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Telomerase, also calledterminal transferase,[1] is aribonucleoprotein that adds a species-dependenttelomere repeat sequence to the3' end oftelomeres. A telomere is a region of repetitivesequences at each end of thechromosomes of mosteukaryotes. Telomeres protect the end of the chromosome fromDNA damage or from fusion with neighbouring chromosomes. The fruit flyDrosophila melanogaster lacks telomerase, but instead usesretrotransposons to maintain telomeres.[2]
Telomerase is areverse transcriptaseenzyme that carries its ownRNA molecule (e.g., with the sequence 3′-CCCAAUCCC-5′ inTrypanosoma brucei)[3] which is used as a template when it elongates telomeres. Telomerase is active ingametes and mostcancer cells, but is normally absent in mostsomatic cells.
The existence of a compensatory mechanism for telomere shortening was first found by Soviet biologistAlexey Olovnikov in 1973,[4] who also suggested the telomere hypothesis ofaging and the telomere's connections to cancer and perhaps some neurodegenerative diseases.[5]
Telomerase in theciliateTetrahymena was discovered byCarol W. Greider andElizabeth Blackburn in 1984.[6] Together withJack W. Szostak, Greider and Blackburn were awarded the 2009Nobel Prize inPhysiology or Medicine for their discovery.[7] Later thecryo-EM structure of telomerase was first reported inT. thermophila, to be followed a few years later by the cryo-EM structure of telomerase in humans.[8]
The role of telomeres and telomerase incell aging andcancer was established by scientists atbiotechnology companyGeron with the cloning of theRNA and catalytic components of human telomerase[9] and the development of apolymerase chain reaction (PCR) based assay for telomerase activity called the TRAP assay, which surveys telomerase activity in multiple types of cancer.[10]
Thenegative stain electron microscopy (EM) structures of human andTetrahymena telomerases were characterized in 2013.[11][12] Two years later, the first cryo-electron microscopy (cryo-EM) structure of telomerase holoenzyme (Tetrahymena) was determined.[13] In 2018, the structure of human telomerase was determined through cryo-EM by UC Berkeley scientists.[14]
The main catalytic protein in telomerase istelomerase reverse transcriptase, 1132amino acids long in humans.[15] The protein consists of fourconserved domains (RNA-Binding Domain (TRBD), fingers, palm and thumb), organized into a "right hand" ring configuration that shares common features withretroviral reverse transcriptases, viralRNA replicases andbacteriophage B-family DNA polymerases.[16][17]
TERT proteins from many eukaryotes have been sequenced.[18]
The full human telomerase complex (the holoenzyme) consists of: one copy each oftelomerase RNA (TR or TERC, 452 nucleotides), TERT, andTCAB1, plus two copies of the H/ACA ribonucleoprotein subcomplex. The H/ACA subcomplex consists of two copies each ofdyskerin (DKC1),NHP2,NOP10 andGAR1. Each copy of the H/ACA subcomplex binds to a hairpin structure on the telomerase RNA, a feature specific tovertebrates. 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.[14] The presence of TPP1 andPOT1 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 ofTetrahymena.[14] Previous experiments on catalytically active complex extracted from immortal cells indicated twomolecules each of human TERT, telomerase RNA, anddyskerin (DKC1).[19][20]
For comparison, theTetrahymena telomerase complex consists of: one copy each of TR (TER), TERT, p65 in the core; one copy of p50 (homolog of humanTPP1) as the connection to the rest of the structure; one copy each of Teb1 (paralogous to humanRPA70), Teb2, and Teb3 (altogether aRPA-like complex); and one copy each of p75, p45, p19 (aCST complex).[13]
Theshelterin proteinTPP1 is both necessary and sufficient to recruit the telomerase enzyme to telomeres, and is the only shelterin protein in direct contact with telomerase.[21]
By using TERC, TERT can add a six-nucleotide repeating sequence, 5'-TTAGGG (in vertebrates; the sequence differs in other organisms) to the 3' strand of chromosomes. These TTAGGG repeats (with their various protein binding partners) are called telomeres. The template region of TERC is 3'-CAAUCCCAAUC-5'.[22]
Telomerase can bind the first few nucleotides of the template to the last telomere sequence on the chromosome, add a new telomere repeat (5'-GGTTAG-3') sequence, let go, realign the new 3'-end of telomere to the template, and repeat the process. Telomerase reversestelomere shortening.
Telomerase restores short bits of DNA known astelomeres, which are otherwise shortened after repeated division of a cell viamitosis.
