Heat stress induces hsc70/nuclear topoisomerase I complex formation in vivo: evidence for hsc70-mediated, ATP-independent reactivation in vitro.
We previously demonstrated that in murine T cells thermotolerance correlated with heat shock protein 70 (hsp70) synthesis and protection of nuclear type I topoisomerase (topo I). Topo I activity returned to normal levels following heat stress even in cells not rendered thermotolerant by a prior heat shock. Recovery of topo I activity was not dependent on de novo protein synthesis, suggesting that the cell possesses a pathway(s) for refolding this nuclear protein. In this report we demonstrate that topo I and hsc70, the constitutively produced member of the hsp70 family, associated in vivo during heat stress. That this association may play a physiologically important role in protecting topo I activity from heat stress was suggested by the observation that hsc70 protected topo I from heat inactivation in vitro. hsc70 but not actin also reactivated previously heat-denatured topo I in a dose-dependent fashion. However, refolding of heat-denatured topo I by purified hsc70 was inefficient relative to a hsc70-containing cell lysate. Protection from heat inactivation as well as reactivation by hsc70 did not require exogenous ATP. Similarly, reactivation by the cell lysate was not inhibited by ADP or a nonhydrolyzable analogue of ATP. Thus, our studies suggest that nuclear topo I complexes with hsc70 during heat stress, which may explain, at least in part, why hsp70 proteins accumulate in the nucleus, particularly the nucleolus. This interaction may limit heat-induced protein damage and/or accelerate restoration of protein function in an ATP-independent reaction.
Ciavarra
R P
RP
Department of Microbiology, Eastern Virginia Medical School, Norfolk 23501.
Goldman
C
C
Wen
K K
KK
Tedeschi
B
B
Castora
F J
FJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis*; D002467:Cell Nucleus / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004789:Enzyme Activation; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000096:biosynthesis*; D006358:Hot Temperature; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D013601:T-Lymphocytes / Q000378:metabolism; D059004:Topoisomerase I Inhibitors
D002352:Carrier Proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C494372:Hspa8 protein, mouse; D059004:Topoisomerase I Inhibitors; D000255:Adenosine Triphosphate; D004264:DNA Topoisomerases, Type I