Ubiquitin-Mediated Proteolytic System Plays Diverse Roles in Human Disease

A pharmaceutical milestone seems to have been reached last month when a National Cancer Institute panel approved an anti-tumor drug called PS-341 for government-financed clinical trials. PS-341 is the first agent targeting the ubiquitin-mediated proteolytic system to be headed for human testing, according to ProScript Inc., the small firm in Cambridge, Mass., that developed the drug. Clinical trials at NCI and several medical centers could begin later this summer if the Food and Drug Administration approves an investigational new-drug application for PS-341. STUDYING REVERSE: Ubiquitin's removal may be linked to small-cell tumors, according to research by Douglas Gray of the University of Toronto. Whether the trials succeed, the advent of PS-341 portends a shift in the study of ubiquitin-mediated proteolysis. Though the field has largely focused on the basic mechanisms underlying this cellular garbage-disposal system, a relatively small body of research over the past decade has implicated the system in such common pathologies as cancer, cystic fibrosis, and Alzheimer's disease, as well as in such rare disorders as Angelman syndrome. Some studies are now poised to move into the in vivo phase. "There aren't a tremendous number of labs that are really dedicated to the issue" of links between disease and the ubiquitin system, observes Cecile Pickart, a biochemistry professor at Johns Hopkins University's School of Public Health who has studied ubiquitin since 1982. But she adds, "it's just a matter of time" before a substantial number of scientists get involved. One roadblock to research is that few of the enzymes that select proteins to undergo ubiquitin-mediated degradation are known. The features of a protein that make it a good substrate for these enzymes are also largely unknown. As these details are filled in, Pickart expects the role of the ubiquitin system in disease will become clearer, facilitating drug discovery. STALKING LINKS: Arthur Beaudet of the Baylor College of Medicine is studying the genetic relationships between ubiquitin and Angelman syndrome. Ubiquitin is a highly conserved 76-residue protein in the cytoplasm and nucleus of cells. First purified 24 years ago, it was dubbed "ubiquitous immunopoietic polypeptide" because it caused lymphocyte differentiation and seemed to turn up everywhere--in animal cells, plant cells, bacteria, and yeast (G. Goldstein et al., Proceedings of the National Academy of Sciences , 72:11-5, 1975). Several years later, ubiquitin was identified as the proteolytic agent in a rabbit reticulocyte fraction (K.D. Wilkinson et al., Journal of Biological Chemistry, 255:7529-32, 1980). Ubiquitin-mediated proteolysis is analogous to cutting a log into lumber. A ubiquitin-protein ligase covalently links a lysine residue on a doomed protein (the log) to one end of the ubiquitin molecule (a steel carriage). A huge enzyme complex known as a proteasome (a circular saw) recognizes the protein-ubiquitin combination. After another enzyme removes the ubiquitin for reuse (the steel carriage is likewise reused), the proteasome slices the protein up into short peptides (lumber). "The ubiquitin-proteasome is the anti-ribosome," says Jonathan Yewdell , who has studied proteasome function as chief of the cellular biology section in the Laboratory of Viral Diseases at the National Institute of Allergy and Infectious Diseases. "In other words, the ribosome is chugging out proteins all the time, and the cell has to get rid of a lot of them. Either it doesn't want them anymore, or they weren't made very well in the first place." PS-341, the ProScript drug, inhibits proteasome activity. Taken to an extreme, this blunt action is usually, though not invariably, toxic to cells (R. Glas et al., Nature, 392:618-22, 1998). "The trick is to titrate the amount of inhibition," says Peter Elliott , Senior Director, Pharmacology, at the firm. "The doses that we're using are cytotoxic to dividing cancer cells but not to nondividing normal cells. A cell that's rapidly dividing, like a cancer cell, needs a fair amount of protein turnover and a tight regulation of the cell cycle [another role of the ubiquitin system]. If