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Changes in the heat shock 70 kDa protein level in human neutrophils induced by heat shock

Abstract

Changes in the content of constitutive and inducible proteins of the family of heat shock 70 kDa proteins (HSP70) caused by heat shock in human neutrophils, white blood cells with an atypically short lifespan, which provide a nonspecific defense of the organism against bacterial pathogens, have been studied. An analysis of the intracellular content of the constitutive and inducible HSP70 proteins by flow cytometry revealed a biphasic dynamics of changes in the protein level, which was characterized by an increase in the protein level immediately after heat shock followed by a decrease within 15–30 min after the termination of heat treatment. Because the inhibitor of protein synthesis cycloheximide did not change the dynamics profile, it was assumed that the increase in the HSP70 level is related not to the de novo synthesis of these proteins but to conformational changes of HSP70 molecules and an increased accessibility of some epitopes for antibody binding. Using a panel of antibodies specific to the N-terminal ATP-binding or the C-terminal substrate-binding domains of the protein, it was shown by cell immunofluorescence and flow cytometry that the heat shock-associated increase in the intracellular HSP70 level results from an increased efficiency of the binding of antibodies recognizing the substrate-binding domain. It was also demonstrated that the decrease in the intracellular HSP70 level after the heat shock, may be partially due to a release into the extracellular space of both the constitutive and inducible HSP70 proteins, which is regulated with the involvement of ABC-transporters.

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Abbreviations

mAb:

monoclonal antibody

DPBS:

Dulbecco’s phosphate-buffered saline, pH 7.4

HS:

heat shock

Hsc:

the constitutive heat shock protein of the 70-kDa family

Hsp 70:

the inducible heat shock protein of the 70-kDa family

HSP70 (HSPA):

the family of 70-kDa heat shock proteins

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Correspondence to E. I. Kovalenko.

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Original Russian Text © A.A. Boyko, S.S. Vetchinin, A.M. Sapozhnikov, E.I. Kovalenko, 2014, published in Bioorganicheskaya Khimiya, 2014, Vol. 40, No. 5, pp. 528–540.

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Boyko, A.A., Vetchinin, S.S., Sapozhnikov, A.M. et al. Changes in the heat shock 70 kDa protein level in human neutrophils induced by heat shock. Russ J Bioorg Chem 40, 488–498 (2014). https://doi.org/10.1134/S1068162014050045

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  • DOI: https://doi.org/10.1134/S1068162014050045

Keywords

  • neutrophils
  • cell stress
  • heat shock 70 kDa proteins
  • Hsp70
  • Hsc70
  • heat shock