SCIENCE & TECHNOLOGY
PROTEIN BREAKDOWN RECOUNTED Chemistry Nobelists describe research path that led to prize-winning ubiquitin findings STU BORMAN, C&EN WASHINGTON
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T WAS THE ROAD NOT TAKEN.
In the late 1970s, hot areas of biochemistry research included such fields as protein synthesis. But graduate student (now professor) Aaron Ciechanover and professorAvram Hershko of Technion-Israel Institute of Technology, in Haifa, were interested in the opposite of protein synthesis: regulated protein breakdown, the process by which cells getridof proteins that are problematic or have outlived their usefulness. Enzymologist Irwin A. Rose ofFox Chase Cancer Center, Philadelphia (now at the University of California, Irvine), later joined them in their quest. And the rest, as they say, is history. Ciechanover, Hershko, and Rose will now share the nearly $1.4 million 2004 Nobel
Oct. 11, page 10). "It'sahappy thing, which I have not begun to digest yet"—no pun intended. Regulated protein degradation is now known to play an important role in cell division, DNA repair, protein synthesis, and the immune system. And disease processes such as inflammation, muscle atrophy, and cancer resultfromdysregulation ofthe degradatfve process. A drug that inhibits the proteasome—Millennium Pharmaceuticals' multiple myeloma agent, Velcade—is thefirstprotein degradation inhibitory agent to have been approved, and others are currently being investigated. Research on the ubiquitin system can be traced back about a halfcentury According to Rose, "The whole thing started with the
tion of proteins requires energy was surprising at the time, since most researchers had believed that, to the extent that protein degradation did take place, it would be an energy-releasing process. In the early 1970s, Hershko and coworkers corroborated Simpson's finding by showing that cellular protein degradation requires the ATP-dependent enzyme tyrosine transaminase. Rose says he and his coworkers "began to investigate the possibility offindingan in vitro cell-free system that would show a requirement for ATP. I tried to do that for a number ofyears without success, because I had chosen cells that were much too complicated. This is an important factor. What you really want to do is get therightsystem ifyou're going to make some progress.'' In 1977, cell biology professor Alfred L. Goldberg and coworkers at Harvard Medical School succeeded by producing a cellfree extractfromreticulocytes (maturing red blood cells) in which energy-dependent protein degradation occurred. Hershko's group, now including Ciechanover, soon began using such extracts to study the degradation process. Separating the cell-free reticulocyte system into fractions caused a loss of its protein-degrad-
UBIQUITINATORS Hershko and Ciechanover (from left in left photo) and Rose are this year's chemistry Nobelists.
Prize in Chemistry for having discovered ubiquitin-mediated protein degradation. In this process, chains ofthe peptide ubiquitin mark unwanted proteins and direct them to a large protein complex, the proteasome, which then breaks them down into peptidefragmentsthat can be reused. "I'm really overwhelmed'' about sharing the Nobel Prize, Ciechanover told C&EN the week of the announcement (C&EN,
realization that ATP {adenosine triphosphate} was required for protein breakdown" —an observation made in liver slices in 1953 by biochemist Melvin V Simpson at Tufts College Medical School, Boston. "Prior to that, there wasn't anyunderstanding of how proteins got broken down. In fact, it was uncertain as to whether proteins turned over in normal cells." Simpson's discovery that the degrada-
ing ability, and subsequent recombining of fractions restored it, leading the researchers to conclude that at least two components—and perhaps three, four, or more—were required for cellular protein degradation. "This was an extremely important discovery," Ciechanover says, "as it was the beginning of a new paradigm. Now we know that there are more than 1,000 components of the ubiquitin sys-
"What you really want to do is get the right system if you're going to make some progress." 26
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Target protein ΑΤΡ^ -Ε2, Ε3
Λ
Ε3
^ΛΜ..^Λ...^υΛ..^Λ
.^Α..^Α...^Δ..^Α Proteasome ν * ί Ι \ \ DEGRADING ACT In ubiquitin-mediated protein degradation, ubiquitin Peptide (Ub) is activated by the enzyme E1 in a reaction that requires energy from ATP. fragments ' Ubiquitin is transferred to a second enzyme, E2, and the E2-ubiquitin complex then transfers ubiquitin to a target protein that has been recognized by a third enzyme, E3. By repetitive ubiquitin additions, a ubiquitin chain forms on the unfortunate protein, which is then directed to the proteasome to meet its fate.
