Proteasomes are protein degradative machines that are found in the nucleus and the cytoplasm. They are not only found in all eukaryotic organisms, but have also been found in archaebacteria. Proteasomes play many roles in the cell's life:
1. They remove abnormal and misfolded proteins from the cell.
2. They are involved in the cell's stress response, where they degrade Ub-conjugated regulatory proteins.
3. As part of the Ub system, they are involved in regulating the cell cycle.
4. They are involved in cellular differentiation (where they degrade transcription factors and metabolic enzymes).
5. They play an important role in the immune system by generating antigenic peptides that are presented by the major histocompatibility complex (MHC) class I molecules (such molecules are studied in Immunology).
In short, proteasome activity is involved in most of the processes that also involve ubiquitin. We know they are essential because the removal of proteasome genes in eukaryotes is lethal.
Proteasomes are cylindrical structures very similar to hsp60 chaperonins (we'll discuss this later). Like all cellular machines, proteasomes use ATP to drive conformational changes in their subunits. ATP hydrolysis is not needed to actually cleave the peptide bonds of a protein, but instead is thought to involve recognition of target proteins, their unfolding, the translocation of the substrate protein into the proteasome's chamber, and/or the opening and closing of proteasome gates.
20S Proteasome Chamber in Archaebacteria
Let's consider the structure of the proteasome core. We will focus on archaebacterial proteasomes because they are simpler. Eucaryotice proteasomes are very similar, yet they employ many more different subunits to construct the chamber.
As noted above, the proteasome core is actually a cylinder like hsp60. It is about the same size as hsp60, namely, 15nm in length and 11 nm in diameter. It is composed of four rings stacked on top of each other (like tires). Consider the following figure below.
Each of the four rings is composed of seven individual protein subunits. The two outer rings are made up of alpha subunits and are proteolytically inactive. Note that they also define the "pore" which allows substrate proteins inside. This pore is roughly 0.13 nm in diameter. The two inner rings are composed of beta subunits and are proteolytically active. Together, these four rings define three chambers within the proteasome, the largest one being in the center and defined by the beta subunits. It is in this chamber that proteins are cleaved into small peptides.
Click here to see the protein structure of the alpha and beta subunits. Note that they are very similar in shape. The biggest difference is that the alpha subunit has an extra alpha helix across the top of the molecule. This helix is part of the "pore" and may help guide substrate proteins inside the chamber. The beta subunit shown is mature. At an earlier point in its life, a prosequence was attached to the N-terminal end. This prosequence masks the proteolytic activity of this subunit when it exists as an individual subunit. During assembly of the proteasome, it is cut away to expose the beta-sheet cleft (which defines the active site). In fact, the N-terminal threonine that is created by such cleaving of the prosequence is the amino acid that is directly responsible for proteolysis.
20S Chamber Function
The proteasome represents a unique type of protease - a threonine protease. Most proteases use other amino acids (like serine) as part of the proteolytic active site. When a substrate protein is unfolded and guided into the middle chamber, peptide bonds are cleaved every 8-9 amino acids. Thus, the proteasome takes a single polypeptide chain containing hundreds of amino acids and converts it into numerous short peptides 8-9 amino acids in length. That this regular size is seen suggests a molecular ruler is involved. More specifically, the polypeptide chain is apparently stretched across the chamber so that it interacts with two proteolytic sites concurrently. The distance between these two sites would be thus bridged by 8-9 amino acids of the substrate protein. However, once cut, it is still not clear how the individual short peptides are removed from the chamber, although some have speculated the proteasome may have side windows that serve this purpose.
Click here to view one model for proteasome assembly. According to this model, individual alpha subunits first bind to individual beta subunits. These dimers are not active. Then, these protein dimers interact with each other to form a ring complex. This activity may be guided by chaperones. Once the rings are formed, two such rings can come together with the concurent removal of the beta subunit prosequences. The cleavage of these subunits not only drives the assembly of the 20S chamber, but also activates the subunits inside the chamber. This assembly process thus protects the cytoplasm from indiscriminate proteolysis.
