Telomerase Catalytic Subunit C-terminal Structure And Function

The main biological function of telomere is protecting the end of chromosome from end-to-end fusion, recombination and degradation. The telomerase RNP is minimally composed of two essential elements called the protein telomerase reverse transcriptase (TERT) and the single stranded RNA (TR)which serves as the template for the addition of the repeated sequence to 3'chromosome ends.In most malignant cells, the maintenance of telomeres is achieved by upregulating the expression of the telomerase enzyme.In human cells, hTERT has been demonstrated as the rate-limiting component of cellular telomerase activity and alteration of single amid acid in its conserved regions can abolish the activity of telomerase. hTERT may play essential roles in promoting the growth of tumor cell . As a result,. The inhibition of the expression of hTERT can trigger tumor cell senescence or appoptosis.The subcellular transportation process of hTERT is an important step of the regulations of hTERT in human cells at post-translational level. Recent studies revealed that hTERT have distinct subcellular location in normal human cells comparing to tumor cells. It was found that the expressed hTERT protein is concentrated within the nucleolar regions of the nucleus in normal human cells. However, in the transformation cells and all tested cancer cells hTERT displays diffuse nuclear distribution outside the nucleolar regions. Interestingly, the hTERT resided outside the nucleolar regions can be induced into nucleolar regions under the condition of chomsome DNA damage.These findings indicate that the mechanisms underlying hTERT nucleolar localization in normal cells may be widely affected during cellular tumorigenesis. But the molecular basis and the biological significances of the regulation of hTERT nucleolar localization are still unknown. The hTERT C-terminal domain is very essential for regulating the subcellar localization behavior of hTERT and synthesis of the telomere DNA. The hTERT C- terminus also display important functions in maintianing the activity of hTERT and inhibiting the apoptosis of cells. Doctor Lin Jian in our team has found that a sequence (965aa -981aa) within the hTERT C-terminus is a novel nucleolar location domain (C-NoLD) essential for mediating the hTERT nucleolar localization behaviors. Mutational of this element is able to completely disrupt hTERT nucleolar translocation in both normal and malignant human cells. Moreover, such mutation also leads to the complete loss of the response of hTERT to the DNA damage-induced nucleolar accumulation.In the present study, we use the genetic engineering techniques to research the structure and function of hTERT C-terminus. Our date show that a sequence (882aa -953aa) of hTERT C-terminus also plays a role in hTERT subcellar localization behavior. Deletion mutations of such sequence can enhance the ability of nuclear localization of hTERT but, at the same time, abolish its response to the DNA damage-induced nucleolar accumulation. However, the hTERT aa882-953, which is different from hTERTaa965-981 NoLD, does not have the nucleolar targeting activity. To elucidate the mechanism on the regulation of the hTERT nucleolar localization under the DNA damaging condition will make a great significance to improve the tumor cell's response to radiotherapy and chemotherapy.The evidence that most cancer cells activate telomerase whereas normal cells are usually devoid of telomerase activity has naturally lead to intensive investigations to detect this protein and its activity for a potential use in cancer diagnosis and to eventually monitor the tumor response to therapy. But it has long been argued that directly inhibition of telomerase may affect proliferation of normal cells, which also require a transient telomerase expression to substain its proliferation potential. It has been reported that the ectopic overexpression of hTERTC27 caused a defect in telomere maintenance in hTERT-positive HeLa cells, which led to senescence-like growth arrest and apoptosis. It had no effect on the cellular telomerase enzymatic activity or telomere length. The in vivo effect was further demonstrated as HeLa cells stably expressing hTERTC27 have significantly lower growth rate and reduced tumorigenicity in nude mice xenografts. But the key amino acids in hTETC27 that mediate its anti-tumor function are still unknown. We found that the hTERT fragment 882-953 has an important function in regulating of the hTERT nucleolocalization, so we presume that this fragment may be the essential structural element invovled in the hTERTC27 anti-tumor action. Ectopic overexpression of this fragment in telomerase positive tumor cells can kill and inhibite the growth of the tumor cells but has no effect on telomerase activity. However,in the absence of hTERTaa882-953 , hTERT aa953-1132 which is the C-terminus of hTERTC27 can't kill the tumor cells.Thus,we suggest that hTERT aa 882-953 is the key sequence of hTERTC27 that mediates its antitumor function.