2000 Albert Lasker Basic Medical Research Award

Ubiquitin system for regulated protein degradation

Once scientists cracked the genetic code in the early 1960s, the field of molecular genetics boomed. Researchers began uncovering myriad mechanisms that govern how proteins are made from their DNA blueprints. Soon every journal contained descriptions of new triggers that flipped genes on and off. Although scientists had known since the 1950s that protein levels reflect a balance between production and destruction, the genetic revolution washed out almost all interest in the degradative process. Amidst the tide of inquiry aimed at understanding protein production, however, a small current pushed in the opposite direction. Following trickles of experimental evidence, Avram Hershko and Aaron Ciechanover pursued the idea that cells eliminate proteins with the same degree of sophistication that they manufacture them. At the heart of this process lies ubiquitin, a small protein that targets proteins for destruction. Hershko and Ciechanover elucidated the biochemical pathway that marks proteins, and found that three enzymes act sequentially to accomplish this task. Alexander Varshavsky and Ciechanover then demonstrated that the ubiquitin system for protein degradation works not only in the test tube, but also in living cells, where it plays a key role in regulating cellular growth and division. Varshavsky then discovered the first set of rules that dictates which proteins are destroyed. The discovery of the ubiquitin system has revolutionized scientists' concept of intracellular protein degradation. Unlike early ideas that included the notion of an unregulated protein incinerator inside the cell, current understanding has clarified that protein destruction is a highly complex, temporally controlled, and tightly regulated process. It plays important roles in a broad array of basic cellular events, and when it malfunctions, it causes disease.

ATP requirement: A paradox

As a postdoctoral fellow in San Francisco in the late 1960s, Hershko discovered a curious feature about the degradation of a particular protein. Destroying this protein demanded ATP, the cell's fuel, yet breaking down proteins liberates energy. Furthermore, the enzymes that chew up proteins, proteases, perform their job without energy input. Hershko wondered why an inherently energy-producing reaction consumed ATP. Intrigued by this paradox, he pursued the problem when he returned to the Technion-Israel Institute of Technology in Haifa and set up his own lab.

Scientists knew that cells broke down their own proteins under certain conditions: to dispose of defective proteins and to use them for food when starved. Researchers also realized that destruction as well as production could control protein concentrations, although they didn't appreciate the extent to which cells would obliterate proteins as a way to regulate cellular activities.

Protein degradation was thought to occur in the lysosome, a pouch inside the cell that contains a multitude of protein-destroying enzymes. Interfering with the activities of the lysosome, however, thwarted predominantly the digestion of proteins added to the outside of cells — not those already inside. Moreover, some proteins in cells remained stable for long periods of time, while others disappeared rapidly. If the lysosome indiscriminately demolished proteins, these classes should not be distinguishable. These observations suggested that multiple protein degradation pathways existed, and that only some of them passed through the lysosome. Although scientists understood that ATP was required to maintain the toxic lysosomal environment, no one knew why ATP would be required for the non-lysosomal pathway.

A death tag

To study protein degradation, Hershko wanted to split cells open so he could separate and identify the individual components required. He took advantage of an observation made by Alfred Goldberg of Harvard Medical School, who had shown that extracts of immature red blood cells require ATP to break down abnormal proteins. Because these cells don't contain lysosomes, destruction had to occur by the other pathway. In addition, these cells destroy many proteins as they make their way from their immature lives, in which they perform many tasks, to mature cells specialized to carry hemoglobin. Hershko reasoned that they would serve as a plentiful source of the enzymes that were involved in the ATP-dependent, non-lysosomal protein destruction system.

In 1977, Ciechanover entered Hershko's lab as a graduate student, and joined attempts to understand this process. By attaching radioactive tags to a protein, the researchers could analyze its fate in the cell extract. If the protein remained intact, the radioactive labels would stay bound to a full-sized protein; if it broke down, the labels would end up associated with smaller protein fragments. The researchers separated the blood cell contents into two fractions and found that neither by itself could promote protein degradation. They regenerated the process only by mixing the two components. This result suggested that the reaction required more than one factor.

