Got Proteasomes?

Photo: Courtesy of Boston Biochem PURITY CHECK: Boston Biochem CEO Francesco Melandri (right) and research associate Nick Crawford assess the purity of a protein using SDS-PAGE. Researchers expend a lot of energy studying gene and protein expression. But what happens when the proteins are no longer needed? In the late 1980s Alfred L. Goldberg, professor of cell biology at Harvard Medical School, and Martin C. Rechsteiner, professor of biochemistry at the University of Utah, addressed this question when they discovered proteasomes--large, multienzyme complexes that play a key role in protein breakdown. Their work challenged the assumption that lysosomes were the primary cellular "chop shops" responsible for digesting proteins that the cell no longer needed; indeed, proteasomes degrade the bulk of these proteins. The average human cell contains about 30,000 proteasomes, each of which contains several protein-digesting proteases. These complexes help regulate a whole host of functions including transcription, viral infection, oncogenesis, cell cycle, stress response, ribosome biogenesis, abnormal protein catabolism, neural and muscular degeneration, antigen processing, DNA repair, and cellular differentiation, says Francesco Melandri, chief executive officer of Boston Biochem, a company that specializes in products that help scientists study the ubiquitin-proteasome pathway. Proteasome activity is exquisitely controlled; when it becomes either overzealous (degrading more proteins than it should) or underachieving (neglecting to degrade certain proteins) disease can develop. To aid research into these cellular complexes, scientists have identified a variety of inhibitors, many of which are now commercially available. As a result,researchers can now examine how the proteasome modulates such a wide range of cellular activities. "The whole field has exploded in the last five years," says Goldberg. "These agents have allowed people to discover the regulation of proteins in the cell." FORM FITS FUNCTION When the cell needs to destroy a protein, it usually marks it with a chain of small polypeptides called ubiquitin. This structure acts as a ticket into the proteosome's enclosed proteolytic chamber, and the cell stamps this ticket using three enzymatic activities, E1, E2, and E3. The ATP-dependent E1 enzyme activates ubiquitin and links it to the ubiquitin-conjugating enzyme, E2. The E3 enzyme, an ubiquitin ligase, then links the ubiquitin molecule to the protein. This process repeats itself until the doomed polypeptide trails a long chain of ubiquitin moieties; the proteasome finally degrades the protein into small fragments. The 26S proteasome weighs in at a peptide-crushing 2,400-kDa; its barrel-shaped structure contains a central 20S protein-degrading component with 19S regulatory "caps" on one or both sides. (In some cases, the cell replaces the 19S caps with 11S caps called PA28, which appear to play a role in antigen processing.) Four stacked rings comprise the 20S structure: two central b rings surrounded by two a rings, with each ring consisting of seven proteins. Most of the action occurs at six sites located in the b rings. Two of the sites act like chymotrypsin, which cleaves after hydrophobic residues; two trypsin-like sites cleave after basic residues; and two are like caspase, cleaving after acidic residues. But these enzymes are unique: they belong to the novel class of threonine proteases that use an N-terminal threonine as the nucleophile in the initial attack of the target peptide bond. The k rings have no proteolytic activities, but instead function as regulators. The 19S structures, sometimes called PA700, are gatekeepers that allow only those proteins marked for degradation into the central chamber. Each complex consists of a "base" and a "lid" and contains: binding sites for ubiquitinylated proteins; enzymes to depolymerize the ubiquitin chain; and six ATPases that unfold the proteins, preparing them for entry into the proteasome itself. MANY KEY ROLES The ubiquitin- proteasome pathway degrades 90% of all abnormal, misfolded proteins and all of the short-lived, regulatory proteins in the cell.1 These short-lived proteins, whose half-lives are less than three hours, account for 10% to 20% of all cellular proteins.1 The pathway also breaks down the bulk of longer-lived proteins. All told, the ubiquitin-proteasome pathway is responsible for degrading 80% to 90% of all the cell's proteins.1 As a result, inhibitors do not target individual cellular functions; rather, they affect a broad spectrum of them. Proteasomes play a key role in the generation of antigenic peptides. By degrading intracellular proteins, the proteasomes generate these