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Citation: The Albert Lasker Award for Basic Medical Research

Presented to: Aaron Ciechanover, Avram Hershko, and Alexander Varshavsky

For the discovery and the recognition of the broad significance of the ubiquitin system of regulated protein degradation, a fundamental process that influences vital cellular events, including the cell cycle, malignant transformation, and responses to inflammation and immunity.

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. (see below)

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 (missing word to conclude this sentence?). 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 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.

Citation text by Evelyn Strauss, Ph.D.

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