Study on the structure and function of the C-terminus of the catalytic subunit of telomerase
|School||PLA Military Academy of Medical Sciences|
|Keywords||Telomerase catalytic subunit The nucleolus structure of domain C-NoLD Nucleolus Cancer treatment|
Biological function of telomeres protect the ends of chromosomes, avoid nuclease degradation of the ends of chromosomes and prevent chromosome fusion and rearrangement, plays an important role in maintaining the structure of the genome integrity and stability. Ribonucleoprotein complex with a special reverse transcriptase activity of telomerase by human telomerase reverse transcriptase (Telomerase Reverse Transcriptase, TERT) and telomerase RNA (Telomerase RNA component, TR or TER) template. Its own RNA template by reverse transcriptase synthesis telomeric repeat sequences, and is connected to the chromosome 3 'end. Most tumor cells through the up-regulation of the expression of telomerase to maintain telomere length. hTERT control restrictive component of telomerase activity, hTERT to maintain tumor cell growth is very important, and inhibition of hTERT expression in tumor cells, these cells will senescence or apoptosis. hTERT protein conserved region of a single amino acid change can make changes in telomerase activity or even disappear. hTERT protein intracellular transport is an important post-translational level form of functional regulation. The study found that the nuclear localization of hTERT distribution behavior between normal cells and tumor cells, there is a significant difference. HTERT protein expression in normal cells aggregated distribution nucleolar structure of the nucleus. In transformed cells or tumor cells, hTERT mainly dispersed outside the nucleus nucleoli. Furthermore, the chromosomal DNA damage can induce tumor cells nucleolar outer distribution of hTERT gathered to the nucleolus. These phenomena prompted hTERT nucleolar localization regulation affect the survival of cancerous cells and tumor cells. But on the regulation of hTERT nucleolar localization structural basis, molecular mechanism and biological significance of the tumor is not clear. hTERT C-terminal amino acid sequence has an important role in the regulation of hTERT intracellular localization, and also in the intracellular domain essential for replication of the telomere end. Our laboratory Dr. Lin Jian hTERT C-terminal the new nucleolar localization domain (the C-NOLD) further identification of the conserved basic amino acid residues 965-980 amino acid sequence rich in decision hTERT core Ren positioning of the key signal. The hTERT mutants of the positioning signal sequence mutation, not only in normal cell culture conditions completely lost the ability localized in the nucleoli, and at the same time lose the ability to respond to DNA damage-induced nucleolar localization. In order to further reveal the the hTERT cells localization structure based on the molecular mechanism of regulation of hTERT DNA damage response nucleolar localization. Through the research methods of structure-function relationships, hTERT C end build different gene fragments to the eukaryotic expression the carrier pLEGFPC1 on with green fluorescent protein labels. Comparative analysis of the intracellular distribution of hTERTC terminal fragment, as well as the change in position in case of chromosomal DNA damage. Thus described the molecular mechanism and biological significance of telomerase catalytic subunit nucleolar localization. We found that the C-terminal 882-953aa sequence also has an important role in the regulation of hTERT intracellular localization. Missing the 882-953aa hTERT mutations physical significantly improve the nuclear localization of hTERT, suggesting that the negative regulation of hTERT nuclear localization of the biological effects of the amino acid sequence. It is interesting, but missing the 882-953aa hTERT mutant expression in tumor cells as well as the loss of nucleolar localization of DNA damage-induced reaction. However, we have previously identified hTERTaa965-981 nucleolar localization signal nucleolar targeting signal of hTERT 882-953 amino acid sequence does not exist activity, suggesting the existence of a new molecular mechanism in the regulation of hTERT nucleolar localization hTERTaa882-953 sequence further elucidate the molecular mechanisms in the regulation of hTERT DNA damage response nucleolar localization put chemotherapy treatment response is important to improve and enhance tumor cell. Telomerase differentially expressed in tumor cells and normal cells so that the new target is considered to be indicators of tumor diagnosis and tumor therapy. Telomerase as a target for anticancer drugs in recent years become a hot research cancer treatment. Although telomerase inhibitors efficient, specific and broad-spectrum, advanced and proliferation of tumor, but not completely inhibit the expression of telomerase in normal human cells, but under strict cell cycle-dependent regulation had brief low expression, which is important to maintain cell proliferation ability. Completely inhibited the expression of telomerase in normal cells can rapidly cause the cells to lose the ability to proliferate and advance into the cell replicative senescence, security against telomerase therapeutic strategies cause of concern. In a previous study, we found that over-expression of the C-terminal fragment of hTERT in hTERT positive HeLa cells C27 (882-1132aa), cause cell senescence and apoptosis. The in vivo experiments show stable expression hTERTC27 can inhibit telomerase-positive tumors tumorigenic in nude mice, and hTERTC27 expression does not affect telomerase activity in tumor cells. Mediated hTERTC27 anti-tumor activity of the key amino acid sequence is not yet clear. Since we have found hTERTaa882-953 sequence has an important role in the regulation of hTERT variety of intracellular localization behavior, suggesting its importance on the hTERT-mediated cellular functions. We may therefore proposed hTERTaa882-953 sequence is a key structure of the original anti-tumor activity hTERTC27. In this thesis the karyotype observed and trypan blue staining hTERTaa882-953 on tumor cells in vitro. The built from hTERTC terminal fragment of the lentiviral vector to observe the impact of these fragments of tumor cell growth. TRAP assay detection hTERTaa882-953 tumor cells after infection telomerase activity. The results found that high expression of telomerase-positive tumor cells hTERTaa882-953 sequence acute killing effect on tumor cells, significantly inhibited the growth of tumor cells. With hTERTC27 high expression hTERTaa882-953 sequence did not affect telomerase activity. In addition, the lack of aa882-953 sequence hTERT C-terminal fragment of the loss of the effect of killing tumor cells. Thus we proved hTERT aa 882-953 hTERTC27 inhibit tumor cell growth and function of the main domain. Cancer treatment our findings will provide a new way.