The 20S chamber is simply the core degradative machinery. In eukaryotic cells, the complex is typically associated with what are called 19S caps. The 26S proteasome is thus a complex of the 20S core chamber attached to two 19S caps on each end. Click here to see the 26S proteasome.
It is the 19S caps that tie the proteasome to the Ub system. These caps are composed of about 20 different proteins. Some of these proteins apparently interact with Ub (recall how Ub functions to decrease the rate of dissociation between substrate proteins and the proteasome), although it has yet to be determined which proteins actually interact with Ub. Without the 19S caps, Ub-conjugated proteins are no more likely to be degraded by the proteasome than any other protein. It is also worth noting that the 19S caps appear to be flexibly attached, raising the possibility that movement is involved in the capturing of Ub-conjugated proteins.
The best defined component of the 19S caps involve six proteins with ATPase activity. These six proteins form a ring that sits adjacent to the pore of the proteasome defined by the alpha subunits. ATP hydrolysis is clearly involved with the entry of proteins into the chamber, but the exact mechanism involved remains to be determined.
Put simply, the 20S chamber is the heart of the proteasome that does the degradation. The 19S caps serve to capture and guide proteins into this chamber. 19S caps are not seen in archaebacteria. This is consistent with the fact that neither is Ub found in these bacteria.
Immunoproteasomes as "Hot Rod" Complexes
A very interesting feature of proteasomes is their role in the immune system. Proteasomes can syntheisize short peptide fragments that are then used as antigens in lymphocytes. These antigens are presented on the surface of these cells (through the MHC complex) and play an important role in the cells ability to mount a specific immune response. This role for the proteasome is a demanding one and calls for a "souped-up"
proteasome. In higher vertebrates (not yeast) another complex is used to replace the 19S cap. This complex is know as PA28 (or the 11S cap). This cap is smaller than the 19S cap, but serves to make the proteasome more efficient at generated peptides. It is thought to do so by stretching open the mouth of the proteasome and creating a strain on it so that it is easier for peptides to escape from the chamber. PA28 is a six member ring made up of two different subunits.
Furthermore, in immune cells, some of the beta subunits are replaced by what are called gamma-interferon inducible homologs. These subunits are like the beta subunits, but allow for alterations on the degradation process that better fit the immune system's need. Interferon is a cell hormone that is excreted locally when an infection occurs. Thus, infections cause immune cells to replace certain proteasome parts with more effective ones. The immunoproteasome is one mean proteasome. Click here to see it.
Comparing the proteasome and chaperonin
As the figure below shows, the 20S proteasome is very similar to the hsp60 chaperonin. Both are cylindrical structures of the same size. Both are composed of stacked rings each made up of seven subunits.
Yet in spite of these remarkable similarities, there are important differences. First, the two structures are not evolutionarily related. The amino acid sequences, along with the tertiary structure, of proteasome and chaperone subunits are quite different.
Secondly, the functions are different. Where proteasomes degrade proteins, chaperones provide a protective environment that facilitates proper folding. Of course, it is not surprising that the same conformational solution (the "isolation chamber") is employed for these different functions. In the case of proteasomes, the proteins in the cytoplasm must be protected from the proteolytic processes. Otherwise, indiscriminate degradation would occur. In the case of the chaperones, the unfolded proteins must be protected from the proteins in the cytoplasm. Otherwise, indisciminate aggregation would occur.
When these functional differences are realized, some of the slight differences in architecture are explained. Notice the mouth sizes of both complexes. The diameter of the chaperone's mouth is over three times larger than that of the proteasome. Where a chaperone would not want to greatly restrict access to its chamber, the proteasome would. Thus, the smaller mouth of the proteasome makes sense. Furthermore, remember that the proteasome mouth is complexed to the 19S cap, which addes a further layer of restriction. Hsp60 chaperones do not employ 19S-like caps.