Stressful preconditioning and HSP70 overexpression attenuate proteotoxicity of cellular ATP depletion.

Rat H9c2 myoblasts were preconditioned by heat or metabolic stress followed by recovery under normal conditions. Cells were then subjected to severe ATP depletion, and stress-associated proteotoxicity was assessed on 1) the increase in a Triton X-100-insoluble component of total cellular protein and 2) the rate of inactivation and insolubilization of transfected luciferase with cytoplasmic or nuclear localization. Both heat and metabolic preconditioning elevated the intracellular heat shock protein 70 (HSP70) level and reduced cell death after sustained ATP depletion without affecting the rate and extent of ATP decrease. Each preconditioning attenuated the stress-induced insolubility among total cellular protein as well as the inactivation and insolubilization of cytoplasmic and nuclear luciferase. Transient overexpression of human HSP70 in cells also attenuated both the cytotoxic and proteotoxic effects of ATP depletion. Quercetin, a blocker of stress-responsive HSP expression, abolished the effects of stressful preconditioning but did not influence the effects of overexpressed HSP70. Analyses of the cellular fractions revealed that both the stress-preconditioned and HSP70-overexpressing cells retain the soluble pool of HSP70 longer during ATP depletion. Larger amounts of other proteins coimmunoprecipitated with excess HSP70 compared with control cells deprived of ATP. This is the first demonstration of positive correlation between chaperone activity within cells and their viability in the context of ischemia-like stress.

[1]  H. Kampinga,et al.  Modulation of in Vivo HSP70 Chaperone Activity by Hip and Bag-1* , 2001, The Journal of Biological Chemistry.

[2]  M. Sherman,et al.  Suppression of Stress Kinase JNK Is Involved in HSP72-mediated Protection of Myogenic Cells from Transient Energy Deprivation , 2000, The Journal of Biological Chemistry.

[3]  H. Kampinga,et al.  In Vivo Chaperone Activity of Heat Shock Protein 70 and Thermotolerance , 1999, Molecular and Cellular Biology.

[4]  D. Latchman,et al.  Protection of Neuronal Cells from Apoptosis by Hsp27 Delivered with a Herpes Simplex Virus-based Vector* , 1999, The Journal of Biological Chemistry.

[5]  N. Beaucamp,et al.  Overexpression of hsp70i facilitates reactivation of intracellular proteins in neurones and protects them from denaturing stress , 1998, FEBS letters.

[6]  A. Kabakov,et al.  Early and delayed tolerance to simulated ischemia in heat-preconditioned endothelial cells: a role for HSP27. , 1998, American journal of physiology. Heart and circulatory physiology.

[7]  D. Latchman,et al.  Heat shock proteins: protective effect and potential therapeutic use (review). , 1998, International journal of molecular medicine.

[8]  A. Kabakov,et al.  Protein phosphatase inhibitors and heat preconditioning prevent Hsp27 dephosphorylation, F‐actin disruption and deterioration of morphology in ATP‐depleted endothelial cells , 1998, FEBS letters.

[9]  D. McMillan,et al.  Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and disease. , 1998, Circulation research.

[10]  O. Owen,et al.  Constitutive and inducible hsp70s are involved in oxidative resistance evoked by heat shock or ethanol. , 1998, Journal of molecular and cellular cardiology.

[11]  H. Kampinga,et al.  Hsp70 and Hsp40 Chaperone Activities in the Cytoplasm and the Nucleus of Mammalian Cells* , 1997, The Journal of Biological Chemistry.

[12]  V. Gabai,et al.  Heat Shock Proteins and Cytoprotection: Atp-Deprived Mammalian Cells , 1997 .

[13]  S. Schiaffino,et al.  Binding of cytosolic proteins to myofibrils in ischemic rat hearts. , 1996, Circulation research.

[14]  H. Kampinga,et al.  Thermostability of a nuclear-targeted luciferase expressed in mammalian cells. Destabilizing influence of the intranuclear microenvironment. , 1995, European journal of biochemistry.

[15]  A. Molotkov,et al.  Adaptation of ehrlich ascites carcinoma cells to energy deprivation in vivo can be associated with heat shock protein accumulation , 1995, Journal of cellular physiology.

[16]  D S Latchman,et al.  Differential cytoprotection against heat stress or hypoxia following expression of specific stress protein genes in myogenic cells. , 1995, Journal of molecular and cellular cardiology.

[17]  V L Gabai,et al.  Heat shock-induced accumulation of 70-kDa stress protein (HSP70) can protect ATP-depleted tumor cells from necrosis. , 1995, Experimental cell research.

[18]  M. Kashgarian,et al.  Activation of heat-shock transcription factor by graded reductions in renal ATP, in vivo, in the rat. , 1994, The Journal of clinical investigation.

[19]  H. Kampinga,et al.  Importance of the ATP-binding domain and nucleolar localization domain of HSP72 in the protection of nuclear proteins against heat-induced aggregation. , 1994, Experimental cell research.

[20]  V. Gabai,et al.  Stress-induced insolubilization of certain proteins in ascites tumor cells. , 1994, Archives of biochemistry and biophysics.

[21]  O. Bensaude,et al.  Increased thermal aggregation of proteins in ATP-depleted mammalian cells. , 1994, European journal of biochemistry.

[22]  R. Mestril,et al.  Expression of inducible stress protein 70 in rat heart myogenic cells confers protection against simulated ischemia-induced injury. , 1994, The Journal of clinical investigation.

[23]  William J. Welch,et al.  ATP-induced protein Hsp70 complex dissociation requires K+ but not ATP hydrolysis , 1993, Nature.

[24]  V L Gabai,et al.  Rise in heat‐shock protein level confers tolerance to energy deprivation , 1993, FEBS letters.

[25]  C. Ganote,et al.  Ischaemia and the myocyte cytoskeleton: review and speculation. , 1993, Cardiovascular research.

[26]  K. Nagata,et al.  Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids , 1992, Molecular and cellular biology.

[27]  M. Lovett,et al.  Examining the function and regulation of hsp 70 in cells subjected to metabolic stress , 1992, The Journal of cell biology.

[28]  S. Horie,et al.  Induction of stress proteins in cultured myogenic cells. Molecular signals for the activation of heat shock transcription factor during ischemia. , 1992, The Journal of clinical investigation.

[29]  G. Li,et al.  Heat shock protein hsp70 protects cells from thermal stress even after deletion of its ATP-binding domain. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[30]  A. Fink,et al.  Interaction of hsp70 with unfolded proteins: effects of temperature and nucleotides on the kinetics of binding. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[31]  R. van Wijk,et al.  The isolated neonatal rat-cardiomyocyte used in an in vitro model for 'ischemia'. II. Induction of the 68 kDa heat shock protein. , 1991, Biochimica et biophysica acta.

[32]  M. Morange,et al.  Protein denaturation during heat shock and related stress. Escherichia coli beta-galactosidase and Photinus pyralis luciferase inactivation in mouse cells. , 1989, The Journal of biological chemistry.

[33]  J. M. Burger,et al.  ATP and microfilaments in cellular oxidant injury. , 1988, The American journal of pathology.

[34]  D. Latchman,et al.  Heat shock proteins delivered with a virus vector can protect cardiac cells against apoptosis as well as against thermal or hypoxic stress. , 1999, Journal of molecular and cellular cardiology.

[35]  K. Nagata,et al.  Regulation of thermotolerance and ischemic tolerance. , 1996, EXS.

[36]  R. Morimoto,et al.  Stress-Inducible Cellular Responses , 1996, EXS.