The Ubiquitin System

General Information

Ubiquitin (Ub) is a small protein that is composed of 76 amino acids. This protein is found only in eukaryotic organisms and is not found in either eubacteria or archaebacteria. Among eukaryotes, ubiquitin is highly conserved, meaning that the amino acid sequence does not differ much when very different organisms are compared. For example, there are only 3 differences in the sequence when Ub from yeast is compared to human Ub. This strong sequence conservation suggests that the vast majority of amino acids that make up Ub are essential as apparently any mutations that have occurred over evolutionary history have been removed by natural selection.

Ub is a heat-stable protein that folds up into a compact globular structure. It is found throughout the cell (thus, giving rise to its name) and can exist either in free form or as part of a complex with other proteins. In the latter case, Ub is attached (conjugated) to proteins through a covalent bond between the glycine at the C-terminal end of Ub and the side chains of lysine on the proteins. Single Ub molecules can be conjugated to the lysine of these proteins, or more commonly, Ub-chains can be attached. Conjugation is a process that depends on the hydrolysis of ATP.

Ub is involved in many cell processes. For example, Ub is conjugated to the protein cyclin during the G1 phase of mitosis and thus plays an important role in regulating the cell cycle. Ub conjugation is also involved in DNA repair, embryogenesis, the regulation of transcription, and apoptosis (programmed cell death).

Ub is encoded by a family of genes whose translation products are fusion proteins. The Ub genes typically exist in two states (see the figure below): A. The ubiquitin gene (red) can be fused to a ribosomal protein gene (blue) giving rise to a translation product that is a Ub-ribosomal fusion protein; B. Ub genes can exist as a linear repeat, meaning the translation product is comprised of a linear chain of Ub-molecules fused together (a polyubiquitin molecule). After the fusion proteins are synthesized, another protein called Ub-C-term hydrolase cleaves the fusion proteins at the C-terminal end of Ub. This either liberates an individual Ub and ribosomal protein (as shown in the figure) or liberates a set of Ub monomers from the polyubiquitin (not shown).

Ubiquitin Function

Proteins exist as a linear chain of amino acids. This chain can degrade over time as such a reaction is thermodynamically favorable in an aqueous environment (recall that proteins are synthesized by using energy to drive off a water molecule to form the peptide bond). When proteins degrade over time, this is called protein-turnover. It is the balance between a protein's degradation and its synthesis that determines the concentration of that protein inside the cell.

Studies of protein turnover rates have shown that some proteins are short-lived while others are long-lived. Long-lived proteins constitute the majority of proteins in the cell. Short-lived proteins are typically key regulatory proteins and abnormal proteins (abnormal proteins are often partially unfolded and as we saw in our discussion of chaperones, such proteins are prone to degradation).

Ub functions to regulate protein turnover in a cell by closely regulating the degradation of specific proteins. Such a regulatory role is very important. By regulating protein degradation, cells can quickly eliminate a protein that in turn regulates another function (like a transcription factor that is needed to express a particular gene). Furthermore, this form of control is very effective as the elimination of a particular regulatory protein ensures that the process expressed by the regulatory protein is shut-down. An alternative regulatory strategy used by cells is to simple inactivate proteins (by changing their conformation). Unlike the Ub-linked regulation, such inactivated proteins can mistakenly be reactivated. Of course, Ub-linked regulation is energetically expensive, for if a regulatory protein is needed again, it has to be re-synthesized.

Ub functions in an ATP-dependent fashion. But why is this? We don't need energy (in the form of ATP hydrolysis) to hydrolyze proteins. The reason ATP is required is because machinery is needed to specifically target the proteins that need to be degraded. Ub itself does not degrade proteins. It serves only as a tag that marks proteins for degradation. The degradation itself is carried out by the 26S proteasome (which we will discuss shortly). In short, proteins that are to be degraded are first tagged by conjugating them with Ub and these tagged proteins are then recognized and shuttled to the proteasome for degradation.


The Ubiquitin-Proteasome Pathway

If we were to mix ATP, ubiquitin, and an abnormal protein, we might expect the protein to be conjugated with Ub. But we would be wrong. Something else is needed to attach Ub to such a protein. And what is needed in most cases are three types of enzymes.

1. E1 enzymes known as Ub-activating enzymes. These enzymes modify Ub so that it is in a reactive state (making it likely that the C-terminal glycine on Ub will react with the lysine side-chains on the substrate protein).

2. E2 enzymes known as Ub-conjugating enzymes. These enzymes actually catalyze the attachment of Ub to the substrate protein.

