Characterization of Recombinant REGα, REGβ, and REGγ Proteasome Activators

Abstract

Full-length cDNAs for three human proteasome activator subunits, called REG␣, REG␤, and REG␥, have been expressed in Escherichia coli, and the purified recombinant proteins have been characterized. Recombinant ␣ or ␥ subunits form heptameric species; recombinant ␤ 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␣ and REG␤. These two subunits, alone or in combination, stimulate cleavage after basic, acidic, and most hydrophobic residues in many peptides. Recombinant ␣ and ␤ subunits bind each other with high affinity, and the REG␣/␤ heteromeric complex activates hydrolysis of LLVY-methylcoumaryl-7-amide (LLVY-MCA) and LLE-␤-nitroanilide (LLE-␤NA) more than REG␣ or REG␤ alone. Using filter binding and gel filtration assays, recombinant REG␥ subunits were shown to bind themselves but not ␣ or ␤ subunits. REG␥ differs from REG␣ and REG␤ 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-␤NA by the proteasome. REG␥ binds the proteasome with higher affinity than REG␣ or REG␤ yet with lower affinity than complexes containing both REG␣ and REG␤. In summary, each of the three REG homologs is a proteasome activator with unique biochemical properties.

Expression of Proteasome Activator REGγ in Human Laryngeal Carcinoma and Associations with Tumor Suppressor Proteins

“…Most cellular proteins are degraded through the 26S proteasome after ubiquitination, a way for 26S to recognize proteins that need to be degraded. The alternative proteasomal activator, REG (also known as 11S proteasome) activates the 20S proteasome in an ATP-independent manner and is primarily responsible for ubiquitin-independent protein degradation (Realini et al, 1997). REGα/β can be induced by interferon(IFN)-γ and play an important role in MHC class I antigen presentation.…”

“…The 19S activator binds to 20S proteasome to form the 26S proteasome in an ATP-dependent manner, which mainly performs degradation of proteins in a ubiquitindependent manner. In contrast, the REG activates the 20S proteasome in an ATP-independent manner and is primarily responsible for ubiquitin-independent protein degradation (Realini et al, 1997). There are three REG homologs, called α, β and γ.…”

The proteasome: a protein‐destroying machine

“…It remains to be seen whether a mixture of recombinant PA28␣ and PA28␤ forms a heterohexameric or heteroheptameric complex. Realini et al (1997) reported that recombinant REG␤ and REG␥ (equivalent to PA28␤ and PA28␥, respectively) are capable of stimulating the peptidase activities of the 20S proteasome. Contrary to these results, Song et al (1997) reported that PA28␤ alone has no detectable effect on the peptidase activities of the 20S proteasome but that it appreciably reduces the K m without affecting the V max for the PA28␣-activated proteasome, suggesting an important functional interaction between PA28␣ and PA28␤.…”

Biochemical Analysis of the 20 S Proteasome of Trypanosoma brucei

“…Purification of the 20 S proteasome from T. brucei procyclic form cells was carried out as previously described (33). The human 20 S proteasome, purified from outdated human blood by previously described methods (34), was a kind gift from Prof. Martin Rechsteiner of the University of Utah.…”