you interfere with that, you send it into a downward spiral." Angelman Syndrome Thus far, Angelman syndrome (AS) is the only human disorder known to result from a defect in the machinery of the ubiquitin system. Characterized by mental retardation, seizures, abnormal gait, ataxia, and inappropriate laughter, this rare condition is associated with mutations or deletions in an imprinted gene encoding a ubiquitin-protein ligase (T. Kishino et al., Nature Genetics, 15:70-3, 1997; T. Matsuura et al., Nature Genetics, 15:74-7, 1997). "There are certain parts of the brain where the gene that's inherited from the father is silenced, or turned off, leaving those particular groups of cells entirely dependent on the maternal copy," says Arthur Beaudet , professor of molecular and human genetics at Baylor College of Medicine. "If the maternal copy is defective, there's a problem." The following list shows some highly cited papers relating to ubiquitin-mediated proteolysis. The original paper on the purification of what became known as ubiquitin appeared in 1975, and has been cited more than 300 times to date. The term "ubiquitin" was used first in 1975 in a paper describing the amino acid sequence of the protein. Ubiquitin research has grown at a rapid pace, as shown by the number of papers containing the word "ubiquitin" in the title, measured over four-year intervals since the term's first appearance. (Data source: Web of Science, Institute for Scientific Information, Philadelphia) 1975-197814 1979-198238 1983-198610 1987-1990280 1991-1994500 1995-1998680 Following are some of the most highly cited papers on ubiquitin, as of mid-July: G. Goldstein et al., "Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells," Proceedings of the National Academy Of Sciences, 72[1]:11-15, 1975 Times Cited: 302 D.H. Schlesinger et al., "Complete amino-acid sequence of ubiquitin, an adenylate cyclase stimulating polypeptide probably universal in living cells," Biochemistry, 14:2214-18, 1975 Times Cited: 205 L.T. Hunt, M.O. Dayhoff, "Amino-terminal sequence identity of ubiquitin and nonhistone component of nuclear protein A24," Biochemical and Biophysical Research Communications, 74:650-655, 1977. Times Cited: 89 K.D. Wilkinson et al., "Ubiquitin is the ATP-dependent proteolysis factor-I of rabbit reticulocytes," Journal Of Biological Chemistry, 255:7529-32, 1980. Times Cited: 146 M.H.P. West, W.M. Bonner, "Histone 2B can be modified by the attachment of ubiquitin," Nucleic Acids Research, 8:4671-80, 1980. Times Cited: 105 D. Finley, A. Varshavsky, "The ubiquitin system - functions and mechanisms," Trends in Biochemical Sciences, 10:343-7, 1985 Times Cited: 287 U. Bond, M.J. Schlesinger, "Ubiquitin is a heat-shock protein in chicken-embryo fibroblasts," Molecular and Cellular Biology, 5:949-56, 1985 Times Cited: 293 S. Jentsch et al., "The yeast DNA-repair gene rad6 encodes a ubiquitin-conjugating enzyme," Nature, 329:131-4, 1987. Times Cited: 348 G. Perry et al., "Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer-disease brains," Proceedings of the National Academy Of Sciences, 84:3033-6, 1987. Times Cited: 289 H. Mori et al., "Ubiquitin is a component of paired helical filaments in Alzheimer's disease," Science, 235:1641-4, 1987. Times Cited: 477 M. Glotzer et al., "Cyclin is degraded by the ubiquitin pathway," Nature, 349:132-8, 1991 Times Cited: 819 M. Scheffner et al., "The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of P53," Cell, 75:495-505, 1993. Times Cited: 284 V.J. Palombella et al., "The ubiquitin-proteasome pathway is required for processing the NF-kappa-B1 precursor protein and the activation of NF-kappa-B," Cell, 78:773-85, 1994. Times Cited: 501 A. Ciechanover, "The ubiquitin-proteasome proteolytic pathway," Cell, 79:13-21, 1994 Times Cited: 533 How the loss of the ubiquitin-protein ligase (UBE3A) leads to AS symptoms is unknown. But "there is some rough correlation of those cells that end up deficient, like [those in] hippocampus and cerebellum, with the general findings of the phenotype in the human," Beaudet observes. His lab has been working on developing a UBE3A knockout mouse. Meanwhile, a mouse with a disruption of another ubiquitin-protein ligase gene (Itch) has been found to show