Cell biology

Unchaining the condemned

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Like prisoners, condemned intracellular proteins are shackled with chains to await their fate. These chains are a signal that the proteins must be destroyed and are removed on protein degradation. New work shows how.

To carry out their functions properly, the proteins in our cells must be in the right place at the right time, and at the right concentration. So it's vital that cells achieve the correct balance between protein synthesis and destruction. Although we understand much about how proteins are made, it is only in the past ten years that we have come to appreciate the complexity of their degradation. Like everything else, proteins outlive their usefulness and, whether damaged or just no longer needed, they are often condemned to destruction by the covalent attachment of another protein, called ubiquitin. When this process fails, it has profound consequences for events such as cell division, gene expression and the development of cancer1. Writing on page 403 of this issue and recently in Science, Yao and Cohen2 and Verma and colleagues3 propose that the ubiquitin must be removed for proteins to be rapidly degraded. The enzyme behind this ubiquitin removal might represent another step in protein degradation that is a good drug target.

Ubiquitin is a 76-amino-acid protein that is present in all eukaryotes (loosely, those species, including humans, whose cells have a nucleus). The attachment of a single ubiquitin molecule to an intracellular target protein is often used as a sorting signal, or simply to modify the protein's properties. This 'monoubiquitination' can be reversed by certain cellular deubiquitinating enzymes4. By contrast, the degradation of many — perhaps even most — intracellular proteins is triggered by the addition of several more ubiquitins to the first. The resulting 'polyubiquitin' chain can be recognized by a binding site (or sites) on the cellular executioner — the proteasome.

The proteasome (Fig. 1) is a complex made up of many different proteins; it degrades ubiquitinated proteins and shortens polyubiquitin chains to recycle the ubiquitin. It consists of a large cylindrical particle, with protein-degrading sites in its lumen, and two regulatory particles that cap each end of the cylinder. The regulatory particle binds polyubiquitinated proteins, unfolds them, and feeds them into the lumen of the cylinder, where they are degraded by hydrolysis5.

Figure 1: The proteasome — the cellular protein executioner.
figure1

The proteasome consists of two multiprotein components: the regulatory particle and the cylinder. The regulatory particle itself consists of a base and a lid. Proteins to be degraded are labelled with chains of ubiquitin (Ub); these chains are recognized by the proteasome's lid. The base contains six enzymes that are thought to unfold the substrate protein and move it into the lumen of the cylinder to be degraded. Yao and Cohen2 and Verma et al.3 find that efficient translocation of the protein into the lumen probably requires removal of the polyubiquitin chain by the proposed metalloprotease activity of the Rpn11 subunit, found within the lid. Figure adapted from ref. 5.

Because the protein to be degraded must enter the proteasome's cylinder through a narrow opening in the end, it is likely that the polyubiquitin chain must be removed to allow the whole protein to be fed through the opening and hydrolysed. Moreover, at least in yeast cells, a lack of free ubiquitin is lethal6, so this may be another reason why it must be recycled from substrate proteins.

At least two deubiquitinating enzymes that catalyse the disassembly of polyubiquitin chains are known to be tightly associated with the mammalian proteasome. Neither of these, however, is known to be required for degradation. UCH37 is a member of the ubiquitin carboxy-terminal hydrolase family of enzymes, and cleaves the polyubiquitin chain from its free end, furthest from the attached protein. It has been speculated that this enzyme acts as a molecular clock that slowly shortens ubiquitin chains, allowing the attached protein to be released from the proteasome if there is a delay in efficient degradation7. Thus, clogged proteasomes would be cleared and become available for further use. A second deubiquitinating enzyme, USP14, is a member of the ubiquitin-specific protease family. However, little is known about its role or substrates.

Both of these deubiquitinating enzymes contain a thiol (sulphydryl) group in their active sites, and are inhibited by ubiquitin aldehyde — a substrate analogue that reacts with the thiol group. But nearly ten years ago Hershko and his colleagues8 described another deubiquitinating activity associated with the proteasome that is insensitive to ubiquitin aldehyde. Yao and Cohen2 and Verma et al.3 investigated this activity further. Both groups suggest that the enzyme responsible is a metalloprotease — a metal-ion-dependent protein-cleaving enzyme — and that it links deubiquitination to degradation.

The authors found that an aldehyde-insensitive deubiquitinating activity is tightly associated with purified proteasomes and efficiently removes ubiquitin from model substrates. This activity depends on the main cellular energy store, ATP molecules, and the results imply that ATP-dependent movement of the substrate into the lumen of the proteasome's cylinder may be necessary to properly position the ubiquitin for cleavage.

Yao and Cohen2 also found that, in the absence of the deubiquitination activity, the ubiquitin attached to the substrate was degraded along with the substrate, albeit more slowly. So deubiquitination can become the rate-limiting step in degradation. Using a proteasome inhibitor, Verma et al.3 showed that the deubiquitination activity was required to remove ubiquitin from a substrate that (because the proteasome was inhibited) presumably then became trapped in the lumen of the proteasome. Both studies point to the idea that the movement of substrates into the lumen is coupled with deubiquitination.

The two groups also propose that the best candidate for this aldehyde-insensitive enzyme is the Rpn11 protein, also known as POH1, found in the 'lid' of the regulatory particle. Rpn11's active site has features that suggest it is a metalloprotease. The protein bears little resemblance to other deubiquitinating enzymes but is strikingly similar to a protein found in another multiprotein complex, called the COP9 signalosome, which efficiently removes the ubiquitin-like protein Nedd8 from its conjugate with the cullin protein9. So Rpn11 may define a new family of deubiquitination-type enzymes.

The finding that removal of the polyubiquitin chain is necessary to fully degrade a condemned protein and to recycle single ubiquitins may explain why both groups2,3 observed that mutations of the Rpn11 protein kill yeast cells. However, it remains to be demonstrated that the putative metalloprotease has enzymatic activity in vitro. Another question is what role USP14 plays in the metabolism of proteasome-bound ubiquitin chains. Nonetheless, it is now clear that the unchaining of a condemned protein by deubiquitinating enzymes is a vital part of degradation, and is a potentially exciting new target for the development of drugs that inhibit proteasome function. Such drugs might seem inherently dangerous — after all, protein degradation is an essential cellular event. But in fact some proteasome inhibitors are already in clinical trials and show surprising efficacy in treating multiple myeloma and other cancers of blood cells10. At the doses used, these drugs block about 80% of proteasome activity in blood cells, but do not seem to reach other tissues, where they might be toxic.

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Correspondence to Keith D. Wilkinson.

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Wilkinson, K. Unchaining the condemned. Nature 419, 351–353 (2002) doi:10.1038/419351a

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