title: Ubiquitin aldehyde increases adenosine triphosphate-dependent proteolysis of hemoglobin alpha-subunits in beta-thalassemic hemolysates.
abstract: Two major causes of the anemia in beta-thalassemia are a deficiency in hemoglobin (Hb) beta-subunit (and consequently HbA) synthesis and, due to the resulting excess of Hb alpha-subunits, erythroid cell hemolysis. The hemolytic component might be ameliorated by increasing the intracellular proteolysis of the excess alpha-subunits. Isolated 3H-labeled alpha-chains are known to be degraded primarily by the adenosine triphosphate (ATP)- and ubiquitin (Ub)-dependent proteolysis pathway in unfractionated beta-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 beta-thalassemic donors and 3H-alpha-chains or 3H-alpha-globin as a substrate. Optimum enhancement of ATP-dependent degradation occurred at 0.4 to 1.5 micromol/L Ubal and ranged from 29% to 115% for 3H-alpha-chains and 47% to 96% for 3H-alpha-globin among the four hemolysates. We suggest that Ubal stimulates 3H-alpha-subunit proteolysis by inhibition of an isopeptidase(s) that deubiquitinates, or "edits," Ub-3H-alpha-subunit conjugates, intermediates in the degradative pathway. In control studies, similarly low Ubal concentrations did not enhance the degradation of 3H-alpha2beta2 (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 beta-thalassemia.
authors.affiliation: Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242-1109, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Shaeffer
authors.forename: R E
authors.initials: RE
authors.lastname: Cohen
mesh_terms: D000255:Adenosine Triphosphate / Q000502:physiology*; D019759:Carbon-Nitrogen Lyases; D002474:Cell-Free System; D004353:Drug Evaluation, Preclinical; D010450:Endopeptidases / Q000097:blood*; D005914:Globins / Q000378:metabolism*; D006441:Hemoglobin A / Q000378:metabolism*; D006461:Hemolysis; D006801:Humans; D008190:Lyases / Q000037:antagonists & inhibitors / Q000097:blood; D013268:Stimulation, Chemical; D014452:Ubiquitins / Q000031:analogs & derivatives* / Q000494:pharmacology; D017086:beta-Thalassemia / Q000097:blood* / Q000235:genetics
chemical_list: D014452:Ubiquitins; C051927:ubiquitin-aldehyde; D000255:Adenosine Triphosphate; D005914:Globins; D006441:Hemoglobin A; D010450:Endopeptidases; D008190:Lyases; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase