The proteins at the heart of telomerase

Sydney researchers have made a massive breakthrough in cancer research by identifying the protein composition of telomerase, an enzyme associated with regulating cell senescence but also with over 85 per cent of cancers. Two components were already known, and the third turns out to be dyskerin, an RNA binding protein.

The role of telomerase is to replicate and stabilise telomeres, short DNA sequences that cap the ends of chromosomes to protect them from damage. Telomeres function as a molecular clock, shortening with each division until a signal is sent to the cell to stop dividing.

Telomerase is normally dormant in non-dividing adult cells but is active in renewing cells such as blood cells and cells of the gut, skin and hair follicles. It also plays an active role in uncontrolled cell proliferation and is found in over 85 per cent of human cancers. Telomerase lengthens the telomeres, allowing cancer cells to divide without limit.

While telomerase was discovered almost 20 years ago, its actual composition was undetermined and the enzyme is extraordinarily rare. It was known to have two core components - telomerase RNA (hTR or hTER), which acts as the template for synthesis of the telomere DNA, and telomerase reverse transcriptase (hTERT), which is the catalytic protein component.

Now, a team from Sydney's Children's Medical Research Institute has done two things: it has identified all of the components of telomerase and it has developed a method to purify and amplify the enzyme. The team, led by Dr Scott Cohen, published its results in Science on March 30.

"Telomerase was known to contain two core components - an RNA and a core protein," Cohen said. "It was also known to contain some mixture of other proteins but the identity of these other proteins has remained a mystery for almost 20 years.

"A total of 32 different proteins had been proposed as being part of telomerase. We've found that telomerase isn't 32 proteins - telomerase is just two."

In addition to the telomerase RNA and the hTERT protein, Cohen's team has identified just one other protein, dyskerin, an RNA binding protein.

"Our discovery is rather spectacular," Cohen said. "The primary challenge of human telomerase is that although it is present in a wide range of cancers, within each cancer cell the amount of telomerase is vanishingly small. Telomerase is likely the rarest enzyme in the human cell, so to get enough telomerase to purify it and study it we require a large amount of cancer cells.

"So we elicited the collaboration of Dr George Lovrecz from the CSIRO, who operates a bioreactor and can generate industrial quantities of cancer cells. The quantities we used are thousands of times greater than what cancer researchers normally use."

Cohen has also been able to obtain extremely pure telomerase through an intriguing method he has developed. CMRI researcher Dr Lorel Colgin had previously arranged for a sheep to be vaccinated against human telomerase. The antibodies produced by the sheep proved very useful for partly purifying the enzyme.

Cohen next exploited the ability of telomerase to bind to the end of chromosomes to purify telomerase further. He had noticed a study buried in the scientific literature that reported an unusual property of telomerase. Its affinity for a chromosome depends on the precise nucleotide at the very end. He therefore supplied the partly purified telomerase with exactly the right letters, so it would alter the chromosome end that it was bound to, change its affinity for the chromosome from very strong to very weak, and fall off. This allowed him to collect highly purified telomerase.

The head of CMRI's Cancer Research Unit and senior author of the Science paper, Dr Roger Reddel, said the purification process Cohen has invented "is one of the cleverest I've seen. In about 12 hours he can purify telomerase more than 100-million fold."

Identifying the molecules in the purified enzyme was still a challenge, because the amount of telomerase he extracted from 100 grams of cancer cells was only 100 nanograms. The identification was achieved by Drs Mark Graham, Nicolai Bache and Phil Robinson in CMRI's mass spectrometry unit, which is funded by the popular Jeans for Genes campaign.

The answer was surprisingly simple. Apart from the two components telomerase is already known to contain, there is only one more protein present in the active enzyme.

Cohen used endogenous telomerase from human cells for his research, but in identifying telomerase's composition he has opened the way for research based on synthetic telomerase, allowing further study through x-ray crystallography to determine the precise 3D structure of the enzyme, the starting point for the rational design of drugs to block telomerase action.

See the May/June issue of Australian Life Scientist</> for the full story.