Inhibition of Hsp70 ATPase activity and protein renaturation by a novel Hsp70-binding protein.
A cDNA that codes for an Hsp70-interacting protein (HspBP1) was isolated from a human heart cDNA library using the yeast two-hybrid system. The derived amino acid sequence is unique and therefore represents a new regulator of Hsp70. Northern blots of RNA from human tissues indicate that HspBP1 mRNA has a size of approximately 1.7 kilobase pairs and is present in all tissues analyzed but is most abundant in heart and skeletal muscle. Western blot analysis revealed a protein of approximately 40 kilodaltons detected in cell extracts. The ATPase domain of Hsp70 demonstrated binding to HspBP1. Further experiments showed binding of HspBP1 to Hsp70 and Hsc70 in a total heart extract. HspBP1 (8 microM) inhibited approximately 90% of the Hsp40-activated Hsp70 ATPase activity. HspBP1 prevented ATP binding to Hsp70, and therefore this is the likely mechanism of inhibition. Hsp40-activated ATPase activity is essential for the renaturation activity of Hsp70; therefore, the effects of HspBP1 on renaturation of luciferase in a reticulocyte lysate and a defined system were examined. HspBP1 inhibited renaturation with half-maximal inhibition at 2 microM. These data indicate that we have identified a novel Hsp70-interacting protein that inhibits Hsp70 chaperone activity.
Raynes
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Department of Animal Sciences, University of Arizona, Tucson, Arizona 85721-0038, USA.
Guerriero
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D048868:Adaptor Proteins, Signal Transducing; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D018840:HSP70 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism; D011489:Protein Denaturation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018076:DNA, Complementary; D018840:HSP70 Heat-Shock Proteins; C116331:HSPBP1 protein, human; D012333:RNA, Messenger; D000251:Adenosine Triphosphatases