a spectrum of immunological diseases (W.L. Perry et al., Nature Genetics, 18:143-6, 1998). In some human disorders, a normal ubiquitin system encounters defective proteins that it degrades too much or too little. Cystic fibrosis, for example, is caused by a mutation in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR), a chloride-ion channel. As a result of the most common mutation, "rather than getting to where it's supposed to be and doing its job, the mutant protein gets degraded, and that degradation is mediated, in large part, by the ubiquitin pathway," says Ron Kopito, a professor in the department of biological sciences at Stanford University. The current hypothesis is that mutant CFTR protein fails to fold properly, triggering proteolysis (C.L. Ward et al., Cell, 83:121-7, 1995). But the mutation doesn't affect protein function, suggesting "that maybe in some way, the cell is too zealous in degrading this protein," notes Kopito. Inhibiting the ubiquitin system, however, only decreases degradation of mutant CFTR, leaving the protein's folding efficiency unaffected. Hence, inhibition "doesn't improve the probability of a mutant molecule getting to the plasma membrane and functioning properly," he says. Liddle's syndrome (LS), a rare hereditary form of renal hypertension, appears to result from mutations or deletions in the epithelial sodium-ion channel that likely decrease its ubiquitin-mediated degradation. The ubiquitin-protein ligase targeting this channel has been identified and found to bind to the region that is mutated or deleted in LS (O. Staub et al., EMBO Journal, 15:2371-80, 1996). Unfortunately for researchers, cells contain few copies of the sodium channel and even fewer copies of the ubiquitinated channel, so the link between ubiquitination and LS is still circumstantial, according to Daniela Rotin , an associate professor of biochemistry at the University of Toronto. She notes other complications in the LS story: The disorder probably involves increased openings of the sodium channel as well as defective endocytosis of the channel--which, in turn, may depend on its ubiquitination (L. Hicke, FASEB Journal, 11:1215-26, 1997). Earlier in the 1990s, two findings--one broad, the other more specific--spurred research into links between ubiquitin-mediated proteolysis and cancer. The broad finding was that the ubiquitin system drives the cell cycle (R.W. King et al., Science, 274:1652-9, 1996), an important role in rapidly dividing cancer cells. The specific finding related to a protein, E6, encoded by the human papilloma virus, which causes cervical cancer. E6 binds to a human ubiquitin-protein ligase, thereby targeting the tumor suppressor p53 for ubiquitin-mediated degradation (M. Scheffner et al., Cell, 63:1129-36, 1990; J.M. Huibregtse et al., Molecular and Cellular Biology, 13:775-84, 1993). More recent studies indicate that increased degradation of the putative tumor suppressor p27 could contribute to several forms of cancer. In the test tube, extracts from human colorectal carcinomas with low or absent levels of p27 cause enhanced proteolysis of p27 (M. Loda et al., Nature Medicine, 3:231-4, 1997). This suggests that increased p27 degradation, not reduced gene expression, leads to a low p27 level, a state correlated with earlier death. Other labs have reported similar findings relating to prostate cancer, non-small-cell lung cancer, and lymphoma. "Our current hypothesis is that this has nothing to do with nonspecific proteasomal activity," says Michele Pagano, the assistant professor of pathology at New York University Medical Center who led the colorectal cancer study. "It's perhaps due to an increase in ubiquitination." His group is now seeking the enzyme responsible for p27 ubiquitination. The reverse of ubiquitination--the removal of an attached ubiquitin right before a protein is degraded--may be involved in small-cell tumors and lung adenocarcinomas. One study found these growths had higher mRNA levels of a gene, Unph, that apparently encodes a de-ubiquitinating enzyme (D.A. Gray et al., Oncogene, 10:2179-83, 1995). A later study yielded opposite results (A. Frederick et al., Oncogene, 16:153-65, 1998). "Until we get some decent animal models, we're not going to