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

热休克蛋白70是一种多功能的分子伴侣，其与蛋白质底物的相互作用受ATP水解和ADP-ATP交换的调节。在该基金会的资助下，我们发现，除了ATP酶活性外，纯化的Hsp 70和20 S蛋白酶体，一个新的N-末端亲核水解酶家族，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0.5mM ADP存在下，这些蛋白的ATP水解和ATP合成的速率常数在7.5-8.5的最适pH范围内是相似的.与NDP激酶不同，Hsp 70和20 S蛋白酶体都表现出对ATP作为磷酸供体的相当严格的偏好性和偏向的底物特异性.在反应过程中，这两种蛋白质形成酸不稳定的自磷酸化中间体和核苷二磷酸依赖的去磷酸化的后者，然后发生。这些性质与NDP激酶的性质不相同但相似，并且与腺苷酸激酶和ATP合酶的性质不相似。20 S蛋白酶体由许多低分子量亚基组成，排列成四个环的堆叠，每个环包含七个不同的α-或β-亚基。在这些亚基中，我们鉴定了β-型亚基中的C5和α-型亚基中的C8在γ-磷酸转移反应中被自磷酸化，并被8-叠氮基-[α-^ P] ATP光亲和标记<32>，表明蛋白酶体的C5和C8亚基负责NDP激酶样活性。我们正在试图确定NDP激酶在这些蛋白中的活性位点，并试图确定NDP激酶在Hsp 70的伴侣活性中的作用以及20 S蛋白酶体蛋白水解过程中底物的构象修饰。

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.