Hemoglobin composed the major portion of one of the fractions. After many conventional but unsuccessful attempts to separate the substance required for protein degradation from the abundant hemoglobin, the researchers took an unusual step: They boiled the fraction. Like most proteins, which denature when heated, hemoglobin hardened. In contrast, the portion required for protein degradation remained dissolved and active. In 1978, the researchers purified it and named it ATP-dependent proteolysis factor 1 (APF-1). Later, others showed that APF-1 was ubiquitin.

Thinking that this small, heat-stable protein might activate a protease, they sought the presumptive target enzyme. They labeled APF-1 with a radioactive tag, mixed it with the cellular fraction, and separated the proteins in the extract. In the absence of energy input, the radioactive APF-1 migrated as the small protein it was. But when the researchers added ATP, they saw not one, but multiple radioactive proteins of different sizes. This result suggested that APF-1 was attaching to many proteins in the extract.

Because the cellular fraction contained proteins destined for degradation in addition to enzymes required for the reaction, the team began to suspect that APF-1 was linked to degradation targets rather than to the proteases that destroyed them. To probe this possibility, they added single radioactive proteins known to be good substrates for proteolysis to the APF-1–containing extract. As they hypothesized, the proteins increased by the size of APF-1. Furthermore, each sample produced numerous radioactive proteins, each differing by the size of another APF-1 molecule. These observations indicated that multiple APF-1 molecules bound to individual proteins fated for destruction. Ironically, it appeared that proteins get bigger, not smaller, before they are demolished.

Other surprises revealed themselves. Physical and chemical treatments designed to disrupt loose interactions between proteins did not perturb the association between APF-1 and target proteins. Aided by advice from Irwin Rose, who hosted the Israeli researchers during a sabbatical at the Fox Chase Cancer Center in Philadelphia, they determined that APF-1 was linked to the proteins by the same type of stable bond that holds together amino acids in proteins. This provided a mechanism for their observation that proteins grew before they shrank. From these experiments, Hershko, Rose, and Ciechanover predicted that APF-1 attachment constituted a death signal that directed a protein to a protease.

The researchers did not yet realize that they were working on a previously known protein. Keith Wilkinson, a postdoctoral fellow in the Rose lab, noticed a similarity between Ciechanover's and Hershko's findings and those from a distant scientific realm. His friend Michael Urban, another postdoctoral fellow, knew that a small protein called ubiquitin attached to a DNA-associated protein in the same unusual way that APF-1 bound to proteins destined for destruction. Ubiquitin had been discovered several years earlier, and was known to reside in a wide variety of organisms and tissues (thus the name) — but performed no known function. Wilkinson, Urban, and Arthur Haas showed that APF-1 and ubiquitin were one and the same protein.

Ubiquitination machinery

Hershko and Ciechanover teased apart the extracts to identify the cellular equipment that added ubiquitin to proteins. They discovered that three enzymes were required. The first (E1) activates ubiquitin by forming a high-energy bond with it, using ATP in the process. E1 then transfers ubiquitin to the second enzyme (E2). The third enzyme (E3) unites E2 and a protein, facilitating the transfer of ubiquitin to its target.

In 1985, Hershko showed that the many ubiquitin molecules add to proteins by linking together, and that proteins with the resulting chains are better substrates for degradation than those with single ubiquitins attached at multiple sites. Several years later, Varshavsky showed how the individual ubiquitin molecules hook up in these chains. Scientists now know that the multi-ubiquitin structure provides a molecular handle on proteins bound for destruction. The proteasome, a large apparatus that contains multiple proteases, can presumably grab the multi-ubiquitin chain-bearing proteins by this distinctive handle; ubiquitin therefore provides the molecular ticket to this destruction machine.

Hershko, Ciechanover, and their colleagues had unraveled the mechanism of ubiquitin-dependent degradation in cellular extracts. But molecular machinery doesn't always behave the same in the test tube as it does in an intact cell, so no one knew how relevant these findings were to living creatures. Some of the researchers' observations suggested that the ubiquitin system worked in intact cells. They discovered that when they fed cells an abnormal amino acid that could be incorporated into proteins, ubiquitin latched onto the resulting proteins more efficiently than it attached to normal versions. Furthermore, protein destruction rose significantly in these cells. This correlation between ubiquitination and degradation led the researchers to hypothesize that the cell targeted these abnormal proteins for degradation via the ubiquitin system. But which specific physiological processes, if any, were affected? Achieving a detailed understanding of the design and functional significance of the ubiquitin system would require some means to perturb the ubiquitin system in cells.

Ubiquitin system in living cells

At MIT, Varshavsky's interest in a completely different problem led him, with Ciechanover, to establish the importance of the ubiquitin system in living cells. Varshavsky was trying to understand the function of the DNA-associated protein that also had a ubiquitin attached to it. Scientists in Japan had discovered a particular type of mutant mouse cell that appeared to have a defect related to this protein. At 32 degrees these cells behave normally, while at 39 degrees they stop dividing. Furthermore, the Japanese scientists found that these cells stop making the ubiquitinated form of the DNA-associated protein at the higher temperature.

Varshavsky suspected that the cells might harbor a flaw somewhere in the ubiquitin system. Some substance required for attaching ubiquitin was damaged, he reasoned. It could function tolerably well at the lower, but not at the higher, temperature.

When Ciechanover came to MIT as a postdoctoral fellow in a different lab, he started moonlighting with Varshavsky on this project. He joined Daniel Finley, a graduate student, and together the group showed that extracts made from the mutant cells grown at 32 degrees and then shifted to 39 degrees did not add ubiquitin to proteins. They pinned the defect to an abnormal version of E1, the enzyme that activates ubiquitin for transfer to protein targets.

Next they showed that these cells defective for ubiquitination also lost the ability to destroy short-lived and abnormal proteins. This was the first clear evidence that attaching ubiquitin to proteins is essential for their degradation in a living cell.

When these cells stop growing, they do so in a specific way. While dividing, cells enact a highly ordered sequence of events. Together, these comprise the so-called cell cycle. This organized pathway ensures, for example, that they duplicate their DNA before they split in two. When shifted to the higher temperature, most of the cells with defective E1 enzyme halt at a specific step in this cycle.

At the time, other groups had recently discovered particular proteins that disappear at certain points in the cell cycle. Their destruction enables the cell to proceed on its reproduction pathway. Varshavsky, Finley, and Ciechanover suggested that a failure to destroy one of these proteins in the mutant mouse cells could thwart the cell's ability to move from one step to the next. The researchers also showed that the shift to 39 degrees increased the amounts of certain stress-related proteins. They hypothesized that the ubiquitin system normally targets a protein that sparks production of these stress-related proteins; if the ubiquitin system failed to function, it would not demolish the regulator protein, which would continue to trigger production of the stress-related proteins it controls. Through the work of many labs, both of these hypotheses have since proved true.

Ubiquitin system in many physiological processes

To fully understand what biological processes the ubiquitin system impacts, Varshavsky turned to yeast, where he could more rigorously tie individual genes to particular physiological events. Scientists had known for years that disrupting the activities of a certain protein interfered with the cell's ability to repair DNA, but they had no clues about its enzymatic function. Varshavsky showed that this protein was an E2 enzyme. Subsequently, he made other critical links between the ubiquitin protein degradation system and proteins known to play central roles in vital cellular processes.

The work so far had established that ubiquitin provided a universal flag that condemned a protein to destruction. But what signal determined whether a protein would be ubiquitinated in the first place? Proteins presumably carried other signals that would indicate to one or more E3 enzymes whether to add ubiquitin. With such a system, the cell could obliterate different groups of proteins at particular times or under certain conditions. In 1986, Varshavsky elucidated the first of what has turned out to be a large catalog of signals and systems for degradation. Each of the many E3 enzymes recognizes specific features, and thus targets a small subset of proteins.

The ubiquitin system touches virtually every major physiological process. In addition to governing cell division and response to stress, for example, it controls central regulators of the immune system and embryonic development. Not surprisingly for a system that impacts so many crucial activities, defects in ubiquitination underlie a number of diseases.

Some viruses, for example, exploit the ubiquitin system. Human papilloma virus, which causes cervical cancer, releases cells from their normal growth constraints. It accomplishes this with a protein that attaches to p53, a protein that normally pulls the brakes on uncontrolled cell division. Binding of the human papilloma virus protein renders p53 susceptible to ubiquitin addition by a host E3 enzyme. In this way, the virus fools the cell into thinking that normal p53 is defective, and should be destroyed. Thus, virus-infected cells multiply instead of committing suicide when they should, and the virus gains a permanent host.

