Heat Shock Protein 70 Cytosolic Sequestration by Excess of Free Alpha-Globin Chains Is a Key Mechanism of the Ineffective Erythropoiesis in β-Thalassemia Major Patients

Despite extensive knowledge of the molecular defects causing β-thalassemia major (TM), less is known about the mechanisms responsible of the ineffective erythropoiesis. This latter is characterized by accelerated erythroid differentiation, apoptosis and maturation arrest at the polychromatophilic stage. It explains, at least in part, the profound anemia observed in this disease. Although it has been proposed that both the precipitation of unmatched globin chains as well as the accumulation of unbound iron could lead to oxidative stress and subsequent hemolysis, the mechanism by which apoptosis and maturation arrest are induced remains unclear.

We have previously demonstrated that normal human erythroid cell maturation requires the transcription factor GATA-1 and an transient activation of caspase-3 at the basophilic stage (Zermati Y, J Exp Med 2001). Although GATA-1 is a capsase-3 target, we have shown that it was protected during erythroid maturation by the nucleus translocation of heat shock protein 70 (Hsp70) at the onset of caspase-3 activation (Ribeil JA, Nature 2007). Hsp70 is an universally highly conserved protein chaperone involved in the regulation of protein folding and in the prevention of damaging aggregates formation (Hart HU, Nature 2011). We hypothesized that, in addition to AHSP the cognate chaperone of α-globin chains, Hsp70 could also serve as a secondary chaperone in case of excess of free α-globin chains or aggregates to reduce their toxicity. As a result, this interaction could be detrimental by preventing the nuclear localization of Hsp70 leading to the degradation of GATA-1, maturation arrest and apoptosis in TM erythroid mature cells.

During erythroid maturation of CD34+ derived from TM peripheral blood (n=8) or bone marrow (n=3), we have first shown by confocal microscopy that, in contrast to CD34+ from healthy donor (n=10), Hsp70 nuclear translocation did not occur at the late stage of maturation. As a result, GATA-1 was poorly expressed, end stage erythroid maturation was arrested and apoptosis occurred. Hsp70/α-globin complexes were evidenced by confocal analysis in cytosol (specific co-localization), by immunoprecipitation of lysed differentiated erythroid TM cells in liquid culture, and by yeast two-hydrid experiments. In order to better characterize this interaction, we have demonstrated by molecular modeling, docking and molecular dynamic simulation (3D models of the human full-length Hsp70^1-701generated by homology modeling from crystallographic structures) that α-globin bounds to a highly electronegative cavity formed by NBD, SBD and LID (3 major domains of Hsp70). The Hsp70/α-globin complex is characterized by extensive protein-protein interactions stabilized by multiple hydrogen bonds that we have accurately characterized.

Then, to demonstrate the pathological role of the Hsp70 cytosolic sequestration, by lentivirus transfection of TM CD34+ cells, we have expressed a nucleus-targeted mutant of Hsp70 and a wtHsp70 (overexpressing Hsp70). As expected, both lentivirus increased nuclear Hsp70 expression and rescued GATA-1 expression. They also increased TM erythroid cell maturation (more mature cells -acidophils and reticulocytes- than in control transfected cells -21.5% vs 11,8%, p<0,05) and decreased apoptosis (50% decreased). Transfection with a retrovirus mutant GATA-1 (μGATA-1, uncleavable by actived caspase-3) had similar positive effects on erythroid maturation and cell death. Finally, by flow cytometry analysis we could observe that concomitantly to GATA-1 protection in these experiments the percentage of F cells was significantly increased (+20%) at end stages of maturation. To ensure the specificity of our findings, β-globin gene lentivirus transfection of TM CD34+ cells led to disappearance of Hsp70/α-globin interactions in the cytosol, Hsp70 nuclear localization, GATA-1 protection and normal erythroid maturation.

Our data indicate that, cytosolic Hsp70 sequestration by α-globin chains prevents its nuclear localization and is a key mechanism of the ineffective erythropoiesis observed in β-TM patients. In order to increase nuclear Hsp70 translocation, developing drugs or small molecules that could increase Hsp70 expression or better disrupt the Hsp70/α-globin complex could be a novel approach of targeted therapies to improve erythropoiesis in TM.

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