1、The Nobel Prize in Chemistry 2004 was awarded jointly to Aaron Ciechanover, Avram Hershko and Irwin Rose for the discovery of ubiquitin-mediated protein degradation.Information for the Public6 October 2004A human cell contains some hundred thousand different proteins. These have numerous important f
2、unctions: as accelerators of chemical reactions in the form of enzymes, as signal substances in the form of hormones, as important actors in the immune defence and by being responsible for the cells form and structure. This years Nobel Laureates in chemistry,Aaron Ciechanover,Avram HershkoandIrwin R
3、ose, have contributed ground-breaking chemical knowledge of how the cell can regulate the presence of a certain protein by marking unwanted proteins with a label consisting of the polypeptide ubiquitin. Proteins so labelled are then broken down degraded rapidly in cellular “waste disposers” called p
4、roteasomes.Animation (Plug in requirement: Flash Player 6)Through their discovery of this protein-regulating system Aaron Ciechanover, Avram Hershko and Irwin Rose have made it possible to understand at molecular level how the cell controls a number of very important biochemical processes such as th
5、e cell cycle, DNA repair, gene transcription and quality control of newly-produced proteins. New knowledge of this form of controlled protein death has also contributed to explaining how the immune defence functions. Defects in the system can lead to various diseases including some types of cancer.P
6、roteins labelled for destructionDegradation needs no energy or does it?While great attention and much research have been spent on understanding how the cell controls the synthesis of a certain protein at least five Nobel Prizes have been awarded in this area the reverse, the degradation of proteins,
7、 has long been considered less important. A number of simple protein-degrading enzymes were already known. One example is trypsin, which in the small intestine breaks down proteins in our food to amino acids. Likewise, a type of cell organelle, the lysosome, in which proteins absorbed from outside a
8、re broken down, had long been studied. Common to these processes is that they do not require energy in order to function.Experiments as long ago as the 1950s showed, however, that the breakdown of the cells own proteins does require energy. This long puzzled researchers, and it is precisely this par
9、adox that underlies this years Nobel Prize in Chemistry: that the breakdown of proteins within the cell requires energy while other protein degradation takes place without added energy. A first step towards an explanation of this energy-dependent protein degradation was taken by Goldberg and his co-
10、workers who in 1977 produced a cell-free extract from immature red blood cells, reticulocytes, which catalyse the breakdown of abnormal proteins in an ATP-dependent manner (ATP = adenosine triphosphate the cells energy currency).Using such an extract Aaron Ciechanover, Avram Hershko and Irwin Rose,
11、in a series of epoch-making biochemical studies in the late 1970s and early 1980s, succeeded in showing that protein degradation in cells takes place in a series of step-wise reactions that result in the proteins to be destroyed being labelled with the polypeptide ubiquitin. This process enables the
12、 cell to break down unwanted proteins with high specificity, and it is this regulation that requires energy. As distinct from reversible protein modifications such as phosphorylation (Nobel Prize in Physiology or Medicine 1992), regulation through polyubiquitination is often irreversible since the t
13、arget protein is destroyed. Much of the work was done during a series of sabbatical leaves that Avram Hershko and Aaron Ciechanover of the Technion (Israel Institute of Technology) spent with Irwin Rose at the Fox Chase Cancer Center in Philadelphia, USA.The label is ubiquitinThe molecule that would
14、 later prove to be the label that marks out a protein for degradation was isolated as early as 1975. This 76-amino-acid-long polypeptide was isolated from calf sweetbread and was assumed to participate in the maturation of white blood cells. Since the molecule was subsequently found in numerous diff
15、erent tissues and organisms but not in bacteria it was given the name ubiquitin (from Latinubique,“everywhere”) (fig. 1).Fig 1. Ubiquitin a common polypeptide that represents the “kiss of death”. High resolution image (jpeg 184 kB)The discovery of ubiquitin-mediated protein degradationAfter taking h
16、is doctorate, Avram Hershko had studied energy-dependent protein degradation in liver cells, but decided in 1977 to transfer to the reticulocyte extract described above. This extract contained large quantities of haemoglobin, which upset the experiments. In their attempts to remove the haemoglobin u
17、sing chromatography, Aaron Ciechanover and Avram Hershko discovered that the extract could be divided into two fractions, each inactive on its own. But it turned out that as soon as the two fractions were recombined, the ATP-dependent protein degradation restarted. In 1978 the researchers reported t