The same cellular E3 that human papilloma virus uses is involved in a completely different disease. People with Angelman syndrome — an inherited condition of mental retardation and motor skill problems — carry alterations in the gene that encodes this E3 enzyme. Although no one yet knows which protein(s) this enzyme normally targets for degradation, research suggests that destroying it is important for proper brain development. The accumulation of this as yet unidentified protein (or proteins) in people with Angelman syndrome presumably intoxicates the nervous system and leads to symptoms.

Many more illustrations of ubiquitin's central role in human illness exist. For example, the ubiquitin system plays a key role in mounting the inflammatory response that combats microbial invaders and may, in addition, contribute to autoimmune disorders.

These diseases reflect only a small subset of the physiological processes that utilize ubiquitin to control the concentration of particular proteins. Clearly, regulated protein degradation via the ubiquitin system has risen to great importance since its humble beginnings as an obscure research topic nearly 25 years ago.

by Evelyn Strauss

Key publications of Aaron Ciechanover

Ciechanover, A., Hod, Y., and Hershko, A. (1978). A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes. Biochem. Biophys. Res. Commun. 81, 1100–1105.

Hershko, A., Ciechanover, A., Heller, H., Haas, A. L., and Rose, I. A. (1980). Proposed role of ATP in protein breakdown: conjugation of proteins with multiple chains of the polypeptide of ATP-dependent proteolysis. Proc. Natl. Acad. Sci. USA. 77, 1783–1786.

Ciechanover, A., Elias, S., Heller, H., and Hershko, A. (1982). Covalent affinity purification of I ubiquitin-activating enzyme. J. Biol. Chem. 257, 2537–2542.

Hershko, A., Heller, H., Elias, S., and Ciechanover, A. (1983). Components of ubiquitin-protein ligase system: resolution, affinity purification, and role in protein breakdown. J. Biol. Chem. 258, 8206–8214.

Finley, D., Ciechanover, A., and Varshavsky, A. (1984). Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85. Cell. 37, 43–55.

Ciechanover, A., Finley, D., and Varshavsky, A. (1984). Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85. Cell. 37, 57–66.

Ferber, S. and Ciechanover, A. (1987). Role of arginine-tRNA in protein degradation by the ubiquitin pathway. Nature. 326, 808–811.

Ciechanover, A., Ferber, S., Ganoth, D., Elias, S., Hershko, A., and Arfin, S. (1988). Purification and characterization of arginyl-tRNA-protein transferase from rabbit reticulocytes: its involvement in modification and ubiquitin-dependent degradation of proteins bearing acidic N-termini. J. Biol. Chem. 263, 11155–11167.

Key publications of Avram Hershko

Ciechanover, A., Hod, Y., and Hershko, A. (1978). A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes. Biochem. Biophys. Res. Commun. 81, 1100–1105.

Hershko, A., Ciechanover, A., Heller, H., Haas, A. L., and Rose, I. A. (1980). Proposed role of ATP in protein breakdown: conjugation of proteins with multiple chains of the polypeptide of ATP-dependent proteolysis. Proc. Natl. Acad. Sci. USA. 77, 1783–1786.

Ciechanover, A., Elias, S., Heller, H.. and Hershko, A. (1982). Covalent affinity purification of ubiquitin-activating enzyme. J. Biol. Chem. 257, 2537–2542.

Hershko, A., Heller, H., Elias, S., and Ciechanover, A. (1983). Components of ubiquitin-protein ligase system: resolution, affinity purification and role in protein breakdown. J. Biol. Chem. 258, 8206–8214.

Hershko, A., Leshinsky, E., Ganoth, D.. and Heller, H. (1984). ATP-dependent degradation of ubiquitin-protein conjugates. Proc. Natl. Acad. Sci.USA. 81, 1619–1623.

Hershko, A., Heller, H., Eytan, E.. and Reiss, Y. (1986). The protein substrate binding site of the ubiquitin-protein ligase system. J. Biol. Chem. 261, 11992–11999.

Ganoth, D., Leshinsky, E., Eytan, E., and Hershko, A. (1988). A multicomponent system that degrades proteins conjugated to ubiquitin. Resolution of components and evidence for ATP-dependent complex formation. J. Biol. Chem. 263, 12412–12419.

