The ubiquitous `kiss of death'

Print edition : November 05, 2004

PROTEINS are the building blocks of living things. Known to exist in some hundred thousand different forms, they perform important functions. As enzymes they accelerate chemical reactions and as hormones they carry signals. Proteins form an important part of the immune response system and they ensure cells' form and structure. Much of the research has focussed on understanding the synthesis of the various proteins - as many as five Nobel Prizes have been awarded in this field. Only few researchers were engaged in the reverse phenomenon, namely the degradation of proteins, an equally important process. Now three scientists who did work in this area have been honoured with this year's Nobel Prize in chemistry.

Avram Hershko and Aaron Ciechanover.-SHREM YOSI/REUTERS

When the degradation does not work correctly, certain diseases are caused. Cervical cancer and cystic fibrosis are two examples. Knowledge of ubiquitin-mediated protein breakdown, which the Nobel Prize winning work has enabled, offers an opportunity to develop drugs against these and other diseases.

Aaron Ciechanover and Avram Hershko, both of Technion - Israel Institute of Technology, Haifa, Israel, and Irwin Rose of the University of California, the United States, contributed ground-breaking chemical knowledge during the 1980s of how the cell can regulate the presence of a certain protein with extreme specificity in time, target and space. They showed that unwanted proteins were marked with a label consisting of many molecules of the polypeptide ubiquitin, a protein with 76 amino acids, and were destroyed.

That is, the cell functions also as a highly efficient quality control station where synthesised proteins are destroyed, if they fail the quality check, at a furious rate. The degradation is not indiscriminate, but takes place through a process that is controlled in detail so that the proteins to be broken down at any given moment are specifically identified by giving a `kiss of death', as it were, via the ubiquitin label.

Ubiquitin was first isolated in 1975 from bovine thymus and was believed to participate in the maturation of white blood cells. Since it was subsequently found in numerous other tissues - but not in bacteria - it was named ubiquitin (to imply "found everywhere"). Later, its role in protein degradation - known as proteolysis in technical parlance - became apparent.

Irwin Rose. A file photo.-AP

The protein with the ubiquitin tag is transported to the proteasome, a large enzyme that enables the process of proteolysis, where it is recognised as the key in a lock, which is a signal that a protein is on the way for disassembling. Shortly before the protein is squeezed into the proteasome, the ubiquitin molecule is disconnected for re-use. In the proteasomes, which act as cellular "waste disposers", the break down or degradation of the proteins occurs rapidly. The Nobel Prize for the trio is "for the discovery of ubiquitin-mediated proteolysis".

A number of protein-degrading enzymes were already known and studied. For instance, trypsin breaks down the food proteins to amino acids in the intestines. Similarly, in lysosome, a type of cell organelle, proteins absorbed from outside are broken down. However, these processes do not require energy. In contrast, investigations in the 1950s indicated that degradation of most intracellular protein required metabolic energy. The attention given to the apparently non-regulated break down of certain other proteins in the lysosomes held back progress in the field of energy-dependent proteolysis. What finally led to the discovery of the central pathway of ubiquitin-mediated proteolysis was Hershko's great devotion to understanding the paradox of why intracellular proteolysis required energy.

In 1977, J.D. Etlinger and A.L. Goldberg provided a methodological advance for such studies by demonstrating that degradation of abnormal proteins in a cell-free extract from immature red blood cells (reticulocytes) occurred in an ATP-dependent manner. ATP, or adenosine triphosphate, is the main energy-carrying chemical intermediate in all organisms.

Using such an extract, the three laureates carried out a series of path-breaking biochemical experiments by which they showed that protein degradation occurred in a series of step-wise reactions that resulted in ubiquitin being bonded to proteins to be destroyed. This process enables the cell to break down unwanted proteins with high specificity, and it is this regulation that requires energy.

The discovery of mediation by ubiquitin was, in a sense, serendipitous. The extract that the scientists used contained large quantities of haemoglobin, which "contaminated" the experiments. In their attempts to remove it using chromatography, they discovered that the extract could be divided into two fractions, each by itself inactive. But as soon as the two fractions were recombined, the ATP-dependent protein degradation restarted. They found that the active component of one fraction was a heat-stable polypeptide with a molecular weight of only 9,000, which they called APF-1 (active principle in fraction-1). Later the protein was proved to be ubiquitin.

The actual breakthrough came in 1980, which the trio reported in two publications. Until then, the function of APF-1 was unknown. In the first work they showed that APF-1 was bound covalently - a very stable chemical bond - to different proteins in the extract. In the second, they showed that several APF-1 molecules could be bound to the same target protein. It is this `polyubiquitination' that constitutes the actual labelling or `kiss of death' and forms the triggering signal that leads to degradation in the proteasome.

