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Discovery of chaperone-type nucleoside diphosphate kinase, a novel function of molecular chaperone proteins, and role of the activity in proteolysis

伴侣型核苷二磷酸激酶的发现、分子伴侣蛋白的新功能及其在蛋白水解中的作用

基本信息

批准号：
11670128
负责人：
YANO Mihiro
金额：
$ 2.56万
依托单位：
Institute for Enzyme Research, The University of Tokusahima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
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

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670128/
关键词：
NDPK Molecular chaperone ATPase active site ATP syntase 14-3-3 protein 化学修飾 raf-1 蛋白質分解酵素

项目摘要

Hsp70 and 14-3-3 proteins are multifunctional molecular chaperones 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, Hsp70, 14-3-3 proteins and purified 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.During the reaction, these proteins formed acid-labile autophosphorylated intermediates and nucleoside diphosphate-dependent dephosphorylation of the latters then occurred. 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. In addition, we recently identified the autophosphorylated amino acid residues, T204 and T211, in Hsp70 and a novel ATP binding site, H227, E231, D232, in HSP70. We are now trying to identify the role of NDP kinase in the chaperone activity of Hsp70 and 14-3-3 proteins and the conformational modification of substrates in the processing of proteolysis by 20S proteasome.

Hsp70 和 14-3-3 蛋白是多功能分子伴侣，其与蛋白质底物的相互作用受到 ATP 水解和 ADP-ATP 交换的调节。在该基金会授予的期限内，我们发现，除了 ATP 酶活性外，Hsp70、14-3-3 蛋白和纯化的 20S 蛋白酶体，一个新的 N 端亲核水解酶家族，不含核苷二磷酸 (NDP) 激酶，表现出内在的 ADP-ATP 交换活性。在 5 mM ATP 和 0.5 mM ADP 存在下，这些蛋白质的 ATP 水解和 ATP 合成速率常数在 7.5-8.5 的最佳 pH 范围内相似。在反应过程中，这些蛋白质形成酸不稳定的自磷酸化中间体，然后发生后者的核苷二磷酸依赖性去磷酸化。 20S 蛋白酶体由许多低分子量亚基组成，这些亚基排列成四个环的堆叠形式，每个环包含七个不同的 α 或 β 亚基。在这些亚基中，我们发现β型亚基中的C5和α型亚基中的C8在γ-磷酸转移反应期间被自磷酸化，并用8-叠氮基-[α-^<32>P]ATP进行光亲和标记，表明蛋白酶体的C5和C8亚基负责NDP激酶样活性。此外，我们最近鉴定了 Hsp70 中的自磷酸化氨基酸残基 T204 和 T211，以及 HSP70 中的新 ATP 结合位点 H227、E231、D232。我们现在正试图确定 NDP 激酶在 Hsp70 和 14-3-3 蛋白的伴侣活性中的作用以及 20S 蛋白酶体蛋白水解过程中底物构象修饰的作用。