Telomerase
Telomerase, also called terminal transferase, is a ribonucleoprotein that adds a species-dependent telomere repeat sequence to the 3' end of telomeres. A telomere is a region of repetitive sequences at each end of the chromosomes of most eukaryotes. Telomeres protect the end of the chromosome from DNA damage or from fusion with neighbouring chromosomes. The fruit fly Drosophila melanogaster lacks telomerase, but instead uses retrotransposons to maintain telomeres.
Telomerase is a reverse transcriptase enzyme that carries its own RNA molecule which is used as a template when it elongates telomeres. Telomerase is active in gametes and most cancer cells, but is normally absent in most somatic cells.
History
The existence of a compensatory mechanism for telomere shortening was first found by Soviet biologist Alexey Olovnikov in 1973, who also suggested the telomere hypothesis of aging and the telomere's connections to cancer and perhaps some neurodegenerative diseases.Telomerase in the ciliate Tetrahymena was discovered by Carol W. Greider and Elizabeth Blackburn in 1984. Together with Jack W. Szostak, Greider and Blackburn were awarded the 2009 Nobel Prize in Physiology or Medicine for their discovery. Later the cryo-EM structure of telomerase was first reported in T. thermophila, to be followed a few years later by the cryo-EM structure of telomerase in humans.
The role of telomeres and telomerase in cell aging and cancer was established by scientists at biotechnology company Geron with the cloning of the RNA and catalytic components of human telomerase and the development of a polymerase chain reaction based assay for telomerase activity called the TRAP assay, which surveys telomerase activity in multiple types of cancer.
The negative stain electron microscopy structures of human and Tetrahymena telomerases were characterized in 2013. Two years later, the first cryo-electron microscopy structure of telomerase holoenzyme was determined. In 2018, the structure of human telomerase was determined through cryo-EM by UC Berkeley scientists.
Structure
The catalytic protein
The main catalytic protein in telomerase is telomerase reverse transcriptase, 1132 amino acids long in humans. The protein consists of four conserved domains, organized into a "right hand" ring configuration that shares common features with retroviral reverse transcriptases, viral RNA replicases and bacteriophage B-family DNA polymerases.TERT proteins from many eukaryotes have been sequenced.
The telomerase complex
The full human telomerase complex consists of: one copy each of telomerase RNA, TERT, and TCAB1, plus two copies of the H/ACA ribonucleoprotein subcomplex. The H/ACA subcomplex consists of two copies each of dyskerin, NHP2, NOP10 and GAR1. Each copy of the H/ACA subcomplex binds to a hairpin structure on the telomerase RNA, a feature specific to vertebrates. There is also a histone H2A-H2B dimer wrapping around the telomeric DNA as it operates and its presence is essential for the functioning of the telomerase complex. The presence of TPP1 and POT1 is not detected in the cyro-EM study, but based on previous studies they should be attached to TERT's TEN domain in a way analogous to p50 and Teb1 of Tetrahymena. Previous experiments on catalytically active complex extracted from immortal cells indicated two molecules each of human TERT, telomerase RNA, and dyskerin.For comparison, the Tetrahymena telomerase complex consists of: one copy each of TR, TERT, p65 in the core; one copy of p50 as the connection to the rest of the structure; one copy each of Teb1, Teb2, and Teb3 ; and one copy each of p75, p45, p19.
Mechanism
The shelterin protein TPP1 is both necessary and sufficient to recruit the telomerase enzyme to telomeres, and is the only shelterin protein in direct contact with telomerase.By using TERC, TERT can add a six-nucleotide repeating sequence, 5'-TTAGGG to the 3' strand of chromosomes. These TTAGGG repeats are called telomeres. The template region of TERC is 3'-CAAUCCCAAUC-5'.
Telomerase can bind the first few nucleotides of the template to the last telomere sequence on the chromosome, add a new telomere repeat sequence, let go, realign the new 3'-end of telomere to the template, and repeat the process. Telomerase reverses telomere shortening.
Clinical implications
Aging
Telomerase restores short bits of DNA known as telomeres, which are otherwise shortened after repeated division of a cell via mitosis.In normal circumstances, where telomerase is absent, if a cell divides recursively, at some point the progeny reach their Hayflick limit, which is believed to be between 50 and 70 cell divisions. At the limit the cells become senescent and cell division stops. Telomerase allows each offspring to replace the lost bit of DNA, allowing the cell line to divide without ever reaching the limit. This same unbounded growth is a feature of cancerous growth.
Embryonic stem cells express telomerase, which allows them to divide repeatedly and form the individual. In adults, telomerase is highly expressed only in cells that need to divide regularly, especially in male sperm cells, but also in epidermal cells, in activated T cell and B cell lymphocytes, as well as in certain adult stem cells, but in the great majority of cases somatic cells do not express telomerase.
A comparative biology study of mammalian telomeres indicated that telomere length of some mammalian species correlates inversely, rather than directly, with lifespan, and concluded that the contribution of telomere length to lifespan is unresolved. Telomere shortening does not occur with age in some postmitotic tissues, such as in the rat brain. In humans, skeletal muscle telomere lengths remain stable from ages 23 –74. In baboon skeletal muscle, which consists of fully differentiated postmitotic cells, less than 3% of myonuclei contain damaged telomeres and this percentage does not increase with age. Thus, telomere shortening does not appear to be a major factor in the aging of the differentiated cells of brain or skeletal muscle. In human liver, cholangiocytes and hepatocytes show no age-related telomere shortening. Another study found little evidence that, in humans, telomere length is a significant biomarker of normal aging with respect to important cognitive and physical abilities.
Some experiments have raised questions on whether telomerase can be used as an anti-aging therapy, namely, the fact that mice with elevated levels of telomerase have higher cancer incidence and hence do not live longer. On the other hand, one study showed that activating telomerase in cancer-resistant mice by overexpressing its catalytic subunit extended lifespan. A study found that long-lived subjects inherited a hyperactive version of telomerase.
Premature aging
Premature aging syndromes including Werner syndrome, Progeria, Ataxia telangiectasia, Ataxia-telangiectasia like disorder, Bloom syndrome, Fanconi anemia and Nijmegen breakage syndrome are associated with short telomeres. However, the genes that have mutated in these diseases all have roles in the repair of DNA damage and the increased DNA damage may, itself, be a factor in the premature aging. An additional role in maintaining telomere length is an active area of investigation.Cancer
In vitro, when cells approach the Hayflick limit, the time to senescence can be extended by inactivating the tumor suppressor proteins p53 and Retinoblastoma protein. Cells that have been so-altered eventually undergo an event termed a "crisis" when the majority of the cells in the culture die. Sometimes, a cell does not stop dividing once it reaches a crisis. In a typical situation, the telomeres are shortened and chromosomal integrity declines with every subsequent cell division. Exposed chromosome ends are interpreted as double-stranded breaks in DNA; such damage is usually repaired by reattaching the broken ends together. When the cell does this due to telomere-shortening, the ends of different chromosomes can be attached to each other. This solves the problem of lacking telomeres, but during cell division anaphase, the fused chromosomes are randomly ripped apart, causing many mutations and chromosomal abnormalities. As this process continues, the cell's genome becomes unstable. Eventually, either fatal damage is done to the cell's chromosomes, or an additional mutation that activates telomerase occurs.With telomerase activation some types of cells and their offspring become immortal, thus avoiding cell death as long as the conditions for their duplication are met. Many cancer cells are considered 'immortal' because telomerase activity allows them to live much longer than any other somatic cell, which, combined with uncontrollable cell proliferation is why they can form tumors. A good example of immortal cancer cells is HeLa cells, which have been used in laboratories as a model cell line since 1951.
While this method of modelling human cancer in cell culture is effective and has been used for many years by scientists, it is also very imprecise. The exact changes that allow for the formation of the tumorigenic clones in the above-described experiment are not clear. Scientists addressed this question by the serial introduction of multiple mutations present in a variety of human cancers. This has led to the identification of mutation combinations that form tumorigenic cells in a variety of cell types. While the combination varies by cell type, the following alterations are required in all cases: TERT activation, loss of p53 pathway function, loss of pRb pathway function, activation of the Ras or myc proto-oncogenes, and aberration of the Protein phosphatase 2. That is to say, the cell has an activated telomerase, eliminating the process of death by chromosome instability or loss, absence of apoptosis-induction pathways, and continued mitosis activation.
This model of cancer in cell culture accurately describes the role of telomerase in actual human tumors. Telomerase activation has been observed in ~90% of all human tumors, suggesting that the immortality conferred by telomerase plays a key role in cancer development. Of the tumors without TERT activation, most employ a separate pathway to maintain telomere length termed Alternative Lengthening of Telomeres. The presence of this alternative pathway was first described in an SV40 virus-transformed human cell line, and based on the dynamics of the changes in telomere length, was proposed to result through recombination. However, the exact mechanism remains unclear.
Elizabeth Blackburn et al., identified the upregulation of 70 genes known or suspected in cancer growth and spread through the body, and the activation of glycolysis, which enables cancer cells to rapidly use sugar to facilitate their programmed growth rate.
Approaches to controlling telomerase and telomeres for cancer therapy include gene therapy, immunotherapy, small-molecule and signal pathway inhibitors.