Discovering how the body carries out quality control has earned three scientists the 2004 Nobel Prize in chemistry. Karen Harries-Rees looks at their work.

Discovering how the body carries out quality control has earned three scientists the 2004 Nobel Prize in chemistry. Karen Harries-Rees looks at their work.

Regulated protein degradation is key to hundreds of biochemical reactions and understanding the process has opened up opportunities to develop drugs to fight diseases such as cancer and cystic fibrosis.

It is for their work in discovering this process that Aaron Ciechanover, Avram Hershko and Irwin Rose have been jointly awarded the 2004 Nobel prize in chemistry. ’I am happy that I can speak at all. I am not myself for sure,’ said an elated Ciechanover, speaking to the Royal Swedish Academy of Sciences shortly after the announcement.

Ciechanover explained the work: ’We [humans] are not a building that stays still, we are all the time exchanging our proteins, synthesising and destroying them. Some proteins get spoilt. We discovered the process by which the body exercises quality control,’ he told Israeli newspaper Haaretz.

The three scientists went against the stream of research, which in the late 1970s and early 1980s was concentrating on explaining how the cell produces proteins. They discovered one of the cell’s most important cyclical processes, regulated protein degradation. Their work has made it possible to understand biochemical processes such as the cell cycle, DNA repair, gene transcription and quality control of newly-produced proteins.

Prior to their work, scientists knew about a number of simple protein-degrading enzymes, such as trypsin which breaks down proteins in food to amino acids in the small intestine. These processes need no energy. But experiments in the 1950s had showed that the breakdown of the cell’s own proteins does need energy. A first step towards explaining this was made by Alfred Goldberg and his co-workers in 1977 when they produced a cell-free extract from immature red blood cells, reticulocytes, which catalyse the breakdown of abnormal proteins in an energy-dependent manner.

Ciechanover, Hershko and Rose used such an extract in their work. They showed that protein degradation in cells takes place in a series of step-wise reactions that result in the proteins that are to be destroyed being labelled with the polypeptide ubiquitin, dubbed the kiss of death. This process enables the cell to be very specific about which proteins it breaks down. It is this regulation that needs energy.

The three scientists did much of this work during a series of sabbatical leaves that Hershko and Ciechanover spent with Rose at the Fox Chase Cancer Center in Philadelphia, US. The reticulocyte extract contained large quantities of haemoglobin. While trying to remove the haemoglobin using chromatography, the scientists found the extract could be divided into two fractions that, when combined, restarted the energy-dependent protein degradation. They identified the active component in one of the fractions, which later proved to be ubiquitin.

The breakthrough came in 1980 and was described in two papers published in the Proceedings of the National Academy of Sciences of the USA. Until this point the role of ubiquitin (or APF-1 as it was called at the time) was not known.

Ciechanover, Hershko and Rose showed that APF-1 was bound covalently to various proteins in the extract. They went on to demonstrate that many of APF-1 molecules could be bound to the same target protein - a process called polyubiquitination. We now know that this is the triggering signal that leads to the protein’s degradation in the proteasome - the cell’s waste disposal system.

Between 1981 and 1983 the three scientists and their post docs and students continued to work in this area and developed the multi-step ubiquitin-tagging hypothesis.

This was based on three newly-discovered enzymes called E1, E2 and E3. The process works by the first enzyme, E1, activating the ubiquitin molecule using ATP energy. The ubiquitin molecule is then transferred to the second enzyme, E2. The third enzyme, E3, can recognise the protein that is to be destroyed and the E2-ubiquitin protein binds so near to the protein target that the label can be transferred from E2 to the target protein. This is repeated several times until the protein has a short chain of ubiquitin molecules. The proteasome recognises this chain and lets it in, where it is destroyed.

All the research so far had been carried out in cell-free systems. Next, work progressed to study what happened in a cell. Work by Hershko and his co-workers showed that cells break down faulty proteins using this system. Later research has shown that up to 30 per cent of newly-synthesised proteins fall foul of the cell’s quality control process and are broken down in this way.

