5.15: The Proteasome Structure

The ubiquitin-proteasome pathway is a well-known mechanism utilized by eukaryotic cells to remove cytoplasmic proteins that are misfolded, damaged, or no longer needed. In this pathway, the protein that needs to be eliminated undergoes a process called ubiquitination, where a chain of ubiquitin molecules is attached to the 48th lysine residue of the target protein. This ubiquitin modification helps the proteasome distinguish between a target protein and a healthy protein.

The proteasome is an ATP-driven, large multi-subunit protease (26S) found in the nucleus and cytoplasm of all eukaryotes. The 26S proteasome comprises a central hollow cylindrical core (the 20S) and ring-shaped protein caps (19S). The 20S core particle is barrel-shaped, containing stacked rings with proteolytic active sites lined inside. The 19S caps act as gatekeepers and regulatory units of the proteasome. They are present at one or both ends of the core.

Caps have ubiquitin receptors, ubiquitin hydrolase, and unfoldase, each with a unique function. The ubiquitin receptor recognizes and allows ubiquitin-tagged proteins into the core, the hydrolase deubiquitinase cleaves ubiquitin from the substrate, and the unfoldase protein unfolds target proteins as they enter the protein core.

The hexameric unfoldases create a ring structure in the proteasome cap and belong to a large class of AAA proteins. Their mechanistic action is similar to other ATP-dependent helicases that cause DNA unwinding. The ATP-bound unfoldase ring binds a folded target protein held in place by its ubiquitin tag at the proteasomal cap. Next, a conformational change, driven by ATP hydrolysis, pulls the target protein substrate into the 20S core and strains the ring structure. As a result, when a protein is subjected to pulling forces, it can undergo two possible outcomes— it may either undergo partial unfolding and enter the cylindrical core, or it can retain its structure while partially retracting.

Highly stable substrates generally require hundreds of cycles of ATP hydrolysis before being pulled through the AAA ring. Once de-ubiquitylated and unfolded, the target protein is exposed to proteases inside the proteasomal core for digestion. In contrast to free cellular proteases, which quickly dissociate after their action on the target protein, the proteasome core holds the target protein until it is fully digested into smaller peptides. These small peptides are then released into the cytosol for further processing.

The 26S proteasome, is a large protein complex that degrades misfolded proteins through the ubiquitin-proteasome pathway.

Each proteasome consists of two major types of subunits—the 19S caps and the 20S core.

The 19S subunits are large protein caps attached at one or both ends of the core that act to regulate proteasome activity.

The cap recognizes a small protein called ubiquitin and only allows the entry of proteins with covalently-linked ubiquitin into the core.

Deubiquitinase is an enzyme in the cap that removes ubiquitin from the protein substrate before it enters the 20S core. 

Additionally, the cap contains an ATPase enzyme that helps unfold the target protein using the energy from ATP hydrolysis.

The 20S core particle is a hollow cylinder composed of multiple alpha and beta protein subunits assembled as a stack of rings.

Some of the proteins in the rings are proteases, enzymes responsible for the break down of proteins that enter the proteasome core.