Studies during the 1960s and 1970s suggested that some of the excess α-subunits are destroyed by intracellular proteolysis, although the biochemical mechanism and a basis for its inefficiency in α-subunit degradation were unknown. During the 1980s one of us1, 2 showed that the excess, newly synthesized 3H-α chains (α-subunits with associated heme groups) in intact β-thalassemic reticulocytes or their unfractionated hemolysates were degraded primarily by the ATP-and ubiquitin-dependent proteolysis pathway. In this major proteolysis system (FIG. 1), the carboxyl terminus of ubiquitin (Ub), a polypeptide of 8,565 Da, is activated in an ATP-dependent reaction. Through a series of enzymatic steps, activated Ub molecules are covalently linked by an “isopeptide” bond to the ε-amino group (s) of one or more lysine residues of the protein destined for proteolysis (reviewed by Hershko and Ciechanover5). These Ub-protein conjugates are recognized through their Ub “tag” by the 26S proteasome, a multi-subunit protease complex, and the protein substrate moiety is then degraded in a process that also utilizes ATP. The Ub components of the Ub-protein conjugates are released to the cytoplasmic pool and thus serve as catalysts in the proteolysis reaction.

The cytoplasm that catalyzes the degradation of ubiquitinated proteins also contains several Ub-protein hydrolases or “isopeptidases.” One (or a subset) of these enzymes cleaves the isopeptide bond between the carboxyl terminus of Ub and the ε-amino group (s) of the conjugated protein or, in the case of a polyUb chain, of the neighboring Ub monomer. This type of isopeptidase deubiquitinates or “disassembles” the conjugate and thus can play an “editing” role (FIG. 1) in the proteolysis scheme. Studies by one of us and his colleagues6, 7 showed that addition of submicromolar amounts of ubiquitin aldehyde (Ubal), a potent inhibitor of many isopeptidases, greatly increased the levels of the conjugates of cytochrome c and ribonuclease A substrates in crude ubiquitination reaction mixtures. Presumably, this synthetic compound, a Ub derivative with the C-terminal carboxyl replaced by an aldehyde group, 8 inhibited isopeptidases that disassembled these conjugates.