Claudio Realini

KEKE MOTIFS. PROPOSED ROLES IN PROTEIN-PROTEIN ASSOCIATION AND PRESENTATION OF PEPTIDES BY MHC CLASS I RECEPTORS

A stretch of 28 ‘alternating’ lysine (K) and glutamate (E) residues is found in an activator of the multicatalytic protease. Such ‘KEKE sequences’ are also present in subunits of the multicatalytic protease, in subunits of the 26S protease and in a variety of chaperonins. We propose that KEKE regions promote association between protein complexes. Furthermore, they may contribute to the selection of peptides presented on MHC Class I receptors.

Structure of the proteasome activator REGα (PA28α)

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, REGα, REGβ and REGγ (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. REGα and REGβ preferentially form a heteromeric complex, although purified REGα forms a heptamer in solution and has biochemical properties similar to the heteromeric REGα/REGβ complex,. We have now determined the crystal structure of human recombinant REGα at 2.8 Å resolution. The heptameric barrel-shaped assembly contains a central channel that has an opening of 20 Å diameter at one end and another of 30 Å diameter at the presumed proteasome-binding surface. The binding of REG probably causes conformational changes that open a pore in the proteasome α-subunits through which substrates and products can pass.

Characterization of recombinant REGα, REGβ, and REGγ proteasome activators

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

Proteasome activator 11S REG or PA28: recombinant REG alpha/REG beta hetero-oligomers are heptamers.

The proteasome activator 11S REG or PA28 is a conical molecule composed of two homologous subunits, REG alpha and REG beta. Recombinant REG alpha forms a heptamer, whereas recombinant REG beta is a monomer. When mixed with REG beta, a monomeric REG alpha mutant (N50Y) forms an active hetero-oligomer in which the molar ratio of REG beta to REG alpha(N50Y) is close to 1.3. This apparent stoichiometry is consistent with the REG alpha(N50Y)/REG beta hetero-oligomer being a heptamer composed of three alpha and four beta subunits. Chemical cross-linking of the alpha/beta oligomers revealed the presence of REG alpha-REG beta and REG beta-REG beta dimers, but REG alpha-REG alpha dimers were not detected. The mass of the REG alpha(N50Y)/REG beta hetero-oligomer determined by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) is 194 871 +/- 40 Da in good agreement with the theoretical mass of 194 856 Da for an alpha 3 beta 4 heptamer. Hexamers were not observed in the mass spectrum. For wild-type REG subunits coexpressed in bacteria cells at an apparent beta/alpha molar ratio of approximately 1.2, the resulting hetero-oligomers observed by ESI-TOF MS were again predominantly alpha 3 beta 4 heptamers, with trace amounts of alpha 4 beta heptamers also present. On the other hand, the mass spectrum contained a mixture of alpha 7, alpha 6 beta 1, alpha 5 beta 2, and alpha 4 beta 3 heptamers when the REG beta/REG alpha ratio was 0.1. Thus, formation of heptamers is an intrinsic property of recombinant REG alpha and REG beta subunits. On the basis of these results, we propose that 11S REG purified directly from eukaryotic cells is also heptameric, likely alpha 3 beta 4 or a mixture of alpha 3 beta 4 and alpha 4 beta 3 species.

Human lymphoblast and erythrocyte multicatalytic proteases: differential peptidase activities and responses to the 11S regulator

The multicatalytic protease (MCP) or 20S proteasome was purified from human red blood cells and two lymphoblastoid cell lines, 721.45 which constitutively expresses protease subunits LMP2 and LMP7, and 721.174 in which genes for these subunits are deleted. Each MCP was assayed using a series of fluorogenic peptides. The hydrophobic peptides gGGF‐MCA, sRPFHLLVY‐MCA and sLY‐MCA were particularly good substrates for 721.45 MCP as compared to the enzyme from 721.174 and red blood cells. In addition, hydrolysis of gGGF‐MCA and sLY‐MCA was activated by human red blood cell and recombinant regulators to a greater extent using MCP from 721.45 lymphoblasts. Thus, LMP2/LMP7 and regulator appear to act synergistically in the enhanced degradation of gGGF‐MCA and sLY‐MCA by the multicatalytic protease.

Potential immunocompetence of proteolytic fragments produced by proteasomes before evolution of the vertebrate immune system

To generate peptides for presentation by major histocompatibility complex (MHC) class I molecules to T lymphocytes, the immune system of vertebrates has recruited the proteasomes, phylogenetically ancient multicatalytic high molecular weight endoproteases. We have previously shown that many of the proteolytic fragments generated by vertebrate proteasomes have structural features in common with peptides eluted from MHC class I molecules, suggesting that many MHC class I ligands are direct products of proteasomal proteolysis. Here, we report that the processing of polypeptides by proteasomes is conserved in evolution, not only among vertebrate species, but including invertebrate eukaryotes such as insects and yeast. Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes. Moreover, many major dual cleavage peptides produced by invertebrate proteasomes have the length and the NH2 and COOH termini preferred by MHC class I. Thus, the ability of proteasomes to generate potentially immunocompetent peptides evolved well before the vertebrate immune system. We demonstrate with polypeptide substrates that interferon γ induction in vivo or addition of recombinant proteasome activator 28α in vitro alters proteasomal proteolysis in such a way that the generation of peptides with the structural features of MHC class I ligands is optimized. However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis. The data suggest that proteasomes may have influenced the evolution of MHC class I molecules.