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

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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.