In normal circumstances, where telomerase is absent, if a cell divides recursively, at some point the progeny reach theirHayflick limit,[23] which is believed to be between 50 and 70 cell divisions. At the limit the cells become senescent andcell division stops.[24] 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 ofcancerous growth.[25]
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 malesperm cells,[26] but also inepidermal cells,[27] in activatedT cell[28] andB cell[29]lymphocytes, as well as in certainadult stem cells, but in the great majority of casessomatic cells do not express telomerase.[30]
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.[31] Telomere shortening does not occur with age in somepostmitotic tissues, such as in the rat brain.[32] In humans, skeletal muscle telomere lengths remain stable from ages 23 –74.[33] In baboon skeletal muscle, which consists of fullydifferentiated postmitotic cells, less than 3% ofmyonuclei contain damaged telomeres and this percentage does not increase with age.[34] 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 andhepatocytes show no age-related telomere shortening.[35] Another study found little evidence that, in humans, telomere length is a significantbiomarker of normal aging with respect to important cognitive and physical abilities.[36]
Some experiments have raised questions on whether telomerase can be used as ananti-aging therapy, namely, the fact that mice with elevated levels of telomerase have higher cancer incidence and hence do not live longer.[37] On the other hand, one study showed that activating telomerase in cancer-resistant mice by overexpressing its catalytic subunit extended lifespan.[38] A study found that long-lived subjects inherited a hyperactive version of telomerase.[39]
Premature aging syndromes includingWerner syndrome,Progeria,Ataxia telangiectasia, Ataxia-telangiectasia like disorder,Bloom syndrome,Fanconi anemia andNijmegen breakage syndrome are associated with short telomeres.[40] However, the genes that have mutated in these diseases all have roles in therepair of DNA damage and the increased DNA damage may, itself, be a factor in the premature aging (seeDNA damage theory of aging). An additional role in maintaining telomere length is an active area of investigation.
In vitro, when cells approach theHayflick limit, the time to senescence can be extended by inactivating thetumor suppressor proteinsp53 andRetinoblastoma protein (pRb).[41]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[42] and chromosomal integrity declines with every subsequent cell division. Exposed chromosome ends are interpreted as double-stranded breaks (DSB) in DNA; such damage is usuallyrepaired 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 divisionanaphase, the fused chromosomes are randomly ripped apart, causing manymutations and chromosomal abnormalities. As this process continues, the cell's genome becomes unstable. Eventually, either fatal damage is done to the cell's chromosomes (killing it viaapoptosis), or an additional mutation that activates telomerase occurs.[41]
With telomerase activation some types of cells and their offspring becomeimmortal (bypass theHayflick limit), thus avoiding cell death as long as the conditions for their duplication are met. Manycancer cells are considered 'immortal' because telomerase activity allows them to live much longer than any other somatic cell, which, combined with uncontrollable cell proliferation[43] is why they can formtumors. A good example of immortal cancer cells isHeLa cells, which have been used in laboratories as a modelcell 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 thetumorigenicclones 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 theRas ormycproto-oncogenes, and aberration of theProtein phosphatase 2 (PP2A).[44] 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 continuedmitosis activation.
This model ofcancer in cell culture accurately describes the role of telomerase in actual human tumors. Telomerase activation has been observed in ~90% of all human tumors,[45] suggesting that the immortality conferred by telomerase plays a key role in cancer development. Of the tumors without TERT activation,[46] most employ a separate pathway to maintain telomere length termedAlternative Lengthening of Telomeres (ALT).[47] 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 throughrecombination.[48] However, the exact mechanism remains unclear.
Elizabeth Blackburnet al., identified the upregulation of 70 genes known or suspected in cancer growth and spread through the body, and the activation ofglycolysis, which enables cancer cells to rapidly use sugar to facilitate their programmed growth rate (roughly the growth rate of a fetus).[49]
Approaches to controlling telomerase and telomeres for cancer therapy includegene therapy,immunotherapy, small-molecule and signal pathway inhibitors.[50]
The ability to maintain functionaltelomeres may be one mechanism that allowscancer cells to growin vitro for decades.[51] Telomerase activity is necessary to preserve many cancer types and is inactive insomatic cells, creating the possibility that telomerase inhibition could selectively repress cancer cell growth with minimal side effects.[52] If a drug can inhibit telomerase in cancer cells, the telomeres of successive generations will progressively shorten, limiting tumor growth.[53]
Telomerase is a goodbiomarker for cancer detection because most human cancer cells express high levels of it. Telomerase activity can be identified by its catalytic protein domain (hTERT).This[clarify] is therate-limiting step in telomerase activity. It is associated with many cancer types. Various cancer cells andfibroblasts transformed with hTERTcDNA have high telomerase activity, while somatic cells do not. Cells testing positive for hTERT have positive nuclear signals. Epithelial stem cell tissue and its early daughter cells are the only noncancerous cells in which hTERT can be detected. Since hTERT expression is dependent only on the number of tumor cells within a sample, the amount of hTERT indicates the severity of cancer.[54]
