Proteasome - Enzymes

The 26S Proteasome (~2500 kDa) is a multicatalytic enzyme with a highly ordered structure composed of at least 32 different subunits arranged in two sub-complexes (a 20S core and a 19S regulator). The 20S core (700 kDa) is composed of 4 rings of 28 non-identical subunits (2 rings are composed of 7 alpha subunits and 2 rings are composed of 7 beta subunits). Proteolysis occurs within the hollow 20S core which can hydrolyze small peptide substrates, short and unstructured polypeptides and occasionally some proteins with hydrophobic or misfolded patches in vitro. Purified 20S has latent peptidase activity which can be activated chemically by the addition of low amounts of SDS or by the association of regulatory particles (19S or 11S regulatory particles). The 19S regulator is composed of a base (containing 6 ATPase and 2 non-ATPase subunits) and a lid (containing up to 10 non-ATPase subunits). This regulatory particle is required for the degradation of poly-ubiquitinated substrates. The 19S 10-protein base binds directly to the 20S α ring and requires ATP binding, while the 19S 9-protein lid binds to poly-ubiquitin chains. Subunits in the 19S collectively contain Ub receptors, deubiquitinating and ATPase activities. This particle recognizes Ub chains, binds to the substrate, unravels its tertiary structure, aids in translocating it through a gated channel into the 20S core, and also disassembles poly-ubiquitin chains.

The immuno proteasome is a modified form of the constitutive 20S that has the essential function of processing class I MHC peptides. During immunoproteasome assembly, the constitutive catalytic Β subunits (Delta, Z and X) are replaced by the inducible subunits LMP2, LMP7 and MECL, the former two being encoded by MHC. The immuno-proteasome functions with an alternate regulator, referred to as the 11S regulator (PA28) that replaces the 19S regulator. This non-ATPase activator complex enhances the generation of class I binding peptides by altering the cleavage pattern of the proteasome. The PA28 complex is constitutively expressed in antigen-presenting cells and its expression is up-regulated by interferon-y. The 11S proteins are not ATPases and form a heptameric structure that promotes the degradation of short peptides but not whole proteins. There are three different PA28 proteins subunits have been identified and they include: PA28α(PSME1) PA28Β(PSME2)and PA28y(PSME3) which can bind to the 20S α ring via C-terminal sequences, and induced conformational changes in the 20S to open the core structure. Each activator complex confers unique protease specificity to the 20S.

The proteolytic activity of the 20S proteasome can be determined in vitro by using a number of small fluorescent peptide substrates, and can be inhibited by various natural product and peptide inhibitors. There are peptide substrates conjugated to fluorophores for the selective measurement of the tryptic (Β2), chymotryptic (Β5) or PGPH activities (Β1) which cleave peptide bonds after basic or acidic residues respectively. The proteasome also possesses two other proteolytic activities which are less well-characterized, ie., branched-chain amino acid-preferring (BrAAP) and small neutral amino acid-preferring (SNAAP) activities. Proteasome inhibitors are broadly categorized as synthetic analogs or natural products and each type has differential specificities. Synthetic inhibitors are peptide based and include derivatives of benzamides, α-ketoamides and -ketoaldehydes, aldehydes (eg. MG132), vinyl sulfones and boronic acids. Peptide aldehydes (such as MG132) are widely used .but they also inhibit lysosomal and calcium activated proteases, and vinyl sulphone based inhibitors also inhibit intracellular cysteine proteinases. PS-341 (Bortezomib) is a dipeptide boronic acid that is a reversible inhibitor of the chymotryptic activity of the proteasome. It is the first therapeutic proteasome inhibitor to be tested in humans and is now marketed as Velcade (produced by Millenium Pharmaceuticals) used in chemotherapy for multiple myeloma. Natural product inhibitors include linear peptide epoxyketones, peptide macrocylesy-lactam thiol ester and epipolythiodioxopiperazine toxin. The natural products lactacystin (Β-lactone as the active form) is a Streptomyces lactacytinaeus metabolite that irreversibly modifies the N-terinal threonine of Βsubunits. Lactacystin however, also inhibits cathepsine A and THP1. Epoxomoycin Epoxomoycin is a linear expoxyketone derived from an actinomycete strain (No.Q996-17) and is highly specific for the 20S.