By around 1983, scientists knew the biochemical mechanisms of ubiquitin-labelled protein destruction but its physiological significance was not yet fully understood.

To get a better idea of which reactions in a cell depend on the ubiquitin system, a mutated mouse cell, with a protein that was sensitive to temperature, was used. At lower temperatures the cell functioned as it should. But cells cultured at higher temperatures stopped growing and showed defective DNA synthesis. The heat-sensitive protein was the enzyme E1 that is responsible for activating the ubiquitin, showing that ubiquitin activation is necessary for the cell to function and that controlled protein breakdown probably takes part in controlling the cell cycle, DNA replication and chromosome structure.

Since the 1980s, ubiquitin-mediated protein degradation’s role has been identified in a number of physiological processes. One such is in DNA repair and cancer. The levels of the tumour-suppressor protein p53 are kept low in a normal cell by continual synthesis and degradation. The breakdown is regulated through ubiquitination and the E3 enzyme forming a complex with the protein p53. Following DNA injury, p53 can no longer bind to the E3 enzyme. So, the breakdown stops and the level of p53 rises, interrupting the cell cycle to allow time for DNA damage to be repaired. If the damage is too extensive the cell triggers programmed cell death (apoptosis). The p53 protein is mutated in 50 per cent of all human cancers.

Infection with human papilloma virus correlates strongly to the occurrence of cervical cancer. The virus avoids the protein p53 control system by one of its proteins activating and changing the recognition pattern of a particular E3 enzyme, which is tricked into ubiquitinating p53, which is then destroyed. The infected cell can not repair DNA damage or trigger cell death in the normal way, allowing DNA mutations to increase in number, eventually leading to the development of cancer.

Regulated protein destruction also has a role in cystic fibrosis, which is caused by a non-functioning plasma membrane chloride channel CFTR (cystic fibrosis transmembrane conductance regulator). Most people with cystic fibrosis have the same genetic damage - the loss of the amino acid phenylalanine in the CFTR protein. This mutation causes faulty folding of the protein. The incorrectly folded protein is destroyed through ubiquitin-mediated protein breakdown instead of being transported out to the cell wall. Without a functioning chloride channel the cell cannot transport chloride ions through its membrane, resulting in, for instance, the build up of secretions in the lungs.

The ubiquitin system is the subject of pharmaceutical research. Drugs could be developed to prevent degradation of specific proteins or to trigger the system to destroy unwanted proteins.

One drug currently in clinical trials is the proteasome inhibitor Velcade, developed by Millennium Chemicals, which has been approved by the US Food and Drug Administration for the treatment of the blood cancer multiple myeloma. Many more are likely to follow.

Further Reading

A Ciechanover et al, Proc. Natl. Acad. Sci. USA, 1980, 77, 1365

A Hershko et al, Proc. Natl. Acad. Sci. USA, 1980, 77, 1783

A Ciechanover et al, Proc. Natl. Acad. Sci. USA, 1981, 78, 761

A Hershko et al, J. Biol. Chem, 1983, 258, 8206

The laureates

Aaron Ciechanover

Professor at the Unit of Biochemistry and director of the Rappaport Family Institute for Research in Medical Sciences, Technion (Israel Institute of Technology), Haifa, Israel

Born:1947 Haifa, Israel

Doctor?s degree in medicine in 1981 at the Technion

Avram Hershko

Distinguished professor at the Rappaport Family Institute for Research in Medical Sciences, Technion, Haifa, Israel

Born: 1937 Karcag, Hungary

Doctor’s degree in medicine in 1969 at the Hadassah Medical School of the Hebrew University, Jerusalem

Irwin Rose

Specialist at the Department of Physiology and Biophysics, College of Medicine, University of California, Irvine, US

Born: 1926 New York, US

Doctor’s degree in 1952 at the University of Chicago, US