Dissecting the assembly pathway of the 20S proteasome.

Proteasomes reach their mature active state via a complex cascade of folding, assembly and processing events. The Rhodococcus proteasome offers a means to dissect the assembly pathway and to characterize intermediates; its four subunits (alpha1, alpha2, beta1, beta2) assemble efficiently in vitro with any combination of alpha and beta. Assembly studies with wild-type and N-terminally truncated beta-subunits in conjunction with refolding studies allowed to define the role of the propeptide which is two-fold: It supports the initial folding of the beta-subunits and it promotes the maturation of the holoproteasomes.

Active site-directed inhibitors of Rhodococcus 20 S proteasome. Kinetics and mechanism.

We have studied the mechanism of inhibition of the recombinant Rhodococcus proteasome by four different chemical classes of active site-directed small molecule inhibitors. Clasto-lactacystin beta-lactone is a time-dependent inhibitor of the Rhodococcus proteasome's ability to hydrolyze Suc-Leu-Leu-Val-Tyr-AMC, a substrate for this proteasome's single type of active site, and proceeds with a kinact/[I] of 1,700 M-1 s-1. Using peptide mapping of tryptic digests, LC/MS, and amino acid sequence analysis, we have established that the Ogamma of the hydroxyl group on the N-terminal threonine of the beta-subunit is the sole modification made by the beta-lactone. Active site titrations of the Rhodococcus proteasome with reversible peptide aldehydes show the expected stoichiometry of one inhibitor molecule per beta-subunit. Prior modification with beta-lactone completely abrogates the binding of peptidyl boronic acid inhibitors, suggesting that these inhibitors also inactivate the enzyme by reacting with the Ogamma moiety on Thr1. High performance liquid chromatography analysis of peptidyl vinyl sulfone-modified intact Rhodococcus proteasome beta-subunit and its tryptic peptides suggests that the peptidyl vinyl sulfone modifies a residue in the N-terminal 20 amino acids. This modification is also blocked by prior treatment with beta-lactone.

Eubacterial proteasomes.

Proteasomes are large, multisubunit proteases with highly conserved structures. The 26S proteasome of eukaryotes is an ATP-dependent enzyme of about 2 MDa, which acts as the central protease of the ubiquitin-dependent pathway of protein degradation. The core of the 26S complex is formed by the 20S proteasome, an ATP-independent, barrel-shaped protease of about 700 kDa, which has also been detected in archaebacteria and, more recently, in eubacteria. Currently, the distribution of 20S proteasomes in eubacteria appears limited to the actinomycetes, while most other eubacteria contain a related complex of simpler structure.

Subunit topology of the Rhodococcus proteasome.

The 20S proteasome, isolated from the nocardioform actinomycete Rhodococcus erythropolis strain NI86/21, is built from two alpha-type and two beta-type subunits. In order to probe the subunit topology, we have set up an expression system which allows coexpression of the genes encoding the alpha- and beta-subunits in all possible combinations. The four respective constructs obtained yielded fully assembled and proteolytically active proteasomes. Biochemical, kinetic and electron microscopy analysis allow us to rule out several of the models which were originally envisaged for the subunit topology of the Rhodococcus proteasome. The experiments further indicate that the assembly pathways of the Rhodococcus and of the Thermoplasma proteasome differ in some important details.

2-D Crystallization of the Rhodococcus 20S Proteasome

The 2D crystallization method using a liquid-liquid interface has been applied to the Rhodococcus 20S proteasome. Two types of ordered arrays were obtained, both large enough for high-resolution analysis. The first one is a hexagonal close-packed array, whereas the second one has fourfold symmetry. By image analysis based on a real space correlation averaging technique, the close-packed array was found to be hexagonally packed but the molecules had complete rotational freedom. The fourfold array is, however, a true crystal with p4 symmetry. Lattice constants are a = b = 20.0 nm and the unit cell of this crystal contains two proteasomes. The diffraction pattern computed from the original picture shows the spots up to (4.5) that correspond to 3.1 nm resolution. After applying an unbending procedure, the diffraction pattern shows spots extending to 1.8 nm resolution.

The proteasome from Thermoplasma acidophilum is neither a cysteine nor a serine protease.

The 20 S proteasome, found in eukaryotes and in the archaebacterium Thermoplasma acidophilum, forms the proteolytic core of the 26 S proteasome which is the central protease of the non-lysosomal protein degradation pathway. Inhibitor studies have indicated that the 20 S proteasome may be an unusual type of cysteine or serine protease and a recent study of the Thermoplasma beta subunit has indicated that it carries the proteolytic activity. We have attempted to obtain information on the nature of the active site by mutating the only cysteine, both histidines and two completely conserved aspartates in the archaebacterial complex as well as all serines of the beta subunit, without decreasing the catalytic activity of the enzyme to any significant extent. Indeed, mutation of the conserved aspartate in the beta subunit increased the activity of the proteasome threefold. We conclude that the proteasome is not a cysteine or serine protease.