6 nM. Competition activity of mutated XREs or HREs with wild type was consistent with their transcription activity. Bending of XRE and HRE induced by binding of the relevant heterodimers was observed with stronger bending of XRE than of HRE. By deletional and mutational analyses, an alanine and three arginine (Ala 8, Arg 9, Arg 11 and Arg 12) residues in the basic sequence of HLF were found to be indispensable for the transcriptional activity

Magnesium Deficiency Causes Loss of Response to Intermittent Hypoxia in Paraganglion Cells*

Magnesium deficiency is suggested to contribute to many age-related diseases. Hypoxia-inducible factor 1α (HIF-1α) is known to be a master regulator of hypoxic response. Here we show that hypomagnesemia suppresses reactive oxygen species (ROS)-induced HIF-1α activity in paraganglion cells of the adrenal medulla and carotid body. In PC12 cells cultured in the low magnesium medium and treated with cobalt chloride (CoCl2) or exposed to intermittent hypoxia, ROS-mediated HIF-1α activity was suppressed. This suppression was due to up-regulation of inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS) that was caused by NF-κB activation, which resulted from ROS and calcium influx mainly through the T-type calcium channels. Induction of tyrosine hydroxylase, a target of HIF-1, by CoCl2 injection was suppressed in the adrenal medulla of magnesium-deficient mice because of up-regulation of IPAS. Also in the carotid body of magnesium-deficient mice, CoCl2 and chronic intermittent hypoxia failed to enhance the tyrosine hydroxylase expression. These results demonstrate that serum magnesium levels are a key determinant for ROS-induced hypoxic responses