Discovery of chaperone-type nucleoside diphosphate kinase activity in Hsp70 and proteasome and its pathophysiological function in these proteins

Hsp70 和蛋白酶体中分子伴侣型核苷二磷酸激酶活性的发现及其在这些蛋白质中的病理生理功能

基本信息

批准号：
11470043
负责人：
KIDO Hiroshi
金额：
$ 9.34万
依托单位：
Institute for Enzyme Research, The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Hsp70 is a multifunctional molecular chaperone whose interactions with protein substrates are regulated by ATP hydrolysis and ADP-ATP exchange. In the period granted by this foundation, we found that, in addition to ATPase activity, purified Hsp70 and 20S proteasome, a new family of N-terminal nucleophile hydrolases, free from nucleoside diphosphate (NDP) kinase, exhibit intrinsic ADP-ATP exchange activity. The rate constants for ATP hydrolysis and ATP synthesis of these proteins were in a similar range at the optimum pH of 7.5-8.5 in the presence of 5 mM ATP and 0.5 mM ADP.Both Hsp70 and 20S proteasome exhibited a considerably strict preference for ATP as a phosphate donor, and a biased substrate specificity, unlike NDP kinase. During the reaction, both proteins formed acid-labile autophosphorylated intermediates and nucleoside diphosphate-dependent dephosphorylation of the latters then occurred. These properties are not identical but similar to those of NDP kinase, and are not similar to those of adenylate kinase and ATP synthase. The 20S proteasome is composed of numerous low molecular mass subunits arranged in a stack of four rings, each containing seven different α- or β-subunits. Among these subunits, we identified that the C5 in the β-type and the C8 in the α-type subunits were autophosphorylated during the γ-phosphate transfer reaction and were photoaffinity labeled with 8-azido-[α-^<32>P] ATP, suggesting that the C5 and C8 subunits of the proteasome are responsible for the NDP kinase-like activity. We are now trying to identify the active sites of NDP kinase in these proteins and also try to identify the role of NDP kinase in the chaperone activity of Hsp70 and the conformational modification of substrates in the processing of proteolysis by 20S proteasome.

HSP70是一种多功能分子链酮，其与蛋白质底物的相互作用受ATP水解和ADP-ATP交换调节。在该基金会批准的时期中，我们发现，除了ATPase活性外，还纯化了HSP70和20S蛋白酶体，这是一个新的N末端核水解酶的家族，不含核外侧二磷酸（NDP）激酶，还暴露了内在的ADP-ATP-ATP交换活动。这些蛋白质的ATP水解和ATP合成的速率在5 mm ATP和0.5 mM ADP的情况下，最佳pH值为7.5-8.5的速率相似。在反应过程中，两种蛋白质均形成酸性自磷酸化的中间体和核苷二磷酸依赖性去磷酸化的后期。这些特性与NDP激酶的特性并不相同，并且与腺苷酸激酶和ATP合酶的特性不同。 20S蛋白酶体由排列在四个环中的众多低分子质量亚基组成，每个亚基都包含七个不同的α-或β-亚基。 Among these subunits, we identified that the C5 in the β-type and the C8 in the α-type subunits were autophosphorylated during the γ-phosphate transfer reaction and were photoaffinity labeled with 8-azido-[α-^<32>P] ATP, suggesting that the C5 and C8 subunits of the proteasome are responsible for the NDP kinase-like activity.现在，我们正在尝试鉴定这些蛋白质中NDP激酶的活性位点，并尝试鉴定NDP激酶在HSP70的伴侣活性中的作用以及底物在20S蛋白酶体处理蛋白水解中的构象修饰。