Molecular shredders: how proteasomes fulfill their role
Introduction
Intracellular protein degradation is one of the most tightly regulated processes in living cells. The basic regulatory principle is to restrict proteolytic action to specific locations that can be accessed only by polypeptides destined for destruction. Well-characterized examples are the proteasomes and other proteases, such as bleomycin hydrolase, ClpAP, DegP, HslU and tricorn (reviewed in [1]). Despite a complete lack of similarity in sequence, structure and mechanism, these proteases have converged to a similar quaternary architecture: the proteolytic subunits associate into oligomeric rings that stack upon each other, yielding barrel-like complexes. The proteolytic sites are buried within the central cavity and are only accessible through narrow gates. This arrangement hinders folded proteins from entering the lumen of the complex. To date, the most fully characterized member of the family of cage-forming proteases is the proteasome. The proteasome is the central protease in nonlysosomal ubiquitin-dependent protein degradation, and is involved in protein quality control, antigen processing, signal transduction, cell cycle control, cell differentiation and apoptosis [2]. The 26S proteasome is a large protein machine (2.5 MDa) that is found in both nuclei and cytoplasm. Its two major subcomplexes are the 670 kDa proteolytic core particle (CP, also known as the 20S proteasome) and the 900 kDa regulatory particle (RP, also known as PA700 or the 19S complex). Eubacteria, which lack ubiquitin, contain at least one proteasome-related system — a miniproteasome termed HslUV [3].
In general, ‘destruction machineries’ need to be controlled by regulatory proteins to maintain cell homeostasis. In the case of the 20S proteasome, degradation of small peptides is provoked by various activators, such as 11S/proteasome activator PA28 [4] or Blm3 [5•], whereas ubiquitinated proteins are recognized, unfolded and translocated into the proteolytic chamber of the proteasome by the action of the PA700/19S regulatory particle 2., 6.. This review aims to explain protease function in the context of the currently known proteasome structures, and to highlight structural features that control protease activity and specificity.
Section snippets
Structural variety of proteasomes
The proteasome from the archaeon Thermoplasma acidophilum represents the prototype for the quaternary structure and topology of the CP 7., 8. (Figure 1). The proteasomal subunits assemble into molecular barrels with a diameter of 100 Å and a length of 160 Å. The CP comprises 28 subunits, which are arranged in four seven-membered rings that stack upon each other, yielding an α7β7β7α7 complex. The two inner rings are solely built of the β-subunits, which delimit the hydrolytic chamber. This central
The proteasome – a threonine protease
As mentioned previously, proteasomes are threonine proteases. Accordingly, a proton acceptor is required to activate the hydroxylic group of Thr1. Although there are several potential acid-base catalysts in close proximity to Thr1, activation seems to arise from its own terminal amino group 15., 16.. The positively charged sidechain of Lys33 might be involved in this process, by lowering the pKa of the Thr1 amino group (Figure 2). The assignment of the N-terminus as the catalytic base in
Inhibiting the proteasome
Proteasome inhibitors have been instrumental in identifying numerous protein substrates and elucidating the importance of the proteasome/ubiquitin pathway in many biological processes; initially, nonspecific cell-penetrating peptide aldehydes were used for this purpose. More recently, it became possible to synthesize compounds with increased potency and selectivity 27., 28.. Furthermore, based on the crystal structures of yeast and bovine liver CP 13., 14.•, molecular modeling can now be used
Regulating access to the proteolytic chamber
In the Thermoplasma and Archaeoglobus CPs, two narrow entry ports, of ∼13 Å diameter, exist at both ends of the cylinder, preventing folded proteins from entering 7., 17.•• (Figure 4a). However, the mechanism by which substrate access is controlled in archaea remains elusive, because a protein complex with regulatory partners has not yet been discovered. Many archaebacteria, such as Methanococcus jannaschii, contain a gene named PAN (proteasome-activating nucleotidase), which is highly
Conclusions
ATP-dependent proteolysis plays an essential role in controlling levels of key regulatory proteins and in the degradation of abnormal polypeptides. In eukaryotes, most proteins in the cytosol and nucleus are eliminated by the ubiquitin/proteasome pathway. A major component within this system is the 26S proteasome, a 2.5 MDa molecular machine consisting of more than 31 different subunits. The 26S proteasome consists of the 20S proteasome, which forms the proteolytic core, and the 19S regulatory
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
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of special interest
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of outstanding interest
Acknowledgements
We would like to thank Timothy Skern and Dara Dunican for helpful discussions and comments on the manuscript.
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