know what's going on," says Douglas Gray, an associate professor of medicine at the University of Toronto and first author of the earlier paper. His lab is accordingly knocking out the homologue of Unph in mice. Muscle-wasting accompanies cancer, as well as many other pathological states, including sepsis and diabetes. Alfred Goldberg , a professor of cell biology at Harvard Medical School, largely blames the underlying phenomenon, increased protein breakdown, on the ubiquitin system (W.E. Mitch, A.L. Goldberg, New England Journal of Medicine, 335:1897-1905, 1996). "This pathway is up-regulated in almost all the major diseases where you get this marked loss of body weight," he says. In a still-unpublished study, his lab found ubiquitination more prevalent in extracts of atrophying muscles from tumor-bearing or septic rats than in control extracts. A known protein-ubiquitin ligase, E3á, seemed to catalyze the reaction, but Goldberg says that there is still no good inhibitor of the enzyme to use in an in vivo experiment. Immunohistochemical studies identified ubiquitin in the neurofibrillary tangles seen in Alzheimer's disease more than 10 years ago (H. Mori et al., Science , 235:1641-4, 1987). Ubiquitin-containing inclusion bodies have since been found in a host of neurodegenerative disorders, including Huntington's and Parkinson's diseases. But little progress has been made in understanding whether ubiquitination contributes to or results from these disorders (A. Alves-Rodrigues et al., Trends in Neurosciences, in press). One problem is the large size and insolubility of the inclusion bodies. As a result, only one protein inside them has ever been conclusively shown to be ubiquitinated: a tau fragment isolated from Alzheimer brains (M. Morishima-Kawashima et al., Neuron , 10:1151-60, 1993). The peptide that forms Alzheimer plaques inhibits ubiquitin-mediated proteolysis in vitro (L. Gregori et al., Journal of Biological Chemistry, 270:19702-8, 1995), but an in vivo finding is lacking. "The view in the field of neurodegeneration is that ubiquitination is something that happens when the cell is dying," says Maria Figueiredo-Pereira, an associate professor of biological sciences at Hunter College of the City University of New York. "I think it can be the other way around--that when ubiquitinated proteins accumulate, that hurts the well-being of the cell, and the cell dies." After she treated an immortalized neuronal cell line with a proteasome inhibitor or with cadmium, an inducer of oxidative stress, ubiquitin-containing inclusion bodies accumulated, in a dose-dependent fashion, in the cells. She has observed the same phenomenon in rat primary neurons. "What makes these proteins aggregate?" Figueiredo-Pereira asks. "Why aren't they being turned over? It could be mutations or oxidative stress or neurotoxic compounds changing their structure and unfolding them. And either they are not being recognized by the degradation machinery, or some of these stimuli themselves might upset that machinery." There are other potential points of impact between the ubiquitin system and human disease. For example, ubiquitination and partial proteasomal degradation of a precursor protein yield a subunit of the transcription factor NF-êB, which is involved in inflammatory and immune responses (V.J. Palombella et al., Cell, 78:773-85, 1994). The ubiquitin system also may generate many of the antigenic peptides that initiate an immune response (K.L. Rock et al., Cell, 78:761-71, 1994). Thus, a recent report suggests how the common Epstein-Barr virus (EBV) evades immune-system surveillance. An EBV protein incorporates a domain that resists ubiquitin-mediated degradation (J. Levitskaya et al., Proceedings of National Academy of Sciences, 94:12616-21, 1997). Researchers will surely uncover much more about the role of ubiquitination in disease. For if protein degradation is the flip side of protein expression, then implications of the ubiquitin system could be as broad as the functions of all the proteins that the system degrades. Douglas Steinberg is a freelance science writer based in New York.

Ubiquitin-Mediated Proteolytic System Plays Diverse Roles in Human Disease

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