Key publications of Alexander Varshavsky

Finey, D., Ciechanover, A., and Varshavsky, A. (1984). Thermolability of ubiquitin- activating enzyme from the mammalian cell cycle mutant ts85. Cell. 37, 43–55.

Ciechanover, A., Finley, D., and Varshavsky, A. (1984). Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85. Cell. 37, 57–66.

Bachmair, A., Finley, D., and Varshavsky, A. (1986). In vivo half-life of a protein is a function of its N-terminal residue. Science. 234, 179–186.

Jentsch, S., McGrath, I. P.. and Varshavsky, A. (1987). The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme. Nature. 329, 131–134.

Bachmair, A. and Varshavsky, A. (1989). The degradation signal in a short-lived protein. Cell. 56, 1019–1032.

Chliu, V., Tobias, I. W., Bachmair, A., Ecker, D., Gonda, D. K., and Varshavsky, A. (1989). A multiubiquitin chain is confined to a specific lysine in a targeted short-lived protein. Science. 243, 1576–1583.

Finley, D., Bartel, B., and Varshavsky, A. (1989). The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature. 338, 394–401.

Johnson, E. S., Gonda, D. K., and Varshavsky, A. (1990). Cis-trans recognition and subunit-specific degradation of short-lived proteins. Nature. 346, 287–291.

E., Ganoth, D., Armon, T., and Hershko, A. (1989). ATP-dependent incorporation of 20S protease into the 26S complex that degrades proteins conjugated to ubiquitin. Proc. Natl. Acad. Sci. USA. 86, 7751–7755.

Award presentation by Michael Brown

Biblical scholars tell us that the Book of Ecclesiastes was written by King Solomon of Jerusalem. The book deals with the fleeting nature of life and includes the famous passage, "For everything there is a season, and a time for every purpose under the heavens: A time to be born and a time to die; A time to plant, and an time to pluck up that which is planted; A time to break down and a time to build up." Solomon was describing things he could see, but his wisdom applies to things we cannot see, including the proteins that make up living cells. Proteins, too, have a time to be born and a time to die. This year's Lasker Basic Science Award is given to three scientists who taught us how proteins die. In so doing, they taught us how cells live.

Let me remind you that proteins are the workers in the beehive of the cell. They catalyze the set of reactions that we call life, and they are traffic cops that regulate the timing of every event. Proteins tell our cells when to be born and when to die.

A turning point in biology was the discovery that proteins are nothing more than strings of chemicals called amino acids. A typical protein has 500 amino acids lined up in a strict order dictated by the genetic code. Indeed, a major purpose of the Human Genome Project is to tell us the order of amino acids in all of our proteins.

The importance of proteins was first appreciated in the 1930s, and biologists soon turned to figuring out how proteins were made. If we can understand how proteins are made and how this information passes from one generation to the next, we will understand life itself. This worldview led to the rise of molecular biology and its three great discoveries: first, recognition of DNA as the hereditary material; second, elucidation of the structure of DNA which told us how it replicates; and third, unraveling of the machinery that turns the DNA code into a protein string.

The molecular biologists gave little thought to the mechanism by which protein strings are disassembled. Indeed, they couldn't believe that their beautiful proteins were ever broken down at all.

And yet, annoying bits of evidence suggested that proteins had a time to die. The most important insight came from Rudolph Schoenheimer right here in New York City. Schoenheimer is not a household word like Watson-Crick, and yet he exposed the other side of the Watson-Crick coin. Schoenheimer fled from Nazi Germany in 1934, and he was given a job by Hans Clarke at Columbia's College of Physicians and Surgeons, one of the few places in the world where isotopes were being made. Schoenheimer fed isotopically labeled amino acids to animals and observed their incorporation into proteins. But the labeled proteins did not live forever. Indeed, the body destroyed its proteins precisely as fast as they were made. In the steady state, if the body made 1,000 molecules of a protein every hour, it also degraded 1,000 molecules every hour. Schoenheimer called this "the dynamic steady state."

Schoenheimer's work did not revolutionize biology — at least not right away. As late as 1955, Jacque Monod, an intellectual icon of molecular biology, proclaimed that "there [is] no conclusive evidence that the protein molecules within the cells of mammalian tissues are in a dynamic steady state." Although Schoenheimer's work was disputed, it laid the foundation for the discovery that we honor today.

