Molecular Machines that Catalyze Intracellular Protein Degradation

“Proteasome” is a modular protease system in which multiple high-molecular weight, multi-subunit complexes are formed from combinatorial binding of multiple, interchangeable protease and regulatory components. The resulting holoenzymes have unique catalytic and regulatory properties based on their composition and this structural and functional diversity probably contributes to the selective participation of the proteasome in diverse cellular processes. The protease component of the system is the 20S proteasome, a 750 kDa cylinder-shaped complex composed of four axially stacked hetero-heptameric rings with an α1-7β1-7β1-7α1-7 architecture.

Three different b-type subunits in each of the two inner rings catalyze peptide bond hydrolysis. Higher eukaryotes have at least three different 20S proteasome subtypes featuring genetically distinct and differentially expressed catalytic b subunits. Most cells constitutively express three catalytic subunits (β1, β2, and β5). In contrast, cells dedicated to immune function express the respective homologs, β1’, β2’, and β5’. The resulting “immunoproteasome” is required for certain immune-specific functions, but immunoproteasomes are also conditionally expressed in non-immune-system cells and may have broader, non-immune-related roles in certain stress responses.

The catalytic centers of β subunits line the surface of a central luminal chamber of the 20S cylinder. Substrate access to the chamber is governed by narrow, gated pores in the center of the outer α-subunit rings. In the absence of regulatory proteins, 20S proteasome is catalytically inert because the gates are constitutively closed. Binding of regulators to cognate sites on the apical face of the α-subunit rings induces gate opening, thereby licensing the resulting 20S/regulator holoenzyme for proteolysis.

Higher eukaryotic cells contain multiple proteasome regulators, including PA700 (aka 19S regulator), PA28αβ, PA28ϒ, PA200, PI31, and p97/VCP, that form holoenzymes with the 20S. Cells also assemble “hybrid-proteasomes” with different regulators on opposite ends of the 20S cylinder [32]. Although holoenzyme diversity expands the potential for proteasome function and regulation, the physiologic roles of most holoenzymes are poorly understood. An exception is the 26S proteasome, a 2.5 MDa holoenzyme featuring PA700 as its regulator. 26S proteasome is the protease of the ubiquitin-proteasome system), and is responsible for the degradation of critical regulatory proteins including master regulators such as p53, HIF, β-catenin, cyclins, c-jun, pVHL, and NFκB (via IκB). The 20-subunit, 750 kDa PA700 complex has properties that mediate the proteasome’s role in ubiquitin-dependent proteolysis of these and many other proteins. For example, two PA700 subunits are polyubiquitin-chain receptors for substrate binding. Six other PA700 subunits are AAA-family ATPases arranged in a heterohexameric ring that binds axially to the 20S a-subunit ring.

In addition to inducing 20S gate-opening and forming the structural connection between 20S and PA700 subcomplexes, these subunits use rounds of ATP binding and hydrolysis to unfold and processively translocate the polyubiquitin-docked protein substrate through the open pore to the 20S’s hydrolytic chamber for proteolysis. Three other PA700 subunits catalytically remove the polyubiquitin chain from the substrate to foster efficient processing.

Despite enormous progress, many fundamental features of the biochemistry and physiology of the proteasome-mediated protein degradation remain poorly understood and the field remains ripe for future discovery. Moreover, it is likely that these future discoveries can be exploited for practical benefit and that new proteasome-based therapeutics will be used to treat a wide range of human diseases.