At its discovery in the 1980s, the proteasome was relegated to the essential but unglamorous role of cellular garbage pail – a last resting place for worn-out, misfolded, or otherwise unwanted proteins. But researchers soon uncovered a host of more specific, and decidedly sexier, roles for the large protein complex. Proteasomal degradation of perfectly good proteins was found to regulate important physiological processes, including cell-cycle progression, inflammatory responses, and antigen presentation.1 Yet now, as aberrant protein accumulation is being implicated in disease states from Alzheimer disease to multiple myeloma, researchers are considering whether simply taking out the trash is more crucial, and more carefully regulated, than once thought.

"We need to take a broader perspective, and look at protein homeostasis and quality control overall," says Richard Morimoto, who studies protein chaperoning and proteasomal degradation at Northwestern University. He maintains that protein homeostasis, the delicate temporal balance between synthesis...


The system offers many potential control points. Proteins are first targeted for proteasomal degradation by covalent attachment of ubiquitin, a small (8.5 kilodalton) – and as the name implies, highly abundant – protein. After the first ubiquitin is added, additional units are linked on in a chain, each connected at lysine 48 of the preceding unit. (Ubiquitin chains form alternate linkages, but these earmark proteins for processes other than degradation.) Numerous enzymes participate in the process: E1s, ubiquitin-activating enzymes; E2s, ubiquitin-carrier proteins; and E3s, ubiquitin ligases. The highly diverse E3s provide most of the system's specificity by recognizing distinct targets.

The polyubiquitinated protein finds its way, or in some cases, is guided by accessory proteins, to one of the many, monstrous (2.5 megadalton) proteasome complexes scattered throughout the cell's cytoplasm and nucleus. At the proteasome, one or sometimes two 19S regulatory subunits are waiting to recognize the protein, unfold it, and guide it into the 20S proteolytic subunit.

The structure of the 20S subunit is well understood from crystallography, says Martin Rechsteiner, the University of Utah professor who discovered the active proteasome complex in 1986. It has a barrel-like structure comprising four stacked rings of α and β subunits. The proteolytic sites reside on three β subunits, whose differing catalytic activities allow protein cleavage after almost any amino acid. By contrast, Rechsteiner says, "The 19S subunit is still structurally a mystery. The functions of only 10 of the 18 to 19 subunits are known. Most have not been crystallized...but a lot of labs are trying," he adds.

Rechsteiner's group is currently occupied with understanding how the 19S subunit regulates proteasome function. They are also investigating several more specialized regulatory subunits that can serve as alternatives to 19S. PA200 is a single polypeptide, found primarily in the nucleus, which is "almost certainly" involved in DNA repair. PA28α and β, which are cytoplasmic complexes of seven 28 kDa subunits, are involved in the processing of certain antigens by immune cells. PA28γ is "still a bit of a mystery," he says. It is most abundant in brain-cell nuclei, and there are hints that it is involved in apoptosis.



© 2005 Elsevier Ltd.

At far left is a side view of the 20S proteasome. Active sites are formed at the N-termini of β1, β2, and β5, each of which has different substrate preferences. At right is a cutaway stereoview showing how the active sites (yellow) are sequestered within the catalytic chamber. (From M. Rechsteiner, C.P. Hill, Trends Cell Biol, 15:27–33, 2005).

Defects anywhere in this highly regulated system can have profound effects on organismal health. Such defects can be global, or they can be quite specific, according to Morimoto. Several inherited syndromes involve specific defects in the proteasome pathway.2 Liddle syndrome, a form of hypertension, is caused by a mutated sodium channel that is no longer recognized by ubiquitination enzymes. Accumulation of the channel leads to excess sodium in the kidney. Von Hippel-Lindau syndrome, which causes a predisposition to certain types of cancer, and Angelman syndrome, a mental retardation disorder, are each caused by defective E3 ligases.

Regardless, it is the more global forms of proteasome dysfunction that are implicated in complex syndromes like neurodegenerative diseases, which often involve accumulation of abnormally folded proteins (for example, the well-known plaques and tangles of Alzheimer disease). Ron Kopito, of Stanford University, directly implicated the proteasome by showing that mutant huntingtin, the culprit in Huntington disease, inhibits proteasome function.3 Christopher Ross' group at Johns Hopkins University School of Medicine showed that mutant α-synuclein, which accumulates in Parkinson disease (PD), has similar effects.4 More recently, Morimoto's group has shown that mutant huntingtin actually gets stuck in the proteasome, with toxic consequences to the cell.5

"This is an important and exciting hypothesis that could unify how we think about degenerative diseases... and could explain why all have similar neurotoxic effects," says Ross. But he cautions that these studies were done in cell models. "No one has shown these effects in vivo," he says, although researchers are currently working on transgenic mouse models.