That discovery begins with another victim of the Nazis, Avram Hershko. Avram was born in 1937, in a small Hungarian village where his father was a schoolteacher. The Nazis sent Avram's mother and father to different concentration camps — one East and one West. For years each of them thought the other was dead. Avram remained with his mother and miraculously they all survived — although most of their relatives did not. In 1950, when Avram was 13, the family emigrated to Israel.

Israel's remarkable attribute is its maintenance of a scholarly tradition even during the most perilous times. Avram attended the Hadassah Medical School of the Hebrew University of Jerusalem, where he earned both an MD and a PhD degree. After serving as a physician in the Israeli army, Avram followed the well-traveled Israeli path of postdoctoral study in the United States. In 1969, he moved to San Francisco, where he worked with the late Gordon Tomkins, a hypnotic figure who mesmerized a cadre of young scientists. Tomkins introduced Avram to the dark side of molecular biology — protein degradation. They studied the regulation of an enzyme in animal cells. They found that the enzyme was degraded much more rapidly than other proteins, and this rapid degradation allowed the enzyme concentration to change rapidly in response to regulatory signals.

In 1972, Avram returned to Israel where he joined the Technion, Israel's MIT. He decided to study protein degradation at the biochemical level. He was fortunate to have a bright graduate student, Aaron Ciechanover. Aaron was a Sabra. Born in Haifa in October 1947, he is one month older than his country. Aaron earned his MD at the Hebrew University, and like Avram he served as an Army physician. In 1976, Aaron entered Avram's laboratory to earn his PhD, and that's when the fireworks began.

Avram and Aaron set up a cell-free system to measure the degradation of proteins in test tubes. They soon learned that multiple enzymes were required. Following the biochemist's classic paradigm, they separated the enzymes, and here they made their revolutionary discovery. When the enzymes selected a protein for destruction, they identified it by attaching a lethal tag. For the unfortunate protein, that tag was a Texas death sentence — there was no appeal. The protein was immediately disassembled into amino acids. The surprising aspect was the nature of the death tag. The tag was a protein — a very small protein called ubiquitin. But how is one protein, ubiquitin, attached to another protein, its target? The ubiquitin-tagging reaction had no precedents in biology so Avram and Aaron were on their own. They no longer had a paradigm to follow.
Over the five years from 1976 to 1981, Avram and Aaron dissected the ubiquitin-tagging reactions methodically, thoroughly, and elegantly. They found that three enzymes were required, acting one after the other. Each enzyme picked up ubiquitin and passed it to the next enzyme in the chain, like relay runners passing a baton. At the end of the line, the protein left holding the ubiquitin was doomed.

Hershko and Ciechanover published their findings in 10 remarkable papers, culminating in a masterful review that put the whole ubiquitin pathway together. Along the way they had help from several colleagues. The most important was Irwin Rose at the Fox Chase Cancer Center in Philadelphia. When the breakthrough work was finished, it was Ciechanover's turn for a postdoctoral fellowship in the United States. He traveled to MIT where he met our third honoree, Alex Varshavsky.

Varshavsky had come to MIT by the most improbable path of all. He was born in 1946 in Moscow and he earned a PhD in biochemistry at Moscow's Institute of Molecular Biology. The story behind Varshavsky's escape from Russia is more riveting than a novel by John Le Carré. Suffice it to say, he used a clever ploy to fool the KGB into allowing him to attend a scientific meeting in Finland. From there he escaped to Sweden, and eventually he ended up at the US consulate in Frankfurt. His visa application was about to be rejected when he had the inspiration to telephone David Baltimore, a Nobel laureate at MIT. He had met Baltimore briefly during David's visit to Moscow and he hoped David would remember him. Whether he remembered him or not, Baltimore responded, and he influenced the State Department to allow Varshavsky to enter the United States. Penniless and without prospects, Varshavsky turned up at MIT. A faculty member, Alex Rich, invited Varshavsky to give an impromptu seminar. His lecture so impressed the faculty that Varshavsky was offered an assistant professorship on the spot.