Unlike reversible protein modifications such as phosphorylation (combination of an organic molecule with phosphate group), polyubiquitination is often irreversible since the target protein is eventually destroyed. Much of the work was done during a series of sabbatical leaves that Ciechanover and Hershko spent with Rose at the Fox Chase Centre in Philadelphia.

ONCE the role of ubiquitin became clear, it paved the way for delineating the sequence of biochemical reactions that achieved it. Between 1981 and 1983, Ciechanover, Hershko and Rose and their students developed the "multi-stage ubiquitin tagging" hypothesis involving three newly discovered enzyme activities they termed E1, E2 and E3. A typical mammalian cell contains one or a few different E1 enzymes, some tens of E2 enzymes and several hundred different E3 enzymes. They showed that, while E1 and E2 assist in the tagging process, it is the specificity of E3 that determines the proteins to be destroyed.

What then is the nature of the molecule proteasome that acts as the "waste disposer"? A human cell contains about 30,000 proteasomes. These are long barrel-shaped structures that can break down practically all poplypeptides to 7-9 amino acids long peptides. The active surface of this protein shredder is inside the barrel, which is shielded from the rest of the cell. The only way into the active surface is through the "lock" mechanism, which recognises the ubiquitin-tagged proteins, denatures them using ATP-derived energy and admits them into the barrel once the ubiquitin molecule is detached. The proteasome itself cannot choose proteins. The E3 enzyme labels the correct target protein with ubiquitin.

All these studies on ubiquitin-mediated degradation were done in cell-free systems. What about its actual physiological function and significance? Hershko and his co-workers used certain immunochemical techniques to demonstrate in detail how the ubiquitin mediation is used to break down faulty proteins. It is now known that up to 30 per cent of newly synthesised proteins in a cell are broken down by the proteasomes, since they do not pass the cell's rigorous quality control.

FURTHER studies by other researchers using mutated cells provided a better insight into the physiological function of the ubiquitin system. A group in Tokyo had isolated a mutated mouse cell in 1980. The mouse-cell mutant contained a protein that, as a result of mutation, became sensitive to temperature. The protein functioned normally at lower, and not at higher, temperatures. Cells cultured at higher temperatures stopped growing. In addition they showed additional problems such as defective DNA synthesis and other malfunctions.

A research group in Boston showed that the heat-sensitive protein was the ubiquitin-activating enzyme E1. It demonstrated that ubiquitin-activation was necessary for normal cell function as well. That is, regulation by ubiquitin was not only necessary for degrading faulty proteins in the cells, but probably essential in the control of cell cycle, DNA replication and chromosome structure. Since the late 1980s, a number of physiologically important substrates for ubiquitin-mediated protein degradation have been identified. They include cell division, DNA repair and important components of the immune defence mechanism.

In recent times, ubiquitin has been found to play an important role in certain extracellular processes in some animal systems as well. These include fertilisation, antibacterial and antifungal activity of adrenal chromaffin cells, programmed death of cells or apoptosis, homing of haematopoietic stem cells into their microenvironment in the bone marrow and spleen and neurological development in the embryo.

The Indian contribution

BESIDES its role in the ATP-mediated degradation of unwanted proteins in the cell, the ubiquitin-proteasome system has also been implicated in a few extra-cellular processes. These include fertilisation in seasquirts, antibacterial activity of chromaffin cells (cells which stimulate production of adrenaline), apoptosis (programmed cell death), homing of haematopoietic stem cells into bone marrow and neural tube development in chick embryo. An Indian research group led by K. Kannan, head of the Biotechnology Department at the Indraprastha University in Delhi, has been involved with the discovery of the last two of these.

Haemotopoietic stem cells are basically the progenitors of all white and red blood cells. They are found in bone marrow. Any defective stem cell can lead to several blood disorders, including leukemia . In 1993, Kannan and his group discovered that ubiquitin molecule acts as a binding agent between the stem cells and the bone marrow. This enables homing of healthy stem cells into bone marrow to cure blood disorders without recourse to radiation and such treatment generally adopted to treat these diseases.

More recently, Kannan's group has shown that there are gross morphological changes in the neural development of the chick embryo after extra-cellular application of uibiquitin. This, he believes, is perhaps owing to some kind of interaction between the extra-cellular ubiquitin and the large population of neural stem cells in the neural tube, as in the case of blood stem cells resulting in the neural development being hampered or delayed. The exact process is not yet clear but, according to Kannan, this has certainly shown that ubiquitin could be a useful probe to study the kinetics of brain development similar to metabolic or enzyme inhibitors in a biochemical pathway.

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