"Transfected systems won't tell everything about what's going on," Morimoto agrees. "In vivo, these diseases take years to decades to affect cells." For this reason, his lab is studying protein homeostasis in a metazoan model, Caenorhabditis elegans. In more acute situations, such as heat shock, cells deal quite well with an excess of abnormally folded proteins. Morimoto suggests that neurodegeneration involves an accumulation of nonspecific events. At some point, he says, the cell recognizes that protein homeostasis cannot be restored; " [It] very slowly grinds to a halt," and may choose to apoptose.

PD is unique among neurodegenerative diseases in that it has a direct connection to the ubiquitin pathway. Autosomal recessive juvenile-onset PD is linked to mutations in parkin, an E3 ligase. However, as Ross points out, it is not clear whether this relates to proteasome function, because parkin catalyzes a lysine-63 linkage, not the type that usually leads to proteasomal degradation. Parkin may be involved in a cell-signaling or vesicle-recycling pathway instead, he says.



© 2004 Elsevier Ltd.

Following processing by an ubiquitin C-terminal hydrolase (UCH), Ub is conjugated to a specific substrate as a chain by the ubiquitin-activating (E1), -conjugating (E2), and -ligating (E3) enzymes. The chain targets the substrate to the 26S proteasome, which comprises one 20S proteolytic complex and two 19S regulatory complexes. (from C.A. Ross, C.M. Pickart, Trends Cell Biol, 14:703–11, 2004)

Research into proteasomal functions in cancer has been stimulated since the discovery and US Food and Drug Administration approval of Velcade, a proteasome inhibitor and promising treatment for multiple myeloma. It has long been known that transformed cells are more susceptible to proteasome inhibitors than normal cells. However, says Robert Orlowski, a University of North Carolina professor who has been involved with Velcade since early clinical trials, "It's not really known why." Transformed cells generally grow rapidly, suggesting a greater need for proteasome activity, he says. But multiple myeloma cells, which are so far the best target for Velcade, usually divide quite slowly. In general though, "cancer cells are more stressed," Orlowski says. They have the large burden of protein synthesis and they accumulate mutated proteins, due to abnormal gene regulation, which may make them more susceptible to proteasome inhibition.

Nevertheless, he says it would be premature to suggest that cancer itself is a disease of protein homeostasis. "There are many ways that changes in proteasome function could contribute to cancer formation, but none are really direct," Orlowski points out. He says that 10 or 20 different cancer-causing pathways are specifically regulated by proteasomal degradation, including the p53 tumor suppressor and the p44/42 MAP kinase pathways. "There is danger in focusing on a single pathway," he warns.

Proteasome inhibitors were first considered in cancer therapy not as anticancer agents but as a treatment for the muscle wasting associated with the disease. Russ Price of Emory University is studying proteasomal function with an eye towards reversing this process, which also occurs in disease states as diverse as starvation, spinal cord injury, and diabetic nephropathy. The actin and myosin fibers of muscle are the body's largest repository of amino acids, and thus are vulnerable to breakdown when there is heavy demand for protein synthesis – to mount an immune response, for example, or for energy, as in diabetes or starvation.

"Loss of lean body mass is a major predictor of mortality and morbidity for many diseases," Price says. "Is there a unique set of signals that are common to all these disorders?" he asks. Thus far, he has found that gene expression for the ubiquitin/proteasome system is "dialed up" in many of these conditions. He is currently unraveling a "complicated symphony of regulatory mechanisms" that control this expression, involving multiple hormones acting as positive and negative regulators.

Academic researchers and drug companies, including Regeneron, continue to take an interest in developing new proteasome inhibitors. Recently, Randall King and collaborators at Harvard Medical School identified a new class of small molecule that binds to ubiquitin and thereby inhibits binding of ubiquitinated proteins to the proteasome.6 But such molecules must be approached with caution. Whether the proteasome is a simple trash pail or a complex regulatory mechanism, fine tuning of its control remains crucial, says Price. " [Proteasomal degradation] is a major protein destruction pathway in cells. You don't want the process happening indiscriminately. You want precise control," he adds.

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