Varshavsky studied histones, which are proteins that bind to DNA and establish the architecture of chromosomes. Histones are the only proteins that carry a ubiquitin tag and live to tell the tale. In the early 1980s, Varshavsky obtained from Japan a mutant clone of hamster cells that had a problem. When grown at elevated temperatures, these cells developed a deficiency of ubiquitinated histones. The cells also had another problem. They could not divide properly. Somehow ubiquitin deficiency stopped the cell cycle. Varshavsky asked a simple question: Was the deficiency of ubiquitinated histones caused by a failure of the enzymes that transfer ubiquitin to proteins? In the most improbable event of this whole story, who should turn up at MIT but Ciechanover, one of only two people in the world who knew how to study the ubiquitin transfer reaction. Ciechanover had not come to MIT to work with Varshavsky — he was working with another professor. Yet, fate brought Varshavsky and Ciechanover together. Even in retrospect, it is nearly impossible to calculate the probability that this meeting would even occur. It was largely the result of the Nazis who got Schoenheimer to New York and Hershko to Haifa and whose destruction of Moscow led eventually to Varshavsky's arrival at MIT. Maybe there is a God who has chosen these people. At MIT Varshavsky, Ciechanover, and graduate student Dan Finley set out to determine whether the mutant hamster cells had a defect in the Hershko/Ciechanover ubiquitin relay. And this is precisely what they found. When grown at elevated temperatures, the hamster cells had a block in the first enzyme of the ubiquitin transfer reaction. This block explained their deficiency of ubiquitinated histones and their failure to divide.

But the ubiquitination defect raised another question: What about protein degradation? Up to this point Hershko and Ciechanover had worked with isolated enzymes. The mutant hamster cells provided an opportunity to learn whether ubiquitination is required for protein degradation in living cells. And indeed that is just what they found. The mutant cells could not degrade their proteins.

The findings in the mutant hamster cells immediately moved ubiquitination to center stage in biology. No longer was this an elegant set of reactions that occurred in test tubes. Ubiquitination was crucial to cellular protein degradation, and it had something to do with cell division.

After making these discoveries in animal cells, Varshavsky switched to yeast, where the ubiquitination pathway could be studied with the full power of genetics. He added important chapters to the ubiquitin story, including the first insights into a mechanism by which cells choose the proteins that are to be degraded. Time prevents me from describing these accomplishments or the later contributions of Hershko and Ciechanover. Suffice it to say, these three scientists established the fundamentals of the ubiquitination system and its role in biology.

Before closing, let me tell you how important ubiquitination has turned out to be. Ubiquitination-triggered protein degradation is at the very core of biology. It influences not only normal cell division, but also the disordered cell division and differentiation that lead to cancer. Ubiquitination is responsible for the inflammatory process that leads acutely to fever, shock and death — and chronically to arthritis and other degenerative diseases. Several human genetic diseases result from defects in ubiquitination. I estimate that 1 out of 10 papers in current biology journals deals with ubiquitination in one system or another. Let me give you just one example.

You all know that cells in the body follow a precise series of reactions in order to divide. Proteins called cyclins control these events. When cyclins drop, it is a signal for the cells to enter the next phase of the cycle. The sudden fall in cyclins is caused by sudden ubiquitination, which leads to their degradation. The degradation allows the cell to enter the next phase of the cycle. We owe our active bodies to the ubiquitin relay that was discovered by Hershko and Ciechanover at the Technion and put into full biological context with Varshavsky.

I hope that this brief introduction gives you a feel for the elegance of the work of our three honorees and its fundamental importance to life. It is with great honor that I present them to you as recipients of the first Albert Lasker Basic Science Award of the new millennium.

Aaron Ciechanover

Nature Medicine Essay


Key publications of Aaron Ciechanover

Ciechanover, A., Hod, Y., and Hershko, A. (1978). A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes. Biochem. Biophys. Res. Commun. 81, 1100–1105.

Hershko, A., Ciechanover, A., Heller, H., Haas, A. L., and Rose, I. A. (1980). Proposed role of ATP in protein breakdown: conjugation of proteins with multiple chains of the polypeptide of ATP-dependent proteolysis. Proc.Natl. Acad. Sci. USA. 77, 1783–1786.

Ciechanover, A., Elias, S., Heller, H., and Hershko, A. (1982). Covalent affinity purification of I ubiquitin-activating enzyme. J. Biol. Chem. 257, 2537–2542.