peptides, which are presented along with the major histocompatibility (MHC) class I molecules on the cell surface. Proteasome inhibitors, including lactacystin and peptide aldehydes, can reduce the production of antigenic peptides, demonstrating the proteasome's involvement in this process.2 However, other antigen-generating pathways may exist, as some antigenic peptides are presented in spite of proteasome inhibitors that block the chymotrypsin-like active site.3 Alternatively, the trypsin-like and caspase-like active sites, which are not inhibited by these inhibitors, participate in antigenic peptide processing. Image: Courtesy of Millenium Pharmaceuticals IMPROVING EFFICACY: Activation of NF-kB requires the proteasome-mediated degradation of its inhibitor, IkB. Preventing IkB degradation and NF-kB activation can induce apoptosis in some types of cancers. In some solid tumors,NF-kB is activated in response to chemotherapy or radiation, sparing treated tumors from apoptosis; in these cases, proteasome inhibition prevents the protective response and improves the effectiveness of treatment. Blocking protein degradation using proteasome inhibitors causes ubiquitin-bound misfolded and damaged proteins to accumulate. Such a buildup can trigger the heat-shock response.3 Cells normally induce this pathway in response to environmental stresses like increased temperature or oxygen radicals, which can cause protein denaturation. The response protects the cell, en-abling it to withstand the effects of high temperatures or toxic agents. Proteasome inhibit-ors, then, can bolster a cell's ability to withstand a volatile environment, and they could be useful in therapies designed to protect tissues against ischemic injury or to maintain organs before transplantation, says Julian Adams, senior vice president of drug discovery, Millennium Pharmaceuticals, Cambridge, Mass. But the inhibitor-induced heat-shock response leads to so many cellular changes that the interpretation of experimental findings could be difficult. THERAPEUTIC ANGLES Proteasome inhibitors have potential pharmaceutical value. Regulatory genes that are modulated by proteasomes include the cyclins, p53, and transcription factor NF-kB. This factor, which plays a central role in such cellular functions as immune and inflammatory responses, apoptosis, and cellular proliferation, is kept in the cytoplasm bound to its inhibitor, IkB. In response to proinflammatory cytokines, IkB is phosphorylated, ubiquitinylated, and degraded. NF-kB is then free to enter the nucleus and jumpstart the inflammatory response. PROTEASOME INHIBITOR SUPPLIERS Affiniti Research Products www.proteasome.com Biomol Research Laboratories www.biomol.com Boston Biochem www.bostonbiochem.com Calbiochem www.calbiochem.com Novagen www.novagen.com Peptides International pepnet.com Proteasomes.com www.proteasomes.com Editor's Note: To the best of our knowledge, this is a comprehensive listing of suppliers. Once in the nucleus, NF-kB promotes the production of interleukins-1 and -6, and survival factors, including inhibitors of apoptosis, which some cancer cells need to continue proliferating. The peptide boronate, PS-341, prevents NF-kB from stimulating the production of these interleukins and other proteins, which makes PS-341 not only a good therapy for cancer, but possibly other diseases caused or exacerbated by inflammation.3 In animal studies, PS-341 has been shown to be effective against arthritis and other inflammatory conditions. PS-341 is the first proteasome inhibitor to enter human clinical trials.4 Its success could usher in other proteasome inhibitor-based drugs. "It's one of the most intriguing new concepts under clinical investigation in cancer," says Adams. The ubiquitin-proteasome pathway, also plays a role in viral replication. Yet the exact role of the ubiquitin-proteasome pathway has yet to be uncovered. The pathway evidently plays a role in virus assembly, as several viruses, such as human immunodeficiency virus type 1, simian immunodeficiency virus, and Moloney murine leukemia virus, contain high levels of free ubiquitin molecules in the mature virion particles.2 Proteasome inhibitors could, therefore, form the basis for a new class of antiviral drugs, and some studies have shown promising results. Researchers have found, for instance, that retroviral budding and maturation are inhibited when they treat infected cells with lactacystin or epoxomicin.2 IMPROVING SPECIFICITY Proteasome inhibitors will continue providing researchers with new insights into how this complex orchestrates so many varied functions. And these insights will surely lead to novel therapeutic agents. But, the very breadth of proteasome-moderated functions that