Characterization of recombinant REG alpha, REG beta, and REG gamma proteasome activators

Full-length cDNAs for three human proteasome activator subunits, called REG alpha, REG beta, and REG gamma, have been expressed in Escherichia coli, and the purified recombinant proteins have been characterized. Recombinant alpha or gamma subunits form heptameric species; recombinant beta subunits are found largely as monomers or small multimers. Each recombinant REG stimulates cleavage of fluorogenic peptides by human red cell proteasomes. The pattern of activated peptide hydrolysis is virtually identical for REG alpha and REG beta. These two subunits, alone or in combination, stimulate cleavage after basic, acidic, and most hydrophobic residues in many peptides. Recombinant alpha and beta subunits bind each other with high affinity, and the REG alpha/beta heteromeric complex activates hydrolysis of LLVY-methylcoumaryl-7-amide (LLVY-MCA) and LLE-beta-nitroanilide (LLE-beta NA) more than REG alpha or REG beta alone. Using filter binding and gel filtration assays, recombinant REG gamma subunits were shown to bind themselves but not alpha or beta subunits. REG gamma differs from REG alpha and REG beta in that it markedly stimulates hydrolysis of peptides with basic residues in the P1 position but only modestly activates cleavage of LLVY-MCA or LLE-beta NA by the proteasome. REG gamma binds the proteasome with higher affinity than REG alpha or REG beta yet with lower affinity than complexes containing both REG alpha and REG beta. In summary, each of the three REG homologs is a proteasome activator with unique biochemical properties.