Hershko, A., Heller, H., Elias, S., and Ciechanover, A. (1983). Components of ubiquitin-protein ligase system: resolution, affinity purification, and role in protein breakdown. J. Biol. Chem. 258, 8206–8214.

Finley, D., Ciechanover, A., and Varshavsky, A. (1984). Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85. Cell. 37, 43–55.

Ciechanover, A., Finley, D., and Varshavsky, A. (1984). Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85. Cell. 37, 57–66.

Ferber, S. and Ciechanover, A. (1987). Role of arginine-tRNA in protein degradation by the ubiquitin pathway. Nature. 326, 808–811.

Ciechanover, A., Ferber, S., Ganoth, D., Elias, S., Hershko, A., and Arfin, S. (1988). Purification and characterization of arginyl-tRNA-protein transferase from rabbit reticulocytes: its involvement in modification and ubiquitin-dependent degradation of proteins bearing acidic N-termini. J. Biol. Chem. 263, 11155–11167.

Avram Hershko

Nature Medicine Essay


Key publications of Avram Hershko

Ciechanover, A., Hod, Y., and Hershko, A. (1978). A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes. Biochem. Biophys. Res. Commun. 81, 1100–1105.

Hershko, A., Ciechanover, A., Heller, H., Haas, A. L., and Rose, I. A. (1980). Proposed role of ATP in protein breakdown: conjugation of proteins with multiple chains of the polypeptide of ATP-dependent proteolysis. Proc. Natl. Acad. Sci. USA. 77, 1783–1786.

Ciechanover, A., Elias, S., Heller, H. and Hershko, A. (1982). Covalent affinity purification of ubiquitin-activating enzyme. J. Biol. Chem. 257, 2537–2542.

Hershko, A., Heller, H., Elias, S., and Ciechanover, A. (1983). Components of ubiquitin-protein ligase system: resolution, affinity purification and role in protein breakdown. J. Biol. Chem. 258, 8206–8214.

Hershko, A., Leshinsky, E., Ganoth, D., and Heller, H. (1984). ATP-dependent degradation of ubiquitin-protein conjugates. Proc. Natl. Acad. Sci. USA. 81, 1619–1623.

Hershko, A., Heller, H., Eytan, E., and Reiss, Y. (1986). The protein substrate binding site of the ubiquitin-protein ligase system. J. Biol. Chem. 261, 11992–11999.

Ganoth, D., Leshinsky, E., Eytan, E., and Hershko, A. (1988). A multicomponent system that degrades proteins conjugated to ubiquitin. Resolution of components and evidence for ATP-dependent complex formation. J. Biol. Chem. 263, 12412–12419.

Alexander Varshavsky

Nature Medicine Essay


Key Publications of Alexander Varshavsky

Finey, D., Ciechanover, A. and Varshavsky, A. (1984) Thermolability of ubiquitin- activating enzyme from the mammalian cell cycle mutant ts85. Cell 37: 43–55.

Ciechanover, A., Finley, D. and Varshavsky, A. (1984) Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85. Cell 37: 57–66.

Bachmair, A., Finley, D. and Varshavsky, A. (1986) In vivo half-life of a protein is a function of its N-terminal residue. Science 234: 179–186.

Jentsch, S., McGrath, I. P. and Varshavsky, A. (1987) The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme. Nature 329: 131–134.

Bachmair, A. and Varshavsky, A. (1989) The degradation signal in a short-lived protein. Cell 56: 1019–1032.

Chliu, V., Tobias, I. W., Bachmair, A., Ecker, D., Gonda, D. K., and Varshavsky, A. (1989) A multiubiquitin chain is confined to a specific lysine in a targeted short-lived protein. Science 243: 1576–1583.

Finley, D., Bartel, B. and Varshavsky, A. (1989) The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature 338: 394–401.

Johnson, E. S., Gonda, D. K. and Varshavsky, A. (1990) Cis-trans recognition and subunit-specific degradation of short-lived proteins. Nature 346: 287–291.

E., Ganoth, D., Armon, T., and Hershko, A. (1989) ATP-dependent incorporation of 20S protease into the 26S complex that degrades proteins conjugated to ubiquitin. Proc. Natl. Acad. Sci. USA 86: 7751–7755.