The expression of hTERT can also be used to distinguishbenign tumors frommalignant tumors. Malignant tumors have higher hTERT expression than benign tumors. Real-timereverse transcription polymerase chain reaction (RT-PCR) quantifying hTERT expression in various tumor samples verified this varying expression.[55]
The lack of telomerase does not affect cell growth until the telomeres are short enough to cause cells to "die or undergo growth arrest". However, inhibiting telomerase alone is not enough to destroy large tumors. It must be combined with surgery,radiation,chemotherapy or immunotherapy.[54]
Cells may reduce their telomere length by only 50-252 base pairs per cell division, which can lead to a longlag phase.[56][57]
A telomerase activatorTA-65 is commercially available and is claimed to delay aging and to provide relief from certain disease conditions.[58][59][60][61][62] This formulation contains a molecule calledcycloastragenol derived from a legume Astragalus membranaceus. Several other compounds have been found to increase telomerase activity:Centella asiatica extract 8.8-fold,oleanolic acid 5.9-fold,astragalus extract 4.3-fold, TA-65 2.2-fold, andmaslinic acid 2-fold.[63]
Immunotherapy successfully treats some kinds of cancer, such asmelanoma. This treatment involves manipulating a human'simmune system to destroy cancerous cells. Humans have two majorantigen identifyinglymphocytes:CD8+cytotoxic T-lymphocytes (CTL) andCD4+helper T-lymphocytes that can destroy cells. Antigen receptors on CTL can bind to a 9-10amino acid chain that is presented by themajor histocompatibility complex (MHC) as in Figure 4. HTERT is a potential target antigen. Immunotargeting should result in relatively few side effects since hTERT expression is associated only with telomerase and is not essential in almost all somatic cells.[64] GV1001 uses this pathway.[50] Experimental drug andvaccine therapies targeting active telomerase have been tested inmouse models, andclinical trials have begun. One drug,imetelstat, is being clinically researched as a means of interfering with telomerase in cancer cells.[65] Most of the harmful cancer-related effects of telomerase are dependent on an intact RNA template.Cancer stem cells that use an alternative method of telomere maintenance are still killed when telomerase's RNA template is blocked or damaged.
Two telomerase vaccines have been developed:GRNVAC1 andGV1001. GRNVAC1 isolatesdendritic cells and the RNA that codes for the telomerase protein and puts them back into the patient to makecytotoxic T cells that kill the telomerase-active cells. GV1001 is a peptide from the active site of hTERT and is recognized by the immune system that reacts by killing the telomerase-active cells.[50]
Another independent approach is to useoligoadenylated anti-telomerase antisenseoligonucleotides andribozymes to target telomerase RNA, leading to the dissociation of the RNA and toapoptosis (Figure 5). The fast induction of apoptosis through antisense binding may be a good alternative to the slower telomere shortening.[56]
siRNAs are small RNA molecules that induce the sequence-specific degradation of other RNAs. siRNA treatment can function similar to traditionalgene therapy by destroying themRNA products of particular genes, and therefore preventing the expression of those genes. A 2012 study found that targeting TERC with an siRNA reduced telomerase activity by more than 50% and resulted in decreased viability of immortal cancer cells.[66] Treatment with both the siRNA and radiation caused a greater reduction in tumor size in mice than treatment with radiation alone, suggesting that targeting telomerase could be a way to increase the efficacy of radiation in treating radiation-resistant tumors.
Blackburn also discovered that mothers caring for very sick children have shorter telomeres when they report that their emotional stress is at a maximum and that telomerase was active at the site of blockages incoronary artery tissue, possibly accelerating heart attacks.
In 2009, it was shown that the amount of telomerase activity significantly increased followingpsychological stress. Across the sample of patients telomerase activity inperipheral blood mononuclear cells increased by 18% one hour after the end of the stress.[67]
A study in 2010 found that there was "significantly greater" telomerase activity in participants than controls after a three-month meditation retreat.[68]
Telomerase deficiency has been linked todiabetes mellitus and impairedinsulin secretion in mice, due to loss ofpancreatic insulin-producing cells.[69]
Mutations in TERT have been implicated in predisposing patients toaplastic anemia, a disorder in which thebone marrow fails to produce blood cells, in 2005.[70]
Cri du chat syndrome (CdCS) is a complex disorder involving the loss of the distal portion of the short arm ofchromosome 5. TERT is located in the deleted region, and loss of one copy of TERT has been suggested as a cause or contributing factor of this disease.[71]
Dyskeratosis congenita (DC) is a disease of thebone marrow that can be caused by some mutations in the telomerase subunits.[72] In the DC cases, about 35% cases areX-linked-recessive on the DKC1 locus[73] and 5% cases areautosomal dominant on the TERT[74] and TERC[75] loci.
Patients with DC have severe bone marrow failure manifesting as abnormalskin pigmentation,leucoplakia (a white thickening of the oral mucosa) andnail dystrophy, as well as a variety of other symptoms. Individuals with either TERC or DKC1 mutations have shorter telomeres and defective telomerase activityin vitro versus other individuals of the same age.[76]
In one family autosomal dominant DC was linked to aheterozygous TERT mutation.[5] These patients also exhibited an increased rate of telomere-shortening, andgenetic anticipation (i.e., the DC phenotype worsened with each generation).
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