Proteasome

The regulation of endogenous proteins or proteostasis was not well known for a large part of the 20th century and protein degradation was initially thought to be nonspecific (1). Studies from the last 25 years however have shown that proteostasis is highly specific and integral to many cellular processes (2, 5). In eukaryotic species, proteostasis is maintained by the ubiquitin-proteasome system (UPS) in which ubiquitin conjugated proteins are recognized and degraded by the 26S proteasome (3, 4). The structure of the 26S proteasome bound to USP14-UbAl (PBD ID: 5GJQ) was reported in August, 2016 at a resolution of 3.5Å, giving clear insight to the structure and function of the proteasome (2, 6). The 26S proteasome is a complex of multiple proteins and is comprised of a 20S core particle (CP) and two 19S regulatory particles (RPs) (2, 4). In its physiological state, the 26S proteasome as well as the dissociated CP and RP subcomplexes exist in equilibrium in the cytoplasm and nucleoplasm (5). The proteasome is also bound to auxiliary deubiquitinating enzymes (DUBs) such as USP14-UbAl (2) which serve to cleave polyubiquitin chains from substrates (2). Protein degradation by the proteasome is ATP and ubiquitin dependent which lends itself well to specificity and regulation in proteostasis. In summary, the 26S proteasome binds to polyubiquitinated substrates, unfolds them and releases free ubiquitin so as to repeat the cycle (2).

The 26S proteasome has a molecular weight of 1724762.97 Da and an isoelectric point of 5.75 (7). The quaternary structure of the proteasome is a hetero 41-mer complex comprised of 48 chains, 34 of which are unique (6). The secondary structure of the proteasome is mainly composed of α-helices and β-strands which impart stability to the protein by minimizing hydrophobic interactions and providing a structural basis for subunit formation. The tertiary structure of the proteasome is a capped barrel. The 20S CP exhibits C2 symmetry and forms a barrel-like structure to form the proteolytic chamber where the catalytic residues are located (2). The CP is composed of four heptameric rings: two stacked β-rings in between two α-rings (2). The two 19S RPs form what is best described as the lid and base to the CP. The base is composed of 10 subunits (Rpt1-6, Rpns 1, 2, 10, 13) and forms a hexameric ring that docks to the CP (2). The lid is a complex assembly of nine subunits (Rpns 3, 5, 6, 7, 8, 9, 11, 12, 15) stabilized by helical bundles and is involved in substrate recognition and deubiquitination (3, 8).

The functional elements of the proteasome include the lid and base of the RP, the Rpn1/Rpn2 complex, a gated channel into the CP and the CP itself (3). The Rpn subunits of the lid exhibit secondary structures including β-strands and α-helices which allow for close association of each subunit. The Rpns associate to form two domains: a PCI domain and a C-terminal helical domain (2, 8). The PCI domain confers stability via intersubunit contacts and the C-terminal helices of the Rpns form a tight bundle which provide a structural basis for the formation of the lid (3, 8). The lid associates to the base and CP via module 1 (Rpns 5, 6, 8, 9) which has attached module 2 (Rpns 3, 7, 12, 15) (9). The Rpt subunits of the base interact via specific hydrogen bonds and van der waals contacts. The Rpt form a hexameric ring that is stabilized by hydrogen bonds from Glu-182 and Arg-339 in Rpt1 to Lys-432 and Glu-426 of Rpt5, respectively (2). Rpns 1 and 2 act as scaffolding proteins and Rpns 10 and 13 are receptors of ubiquitin (3). Rpns 1 and 2 help the proteasome bind to associated proteins such as USP14 (3, 10). The C-terminal tails of the Rpt ring insert into pockets in the α-ring of the CP so as to dock the base to the CP. Hydrophobic-tyrosine-X motifs where X is Lys-418 for Rpt3 and Ala-439 for Rpt5 are responsible for the docking of the base to the CP (2). The Rpt ring also functions as gated channel that folds or unfolds to regulate substrate entry into the CP (2). Stacking heptameric rings in the CP as opposed to hexameric rings gives the proteolytic chamber a larger diameter which in turn allows more space for the catalytic sites to perform more effectively. As with the base, hydrophobic interactions clearly play a role in stabilizing the barrel structure of the CP.

Polyubiquitinated substrates are recognized by Rpns 10 and 13 while the substrate is captured by the Rpt subunits (2). Each Rpt subunit is bound to an ADP molecule which is involved in ATP hydrolysis of substrates (2). USP14 is a deubiquitinating enzyme that is auxiliary to the proteasome but functions to remove ubiquitin chains from substrates (10). Rpn11 is intrinsic to the proteasome and like USP14, it is also a deubiquitinase. However in conjunction with the Rpt ring, ATP powered hydrolysis in the unfolding of the substrate triggers a conformational change which moves Rpn11 to a location above the central pore of the CP (11). It is by this process that the proteasome can feed the unfolded substrate into the proteolytic chamber for degradation. The proteolytic active sites of the CP belong to the β-rings and thus named β1, β2, and β3. Each has a single residue active site in which threonine is the active site (3). The active sites have some specificity as to where they cleave peptide bonds and as such a mixture of proteins are produced by proteolysis rather than single amino acid residues.

A protein similar to the 26S proteasome in humans is the 20S proteasome in complex with PR-957 from Mus musculus (PDB ID: 3UNB) (6). PSI-BLAST is a tool which can list proteins similar to a query protein based on primary structure (12). The degree of similarity is given by the E-value which approaches zero as the sequences of proteins compared to the query protein become more identical. The E-value for the 20S proteasome compared to the 26S proteasome is 6x10^-20(12). Given that both proteins are proteasomes, it follows that they would have similar sequences so as to fold into similar proteolytic complexes. The Dali server can also be used to find similar proteins. The Dali server ranks similarity based on tertiary structure and assigns a Z-score to the resulting proteins of a search query (13). Because the 26S proteasome is a multi-protein complex, the Dali server will not list any results when given the sequence of the entire protein complex. It will however work for individual chains of the proteasome. The 20S proteasome is a hetero 28-mer and exhibits C2 symmetry very similar to the core particle of the 26S proteasome (6). Superimposition of both structures shows that the tertiary structure of the 20S proteasome closely resembles that of the core particle of the 26S proteasome. The 20S proteasome has 2 heptameric β-rings situated in between two heptameric α-rings. The helical character of the subunits in the 20S proteasome also resembles that of the 26S proteasome with its tight bundling and Van der Waals packing. One important difference between the 26S and 20S proteasomes regulation of substrate entry into the proteolytic chamber. Although the 20S proteasome lacks a regulatory particle, its proteolytic chamber is blocked by the α-subunits. The 20S proteasome is different in that the N-termini of the α-subunits block substrate entry as opposed to the C-termini of α-subunits in the 26S proteasome (14).

The degradation, recycling, and refolding of proteins is central to proteostasis and have significant effects on biological systems. Much of the proteins in our body are recycled and so it is clearly important to understand the mechanisms of protein regulation and those specific to the ubiquitin-proteasome system in eukaryotes. Malfunctions in proteostasis have been linked to the genesis of diseases such as Alzheimer’s (15). Such problems may also lead to other chronic disorders and so it is of great importance to adapt proteostasis to the study and perhaps prevention of disease (16).