makes it such an attractive pharmaceutical target also hinders the usefulness of these compounds, because proteasome inhibitors can't target particular proteins, says Melandri. The best use of commercially available proteasome inhibitors is to determine whether a protein is a proteasome target, he says. "Proteasome inhibitors are research tools that point researchers in the right direction," Melandri says. "But these commercially available inhibitors are not protein-target specific, since the fate of all proteins with different functions targeted by the ubiquitin-proteasome pathway is proteasome degradation." Instead, the best targets for drug therapy may be the E2 and E3 enzymes in the ubiqutinylation pathway, he says. As far as researchers know, only one type of E1 enzyme exists, and, there are multiple E2 and E3 enzymes, each of which targets different proteins. "Those are what people are really interested in for therapeutic targets," he says. Laura Lane (lanelaura@hotmail.com) is a freelance writer in San Francisco. References 1. D.H. Lee, A.L. Goldberg, "Proteasome inhibitors: Valuable new tools for cell biologists," Trends in Cell Biology, 8:397-403, 1998. 2. J. Myung et al., "The ubiquitin-proteasome pathway and proteasome inhibitors," Medicinal Research Reviews, 21:245-73, 2001. 3. A.F. Kisselev, A.L. Goldberg, "Proteasome inhibitors: From research tools to drug candidates," Chemistry & Biology, 8:739-58, 2001. 4. D. Steinberg, "Ubiquitin-mediated proteolytic system plays diverse roles in human disease," The Scientist, 12[15]:10,13, July 20, 1998. PROTEASOME INHIBITORS | Laura Lane Because the cell abounds with proteases, it can be difficult to determine which enzyme degrades a given protein. Proteasome inhibitors ease this task considerably: Researchers need only measure the protein's half-life in the presence and absence of an inhibitor to confirm the complex's involvement in protein turnover. The proteasome contains six active sites, but scientists need not inhibit all of them to profoundly reduce protein degradation. Blocking the chymotrypsin-like sites decreases protein degradation significantly, says Craig Crews, an expert in proteasome inhibitors and associate professor of molecular, cellular, and developmental biology and chemistry at Yale University School of Medicine. Inhibition is less effective, on the other hand, when researchers inhibit only the trypsin- or caspase-like sites, so most proteasome inhibitors act on the chymotrypsin-like sites, Crews says. Some of the most widely used proteasome inhibitors are peptide aldehydes. These synthetic inhibitors potently, but reversibly, inhibit the complex's chymotrypsin-like activity. Yet like many inhibitors, the peptide aldehydes do not target the proteasome exclusively; they also inhibit some of the lysosomal cysteine proteases, calpains, and serine proteases of digestive vacuoles. Researchers must confirm, by using other inhibitors, that a particular effect is due to proteasomal inhibition, rather than inhibition of other activities. Examples of the peptide aldehydes include Z-Leu-Leu-Leu-al (MG132), Z-Leu-Leu-nVal-al (MG115), Ac-Leu-Leu-Nle-al (ALLN), and Z-Ile-Glu(OtBu)-Ala-Leu-al (PSI). Members of another class of proteasome inhibitors, the peptide boronates, are both more potent and more selective than the peptide aldehydes. The boronates include the reversible inhibitors Z-Leu-Leu-Leu-Bor (MG262, an analog of MG132), and pyrazylcarbonyl-Phe-Leu-boronate (PS-341). Cambridge, Mass.-based Millennium Pharmaceuticals currently has PS-341 in Phase II clinical trials for cancer patients. Lactacystin is a natural, irreversible, nonpeptide inhibitor that is more selective than peptide aldehydes but less selective than peptide boronates. In an aqueous solution, lactacystin converts to a b-lactone derivative, the inhibitor's active form, which can enter the cell easily. But b-lactone rapidly degrades in water, even if the inhibitor is bound to the proteasome, so the inhibitor's activity decays over time. Peptide vinyl sulfones are synthetic, irreversible proteasome inhibitors. These inhibitors can inhibit cysteine proteases as well as the proteasome; their selectivity depends upon the inhibitor's peptide portion. Epoxyketones, such as expoxomicin, are the most selective proteasome inhibitors. Whereas most other inhibitors react only with the active site's hydroxyl group, epoxyketones react with both the hydroxyl and amino groups of threonine at the proteasome's active site.

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Photo: Courtesy of Boston Biochem
PURITY CHECK: Boston Biochem CEO Francesco Melandri (right) and research associate Nick Crawford assess the purity of a protein using SDS-PAGE.