Redox and Heat Shock Protein Hsp70 Affect the Binding of Erythropoietin RNA Binding Protein to Erythropoietin mRNA

Abstract

Factors affecting complex formation between the cytosolic protein identified in our laboratory to bind to erythropoietin (EPO) mRNA (ERBP) and this mRNA have been investigated. It was observed that reducing agents, dithiothreitol and 2-mercaptoethanol, enhanced ERBP binding activity in a dose-dependent manner. Oxidizing agents such as diamide abolished complex formation. Treatment with the sulfhydryl alkylating agent N-ethylmaleimide also resulted in inhibition of complex formation, suggesting a possible role of sulfhydryl groups in the interaction between this protein and EPO mRNA.

Since heat shock proteins are induced in response to a variety of stresses including hypoxia the role of these proteins in ERBP-EPO mRNA complex formation was studied. Using an electrophoretic mobility shift assay (EMSA) in which human anti-hsc70 was added to ERBP-containing human hepatoma cell (Hep3B) lysates it was found that the ERBP-EPO mRNA complex was supershifted when compared to normal controls. However, no super-shift was observed when hsp70 antibody was added to the reaction. Western blots of these EMSA gels show hsc70 localized to the ERBP-EPO mRNA complex. These findings suggest the involvement of hsc70 in ERBP-EPO mRNA complex formation and perhaps a novel role for hsps in the regulation of Epo mRNA stability. It is possible that hsc70 might be involved in the complex process that controls red blood cell production during oxygen deprivation.

The sections containing redox effects on ERBP have been published (5).