3. E3 enzymes known as Ub-ligases. E3's usually function in concert with E2 enzymes, but they are thought to play a role in recognizing the subtrate protein.

In yeast, there are many types of E1, E2, and E3 enzymes. For example, 13 different E2 enzymes have been found. While they all carry out the conjugation reaction, they are apparently tailored for specific functions. For example, Ubc2 is an E2 enzyme that works in DNA repair, while Ubc3 is also an E2 enzyme that functions to degrade cyclin as part of the cell-cycle.

The general reaction pathway is shown in the figure below. First, Ub is activated by E1 in an ATP-dependent fashion. E2 and E3 then work together to recognize the substrate protein and conjugate Ub to it. Ub can be attached as a monomer or as a previously synthesized chain (as shown). From this point, the ubiquinated protein is shuttled to the proteasome for degradation.


What are the degradation signals?

What determines if a protein gets tagged by Ub and thus marked for degradation? This question cannot yet be fully answered, but scientists have uncovered some interesting clues. Apparently, proteins can contain some form of signal that is recognized by the Ub machinery. Let's consider some of signals thus far discovered.

1. The N-degron. One of the pioneers in Ub research is Alexander Varshavsky. In 1986, Varshavsky carried out an elegant set of experiments (Science 234, 179-186) that showed a correlation between the half-life of a protein and its N-terminal residue. This observation gave rise to the so-called N-end rule where one could generally predict the lifespan of a protein by its N-terminal amino acid. For example, proteins that have Ser as the N-terminal amino acid were long-lived with a half-life of more than 20 hours. In contrast, proteins with Asp as the N-terminal amino have a half-life of only 3 minutes. The mechanism that couples recognition of the N-terminal amino acid and the protein's half-life is still unknown. In is interesting to note, however, that the N-end rule applies to bacteria even though they do not contain ubiquitin.

2. Certain amino acid sequences appear to be signals for degradation. One such sequence is known as the PEST sequence because short stretch of about eight amino acids is enriched with proline, glutamic acid, serine, and threonine. An example is the transcription factor Gcn4p. This protein is 281 amino acids in length and the PEST sequence is found at positions 91-106. The normal half-life of this protein is about 5 minutes. But if the PEST sequence (and only the PEST sequence) is removed, the half-life increases to 50 minutes.

Some signals may also be subject to masking. A signal could be hidden if it is part of a protein-protein interaction. Or it may be masked by covalently attaching phosphate groups to the side chains of certain amino acids. Both of these mechanisms would thus allow for better control, as a proteins degradation signal need only be unmasked to target it for degradation. Such reversible masking appears to be involved in the regulation of both transcription factor and cyclin concentrations.

3. Signals may also be buried in the hydrophobic core. This is why partially folded or abnormal, mutant proteins may be prone to degradation. When such proteins exist in their native state, the signals are hidden and the protein is thus long-lived. But in a partially unfolded state, the signals may be seen by the Ub machinery caused the protein to become tagged by Ub. This reaction appears to be hindered by chaperone activity.

Yet, in spite of much progress, the identification of degradation signals, along with the mechanism by which they are recognized, remains obscure.


How does ubiquitination lead to protein degradation?

Recall that Ub does not itself degrade proteins and instead merely tags proteins for degradation. But it is not entirely accurate to think of Ub as a simple tag, as Ub does appear to be involved in degradation. The proteasome is the structure that actually does the degrading. Ubiquiton's degradation role may simply be to decrease the rate of dissociation between proteasomes and interacting substrate proteins. That is, without Ub, proteins may interact with the proteasome, but quickly dissociate. Ub slows down this dissociation. A substrate protein that is conjugated with Ub-chains is thought to interact with a proteasome for a longer period of time, thus increasing the likelihood that the proteasome will degrade it (we will discuss the proteasome mechanism in the next lecture). In fact, Ub could actually function to tether the substrate protein to the proteasome.

Deubquitination: Another layer of complexity

As if things were not complex enough, there also exists a class of enzymes that function to remove Ub from substrate proteins, thus rescuing them from degradation. There are many types of deubiquitinating enzymes and these are currently being explored. This clearly represents yet one more means of regulating the concentration of proteins in a cell. That is, ubquitinated proteins could be deubiquinated prior to their association with the proteasome, allowing for fine-tuning in concentration regulation. Thus, for a protein to be degraded, not only must it have some type of signal that results in Ub-conjugation, but it must also escape the deubiquinating enzymes. Clearly the cell invests much activity to prevent indiscriminate protein degradation.