Ubiquitin Aldehyde Increases Adenosine Triphosphate–Dependent Proteolysis of Hemoglobin α-Subunits in β-Thalassemic Hemolysates

Abstract

AbstractTwo major causes of the anemia in β-thalassemia are a deficiency in hemoglobin (Hb) β-subunit (and consequently HbA) synthesis and, due to the resulting excess of Hb α-subunits, erythroid cell hemolysis. The hemolytic component might be ameliorated by increasing the intracellular proteolysis of the excess α-subunits. Isolated 3H-labeled α-chains are known to be degraded primarily by the adenosine triphosphate (ATP)- and ubiquitin (Ub)-dependent proteolysis pathway in unfractionated β-thalassemic hemolysates. Our objective was to increase this degradation by targeted intervention. Ub aldehyde (Ubal), a synthetic inhibitor of isopeptidases (proteases that hydrolyze the bond between the Ub polypeptide and its protein adduct), was added to reaction mixtures containing a hemolysate from the blood cells of one of four β-thalassemic donors and 3H-α-chains or 3H-α-globin as a substrate. Optimum enhancement of ATP-dependent degradation occurred at 0.4 to 1.5 μmol/L Ubal and ranged from 29% to 115% for 3H-α-chains and 47% to 96% for 3H-α-globin among the four hemolysates. We suggest that Ubal stimulates 3H-α-subunit proteolysis by inhibition of an isopeptidase(s) that deubiquitinates, or “edits,” Ub-3H-α-subunit conjugates, intermediates in the degradative pathway. In control studies, similarly low Ubal concentrations did not enhance the degradation of 3H-α2β2 (HbA) tetramers or inhibit the activities of methemoglobin reductase and four selected glycolysis pathway enzymes. These and other results may be the basis for a therapeutic approach to β-thalassemia.