The Proteasome Activator 11 S Regulator or PA28

Abstract

The proteasome 11 S regulator (REG) consists of two homologous subunits, REG␣ and REG␤. Each subunit is capable of activating the proteasome, and when combined, they form superactive REG␣/REG␤ complexes. We have previously shown that a highly conserved loop in the REG␣ crystal structure is critical for proteasome activation. We now show that hetero-oligomers formed from REG␣ activation loop mutants and wild-type REG␤ or vice versa are partially active. By contrast, heterooligomers bearing mutations in the activation loops of REG␣ and REG␤ subunits are inactive, demonstrating that both ␣ and ␤ subunits contribute to proteasome activation. We have also characterized REG proteins with mutations near or at their C termini. Partially active REG␣(Y249C) and REG␣(M247V) and an inactive REG␣ subunit bearing five additional C-terminal amino acids formed active hetero-oligomers with REG␤. REG␤ subunits lacking 1, 2, or 9 C-terminal amino acids did not bind or activate the proteasome, but each of these mutants formed partially active hetero-oligomers with the monomer REG␣(N50Y). However, hetero-oligomers formed from REG subunits lacking the last 14 amino acids were unable to bind the proteasome. Thus, C-terminal regions of both ␣ and ␤ subunits are required for hetero-oligomers to bind the proteasome.The proteasome is a large proteolytic enzyme found in all three kingdoms, the archeae, prokaryotes, and eukaryotes (1-7). In higher eukaryotes, the proteasome is believed to play an important role in a variety of cellular processes, including cell cycle progression (8, 9), control of gene expression (10), and antigen presentation (11-13). The proteasome from the archaebacterium Thermoplasma acidophilum is formed from 14 identical ␣ subunits and 14 identical ␤ subunits. To date, 17 distinct subunits have been identified in proteasomes from higher eukaryotes. All can be classified into ␣ or ␤ families based on their homology to the ␣ or ␤ subunits of the Thermoplasma proteasome (13). The quaternary structures of yeast and Thermoplasma proteasomes are quite similar, as revealed by electron microscopy (14 -16) and confirmed by x-ray crystallography (17, 18). The enzymes are composed of four stacked rings with seven subunits in each ring. The two inner rings are formed from ␤ subunits, and the two outer rings consist of ␣ subunits. The proteolytic active sites are located in an inner chamber of the proteasome, with the N-terminal threonines on the ␤ subunits acting as nucleophiles (17)(18)(19). These active sites are not readily accessible from the cytosol, because only two 13-Å pores are present in the ␣ rings of the archaebacterial proteasome, and even these small pores are absent in the crystallized yeast proteasome (17,18). Therefore, some mechanisms must exist to promote substrate access to the proteasome's active sites.Several proteins have been found to bind the proteasome (20). Of these, the 19 S regulatory complex (21-24) and 11 S regulator (REG) 1 , or PA28 (25,26), are best characterized. The 19 S regulatory complex cons...

The Proteasome, a Novel Protease Regulated by Multiple Mechanisms

“…In any case, reconstitution of the ␣ and ␤ subunits restores full and efficient PA28 activity. This result has been explained alternatively by additive activities of each subunit or by modulatory effects of the ␤ subunit on the ␣ subunit (51,53). At least two domains of PA28 are important for its function.…”

“…Recombinant PA28␤ is a monomer. Effects of isolated PA28␤ on proteasome activation have produced conflicting data, whose basis may relate to the very high concentrations of the subunit required to achieve proteasome activation (51,53). In any case, reconstitution of the ␣ and ␤ subunits restores full and efficient PA28 activity.…”

“…At least two domains of PA28 are important for its function. The identity of C-terminal residues (tyrosine in the wild-type proteins) is a critical determinant for binding to the proteasome (48,53). Binding of PA28 to the proteasome is necessary but not sufficient for activation; another domain near the center of the primary structure is required for activation after PA28 binding (53).…”

Structural and Functional Characterizations of the Proteasome-activating Protein PA26 from Trypanosoma brucei

“…The results (data not shown) indicated no detectable hybridization band from any of the DNA digests and thus suggested that a homologous PA26 gene is not present in the genome of any of the four other protozoan pathogens. Recombinant PA26 Can Self-assemble to Form a Heptamer Ring-Recombinant human PA28␣ was reported capable of self-assembly in vitro into a heptamer ring (22,23,37). Its crystal structure showed that the COOH terminus of each subunit protein is important for polymerization as well as interactions between the heptamer ring and mammalian 20 S proteasome, whereas the NH 2 terminus is apparently not involved (23).…”

“…There are three isoforms of PA28: PA28␣, PA28␤, and PA28␥, sharing about 50% amino acid sequence identity (20). The ␣ and ␤ isoforms form a complex with 20 S proteasome in the form of a heptamer ring of three PA28␣ and four PA28␤ or three PA28␤ and four PA28␣ (21,22). The crystal structure of recombinant human PA28␣ has been recently resolved in the form of a self-assembled heptamer ring (23).…”