18、hat the active component of one fraction was a heat-stable polypeptide with a molecular weight of only 9000 which they termed APF-1 (active principle in fraction 1). This protein later proved to be ubiquitin.The decisive breakthrough in the research was reported in two works that Ciechanover, Hershk
19、o and Rose published in 1980. Until that time the function of APF-1 was entirely unknown. In the first work it was shown that APF-1 was bound covalently, i.e. with a very stable chemical bond, to various proteins in the extract.In the second work it was further shown that many APF-1 molecules could
20、be bound to the same target protein; the latter phenomenon was termed polyubiquitination. We now know that this polyubiquitination of substrate proteins is the triggering signal that leads to degradation of the protein in the proteasome. It is this reaction that constitutes the actual labelling, the
21、 “kiss of death” if you will.At a stroke, these entirely unanticipated discoveries changed the conditions for future work: it now became possible to concentrate on identifying the enzyme system that binds ubiquitin to its target proteins. Since ubiquitin occurs so generally in various tissues and or
22、ganisms, it was quickly realised that ubiquitin-mediated protein degradation must be of general significance for the cell. In addition, the researchers guessed that the energy requirement in the form of ATP enabled the cell to control the specificity of the process.The field was now open and between
23、 1981 and 1983 Ciechanover, Hershko, Rose and their post docs and students developed “the multistep ubiquitin-tagging hypothesis” based on three newly-discovered enzyme activities they termed E1, E2 and E3 (fig. 2). We now know that a typical mammalian cell contains one or a few different E1 enzymes
24、, some tens of E2 enzymes and several hundred different E3 enzymes. It is the specificity of the E3 enzyme that determines which proteins in the cell are to be marked for destruction in the proteasomes.Fig 2. Ubiquitin-mediated protein degradation 1. The E1 enzyme activates the ubiquitin molecule. T
25、his reaction requires energy in the form of ATP. 2. The ubiquitin molecule is transferred to a different enzyme, E2. 3. The E3 enzyme can recognise the protein target which is to be destroyed. The E2-ubiquitin complex binds so near to the protein target that the actual ubiquitin label can be transfe
26、rred from E2 to the target. 4. The E3 enzyme now releases the ubiquitin-labelled protein. 5. This last step is repeated until the protein has a short chain of ubiquitin molecules attached to itself. 6. This ubiquitin chain is recognised in the opening of the proteasome. The ubiquitin label is discon
27、nected and the protein is admitted and chopped into small pieces. High resolution image (jpeg 383 kB)All the studies up to this point had been done in cell-free systems. To be able to study the physiological function of ubiquitin-mediated protein degradation as well, Avram Hershko and his co-workers
28、 developed an immunochemical method. By using antibodies to ubiquitin, ubiquitin-protein-conjugate could be isolated from cells where the cell proteins had been pulse-labelled with a radioactive amino acid not present in ubiquitin. The results showed that cells really break down faulty proteins usin
29、g the ubiquitin system, and we now know that up to 30% of the newly-synthesised proteins in a cell are broken down via the proteasomes since they do not pass the cells rigorous quality control.The proteasome the cells waste disposerWhat is a proteasome? A human cell contains about 30,000 proteasomes
30、: these barrel-formed structures can break down practically all proteins to 7-9-amino-acid-long peptides. The active surface of the proteasome is within the barrel where it is shielded from the rest of the cell. The only way in to the active surface is via the “lock”, which recognises polyubiquitina
31、ted proteins, denatures them with ATP energy and admits them to the barrel for disassembly once the ubiquitin label has been removed. The peptides formed are released from the other end of the proteasome. Thus the proteasome itself cannot choose proteins; it is chiefly the E3 enzyme that does this b
32、y ubiquitin-labelling the right protein for breakdown (fig. 3).Fig 3. The cells waste disposer, the proteasome. The black spots indicate active, protein-degrading surfaces. High resolution image (jpeg 187 kB)More recent researchWhile the biochemical mechanisms underlying ubiquitin-labelled protein d
33、egradation were laid bare around 1983 its physiological significance had not yet been fully understood. That it is of importance in destroying defective intracellular proteins was known but, to proceed, a mutated cell was needed in the ubiquitin system. By studying in detail how the mutated cell dif