tern." A key component of one of the pu rified fractions turned out to be a small heat-stable peptide called APF-1. SUBSEQUENTLY, "Avram went to do a sabbatical at [Rose's} lab," Ciechanover says. Rose was "a superb enzymologist" who was into enzyme mechanisms, including the way some enzymes use ATP, and 'Avram thought Ernie would help him decipher the role ofATP in protein degradation." This first stint led to a series of sabbati cals and summer sessions that Ciechanover and Hershko spent with Rose at Fox Chase Cancer Center. Early on, the researchers found that APF-1 could attach to proteins, and in 1980 they hypothesized that APF1 labeling was a signal that led to degrada tion ofthe labeled protein by a downstream protease. Agroup led by University ofUtah biochemistry professor Martin Rechsteiner, and later another led by Goldberg, found this protease to be a large protein complex called the proteasome. Meanwhile, Gideon Goldstein of New Tfork University School of Medicine and coworkers reported having identified and sequenced a peptide "probably represent ed universally in living cells," and they called it "ubiquitous immunopoietic polypep tide," subsequently shortened to "ubiquitin."Three Fox Chase postdocs—Keith D. Wilkinson, Michael K. Urban, and Arthur L. Haas—reported in 1980 that APF-1 was identical to ubiquitin. "It was an unusual situation," Rose recalls. "Postdocs don't usually publish papers by themselves, but that's how it worked out." To be conjugated to protein substrates, ubiquitin first needs to be activated. "We isolated the three enzymes [called El, E2, and E3} that activate ubiquitin and conju
gate it to proteins," Ciechanover says, "and Ernie was instrumental in deciphering the biochemistry of all the activations." "I don't know why I was put into the whole [Nobel Prize-winning} business my self, but I guess I deserve a little credit," Rose says, particularly for his work on the
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mechanism of the El, E2, and E3 system. "I also did some work on isopeptidases, en zymes that take ubiquitin off proteins and recover it for reutilization." However, Ciechanover calls the isolation and mech anistic elucidation of the El, E2, and E3 system "the core discovery" of ubiquitin-
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SCIENCE & TECHNOLOGY 100
YEARS AGO
Noble-Gas Discovery Garnered 1904 Chemistry Nobel Prize
E
very first-year chemistry sturated nitrogen from air and then dent knows about them nowapassed the dried gas through reddays: those chemically dull elehot magnesium, which absorbed ments that occupy the far right the nitrogen and left behind the column of the periodic table. But it heavier component. Rayleigh rewasn't until the end of the 1800s that moved nitrogen from air samples by the noble gases were discovered and forming nitrogen oxides using electheir place in the periodic table was tric discharges. established. For those accomplishThe purified residue was analyzed ments, Sir William Ramsay was by physical and chemical means, awarded the Nobel Prize in Chemistry and its spectrum was measured. 100 years ago. The studies proved that air indeed contains a component that—prior to Born in Glasgow in 1852, Ramsay Ramsay and Rayleigh's work—had "inherited the taste for chemistry," as been unidentified. In August 1894 at he put it in his 1904 Nobel lecture, a meeting of the British Association, from both sides of his family. His paRamsay and Rayleigh announced ternal grandfather was a chemical the discovery of a new element they manufacturer and came from a long named argon—Greek for "lazy" beline of Scottish dyers. His mother's cause of its chemical inertness. In father was a physician and author of the same year that Ramsay won the medical textbooks. Nobel Prize in Chemistry, Rayleigh As a university student, Ramsay was honored with the prize in focused on organic chemistry. But physics, in part for his discovery of during his tenure at the University of argon. Bristol (1882-87) and later at University College London, Ramsay investiWhile conducting experiments gated stoichiometry, thermodynamthat led to the discovery of argon, A I R A P P A R E N T In this photo, Ramsay is ics, properties of gases, and other Ramsay learned that American scithought to be posing in his laboratory at topics in physical chemistry. entists had observed unidentified University College London, where he was chair gases evolving from uranium minIn the early 1890s, Ramsay puzzled of chemistry from 1887 to 1912. erals when they were heated in sulover a curious observation made by furic acid. He turned his attention to those minerals and others, Lord Rayleigh (John William Strutt). Rayleigh, who was a scienhoping to find argon compounds. Instead, he discovered terrestist at the Royal Institution of Great Britain, found that the densitrial sources of helium, which until that time had only been ty of nitrogen collected from air samples always appeared known to exist in the sun. greater than the density of the gas prepared chemically—for example, through decomposition of nitrogen compounds. A related In his Nobel lecture, Ramsay explained that similarities in the problem had been considered more than 100 years earlier by properties of helium and argon and analysis of the periodic table Henry Cavendish, but was left unsolved. At first, Ramsay and led him to conclude that the two elements "belong to the same Rayleigh worked on the problem independently. Eventually, they natural family ... and there must exist at least three other elecollaborated and communicated regularly, though they continments of the same class." ued working in separate laboratories. Using liquefaction and fractional distillation methods, Ramsay succeeded in isolating from air three new elements in the sumThe two proposed that the discrepancy in gas densities was mer of 1898. He named them krypton ("hidden one"), neon ("new due to the presence of a heavier-than-nitrogen component of air, and they devised experiments to search for it. Ramsay sepaone"), and xenon ("the stranger").—MITCH JACOBY
mediated proteolysis because it revealed the molecular details of how the whole process worked. Others who made important discoveries about the ubiquitin system include cell biology professors Alexander J. \farshavsky of California Institute of Technology and Daniel Fînley of Harvard Medical School Varshavsky, Ciechanover, and Finley found clear evidence that ubiquitination is required for many proteins to be degraded in living cells and that ubiquitin-mediated proteolysis is the major selective proteolytic 28
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system in the cell. ^Varshavsky's group discovered the major physiological functions of the ubiquitin system—in the cell cycle, DNArepair, transcriptional regulation, and stress responses—and identified the first protein degradation signals, features ofproteins that cause them to become targets of ubiquitin conjugation. Varshavsky's lab was also responsible for a number of key discoveries about the El, E2, and E3 system. Research on the biological effects of ubiquitin-mediated proteolysis has continued. For example, several groups have con-
tributed to a better understanding of the role ofprotein degradation in processes like the cell cycle and transcriptional regulation On the day ofthe Nobel announcement, a Nobel Foundation interviewer asked Ciechanover ifhe had any advice for young students of science. He replied that one should select a research problem that's original—like protein degradation was in the 1970s, when the predominant attention of most protein biochemists was focused elsewhere. Then, "just believe in yourself and do it," he said. • HTTP://WWW.CEN-ONLINE.ORG
