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.

The specificity of the 20S proteasome, which degrades many intracellular proteins, is regulated by protein complexes that bind to one or both ends of the cylindrical proteasome structure. One of these regulatory complexes, the 11S regulator (known as REG or PA28), stimulates proteasome peptidase activity and enhances the production of antigenic peptides for presentation by class I molecules of the major histocompatibility complex (MHC). The three REG subunits that have been identified, REGalpha, REGbeta and REGgamma (also known as the Ki antigen), share extensive sequence similarity, apart from a highly variable internal segment of 17-34 residues which may confer subunit-specific properties. REGalpha and REGbeta preferentially form a heteromeric complex, although purified REGalpha forms a heptamer in solution and has biochemical properties similar to the heteromeric REGalpha/REGbeta complex. We have now determined the crystal structure of human recombinant REGalpha at 2.8 A resolution. The heptameric barrel-shaped assembly contains a central channel that has an opening of 20 A diameter at one end and another of 30 A diameter at the presumed proteasome-binding surface. The binding of REG probably causes conformational changes that open a pore in the proteasome alpha-subunits through which substrates and products can pass.

To better understand proteasomal degradation of nuclear proteins and viral antigens we studied mutated forms of influenza virus nucleoprotein (NP) that misfold and are rapidly degraded by proteasomes. In the presence of proteasome inhibitors, mutated NP (dNP) accumulates in highly insoluble ubiquitinated and nonubiquitinated species in nuclear substructures known as promyelocytic leukemia oncogenic domains (PODs) and the microtubule organizing center (MTOC). Immunofluorescence revealed that dNP recruits proteasomes and a selective assortment of molecular chaperones to both locales, and that a similar (though less dramatic) effect is induced by proteasome inhibitors in the absence of dNP expression. Biochemical evidence is consistent with the idea that dNP is delivered to PODs/MTOC in the absence of proteasome inhibitors. Restoring proteasome activity while blocking protein synthesis results in disappearance of dNP from PODs and the MTOC and the generation of a major histocompatibility complex class I–bound peptide derived from dNP but not NP. These findings demonstrate that PODs and the MTOC serve as sites of proteasomal degradation of misfolded dNP and probably cellular proteins as well, and imply that antigenic peptides are generated at one or both of these sites.

Covalent linkage of ADP-ribose polymers to proteins is generally considered essential for the posttranslational modification of protein function by poly(ADP-ribosyl)ation. Here we demonstrate an alternative way by which ADP-ribose polymers may modify protein function. Using a highly stringent binding assay in combination with DNA sequencing gels, we found that ADP-ribose polymers bind noncovalently to a specific group of chromatin proteins, i.e., histones H1, H2A, H2B, H3, and H4 and protamine. This binding resisted strong acids, chaotropes, detergents, and high salt concentrations but was readily reversible by DNA. When the interactions of variously sized linear and branched polymer molecules with individual histone species were tested, the hierarchies of binding were branched polymers greater than long, linear polymers greater than short, linear polymers and H1 greater than H2A greater than H2B = H3 greater than H4. For histone H1, the target of polymer binding was the carboxy-terminal domain, which is also the domain most effective in inducing higher order structure of chromatin. Thus, noncovalent interactions may be involved in the modification of histone functions in chromatin.

Proteasomes can be markedly activated by associating with 19S regulatory complexes to form the 26S protease or by binding 11S protein complexes known as REG or PA28. Three REG subunits, ␣, ␤, and ␥, have been expressed in Escherichia coli, and each recombinant protein can activate human proteasomes. Combining PCR mutagenesis with an in vitro activity assay, we have isolated and characterized 36 inactive, single-site mutants of recombinant REG␣. Most are monomers that produce functional proteasome activators when mixed with REG␤ subunits. Five REG␣ mutants that remain inactive in the mixing assay contain amino acid substitutions clustered between Arg-141 and Gly-149. The crystal structure of the REG␣ heptamer shows that this region forms a loop at the base of each REG␣ subunit. One mutation in this loop (N146Y) yields a REG␣ heptamer that binds the proteasome as tightly as wild-type REG␣ but does not activate peptide hydrolysis. Corresponding amino acid substitutions in REG␤ (N135Y) and REG␥ (N151Y) produce inactive proteins that also bind the proteasome and inhibit proteasome activation by their normal counterparts. Our studies clearly demonstrate that REG binding to the proteasome can be separated from activation of the enzyme. Moreover, the dominant negative REGs identified here should prove valuable for elucidating the role(s) of these proteins in antigen presentation.A major mechanism for controlling viral infections involves cytotoxic T lymphocytes that recognize viral peptides presented on the cell surface by major histocompatability complex class I molecules and lyse the infected cells (1, 2). There is considerable evidence that some presented peptides, at least, are produced by the proteasome (3, 4). Crystal structures of Thermoplasma and yeast proteasomes reveal that they are cylindrical protein complexes, composed of four stacked rings (5, 6). The two end rings consist of catalytically inactive ␣-type subunits, whereas the two inner rings are composed of ␤-type subunits, some of which are catalytically active (7). The protease active sites are located within an inner chamber that is virtually sealed from the particles surface (5, 6). Thus, it seems clear that substrate entry to the sites of peptide bond hydrolysis must be tightly regulated.Two proteasome activators have been identified so far. The proteasome can either associate with a 19S regulatory complex to form the 26S protease, which is capable of degrading intact proteins (8-11), or the proteasome can bind an 11S activator called REG or PA28. This association greatly enhances fluorogenic peptide hydrolysis by the proteasome (12, 13). As isolated from human red blood cells, REG is a hexameric or heptameric ring formed from two homologous subunits, REG␣ and REG␤; these two subunits are, in turn, homologous to KI antigen or REG␥. cDNAs for all three proteins have been expressed in Escherichia coli, and each recombinant protein is capable of activating the proteasome in vitro (14).A variety of evidence suggests that REG is involved in antige...