34、fers from a normal cell under various growth conditions, it was hoped to gain a better idea of what reactions in the cell depend on the ubiquitin system.A mutated mouse cell had been isolated in 1980 by a research group in Tokyo. Their mouse-cell mutant contained a protein that, because of the mutat
35、ion, was sensitive to temperature. At lower temperatures the protein functioned as it should, but not at higher. Cells cultured at the higher temperature stopped growing. In addition, they showed defective DNA synthesis and other erroneous functions at the higher temperature. Researchers in Boston q
36、uickly showed that the heat-sensitive protein in the mutant mouse cell was the ubiquitin-activating enzyme E1. Obviously, ubiquitinactivationwas necessary for the cell to function and reproduce itself at all. Controlled protein breakdown was not only important for degrading incorrect proteins in the
37、 cell but it probably also took part in control of the cell cycle, DNA replication and chromosome structure.Since the late 1980s a number of physiologically important substrates for ubiquitin-mediated protein breakdown have been identified. Only a few of the most important will be mentioned here.Pre
38、vention of self-pollination in plantsMost plants are bisexual, hermaphroditic. Self-pollination leads to a gradual decline in genetic diversity which in the long run can cause the whole species to die out. To prevent this, plants use ubiquitin-mediated degradation to reject “own” pollen. The exact m
39、echanism has not yet been clarified but the E3 enzyme has been encountered and when proteasome inhibitors have been introduced, the rejection has been impaired.Regulation of the cell cycleWhen a cell is to make a copy of itself, many chemical reactions are involved. In a human being, six thousand mi
40、llion base pairs must be duplicated in DNA. These are gathered in 23 chromosome pairs that must be copied. Ordinary cell division, mitosis, and the formation of sex cells, meiosis, have many points of contact with the subjects of this years Nobel Prize. The E3 enzyme responsible, a protein complex t
41、ermed the “anaphase-promoting complex” (APC) checks that the cell goes out of mitosis. This enzyme complex has also proved to play an important role in the separation of the chromosomes during mitosis and meiosis. A different protein complex acts like a rope around the chromosome pair, holding it to
42、gether. At a given signal, the APC labels an inhibitor of a certain protein-degrading enzyme, whereupon the inhibitor is carried to the proteasome and destroyed. The enzyme is released, is activated and cuts the rope around the chromosome pair. Once the rope is gone, the chromosome pair can be separ
43、ated. Incorrect chromosome division during meiosis is the commonest cause of spontaneous miscarriage during pregnancy, and an extra chromosome 21 in humans leads to Downs syndrome. Most malignant tumours have cells with changed numbers of chromosomes as a result of incorrect chromosome division duri
44、ng mitosis.High resolution image (jpeg 230 kB)DNA repair, cancer and programmed cell deathProtein p53 has been dubbed “the guardian of the genome” and it is a tumour-suppressor gene. This means that as long as a cell can produce p53 the development of cancer is hampered. Sure enough, the protein is
45、mutated in at least 50% of all human cancer. The amount of protein p53 in a normal cell is low in consequence of continual production and breakdown. The breakdown is regulated through ubiquitination and the E3 enzyme responsible forms a complex with protein p53. Following DNA injury, protein p53 is
46、phosphorylated and can no longer bind to its E3 enzyme. The breakdown stops and the quantity of p53 in the cell rises rapidly. Protein p53 acts as a transcription factor, i.e. a protein that controls the expression of a certain gene. Protein p53 binds to and controls genes that regulate DNA repair a
47、nd programmed cell death. Raised levels of protein p53 lead first to interruption of the cell cycle to allow time for repair of DNA damage. If the damage is too extensive the cell triggers programmed cell death and “commits suicide”.Infection with human papilloma virus correlates strongly to the occ
48、urrence of cervical cancer. The virus avoids the protein p53 control function through one of its proteins activating and changing the recognition pattern of a certain cellular E3 enzyme, E6-AP, which is tricked into ubiquitinating the protein p53, which is totally destroyed. In consequence of this t
49、he infected cell can no longer repair DNA damage in a normal manner or trigger programmed cell death. The DNA mutations increase in number and this can ultimately lead to the development of cancer.Immune and inflammatory reactionsA certain transcription factor regulates many of the genes in the cell that are important for immune defence and inflammatory reactions. This protein, the transcription factor, occurs bound to an inhibitor protein in the cytoplasm of the ce
