|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1993 : ¥300,000 (Direct Cost : ¥300,000)
Fiscal Year 1992 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1991 : ¥1,200,000 (Direct Cost : ¥1,200,000)
We have previously reported that a novel 40-kDa protein is induced by heat shock and several envionmental stresses in mammalian and avian cells and that the N-terminal amino acid sequence of this 40-kDa protein has homology with the bacterialdna Jheat-shock protein. We nextisolated and characterized a cDNA clone encoding 40-kDa heat-shock protein hsp40 from cDNA expression library of human placentaby immunoscreening with antiidenticalto that of HeLacellhsp40 as determined by amino acid sequencer. Deduced amino acid sequence of the cDNA is homologous to bacterial DnaJheat-shock protein834% identical) and its homologues in yeast such as SCJ1, YDJ1(MAS5), SIS1, SEC63 AND Zuotin(34 - 40% identical), with SIS1 being the most homologous. Nucleotide sequence identity between hsp40 and HDJ1(another human DnaJ homologue9 is more than 98% including 5'and3' noncoding region, suggesting that these two proteins are the same geneproduct. Since there are at least three dnaJ homologues in human [hsp40
(HDJ1), HDJ2(HSDJ), HSJ1] and hsp90 family.
Our hsp40 has JandG/F domains but not C domain. Inyeast DnaJ homologues SIS1 has a domain structure and biochemical properties (MW, pl, heat-inducibility and intracellular localization) similar to human hsp40. Also, Therefor, it is suggested that human hsp40 is ahomologue of yeast SIS1 protein.
Indirect immunofluorescence revealed that hsp40 in Hela cells accumulates in the nuclei, especially in the nucleoli during heat shock and returns to the cytoplasm during recovery period. The kinetics of the accumulation in the nucleoli and subsequent return to the cytoplasm during recovery period. The kinetics of the accumulation hsp40 was colocalized with hsc70 (p73) in heat-shocked HeLa cells as demonstrated by double immunofluorescence staining. Hsp70 in the nucleoli was released by the addition of ATP but not by ADP, GTP, nonhydrolyzableATP, nor high saltbuffer. In contrast, hsp40 was not released from the nucleoli with any of these treatments or any combination of these treatments. Thus, hsp40 was not released from the nucleoli after hsp70 has been released in an ATP-dependent manner. These results suggest that hsp40 (DnaJaggregates in the nuclei and nucleoli of heat-shocked HeLa cells.
次に温熱耐性とhsp40の相関をマウスのSCCVII細胞を用いて調べた。温熱耐性およびhsp40を誘導する条件としては熱ショック(44℃ 30分)、ヒ素処理(100μM 1時間)を用いた。温熱耐性はコロニー形成能、hsp40はウエスタンブロットで定量化した。いずれの条件下においてもhsp40の合成・減衰と温熱耐性の発現・減衰の動態はよく相関していた。このことからhsp70だけでなくhsp40も温熱耐性のマーカーになりうることが示された。