Telomere-associated Proteins Tankyrase And TRF1

Aging, such as growth, is one of the important phenomenona in life. Telomere plays importmant role on control chromosomal stability. Telomere length is an important marker of senescence, death and cancer.Telomerase and telomere-associated proteins can regulate telomere length. TRF1, telomere repeat binding factor 1, can bind to telomeric dsDNA by its Myb domain and strengthen telomeric complex as an "closed structure" so that telomerase can not access to telomeric end. Being a negative regulator of telomere length, Long-term overexpression of TRF1 in telomerase -positive tumor cells results in a gradual and progressive telomere shorting. Tankyrase, a poly (ADP-ribose) polymerase (PARP) at human telomere, can add poly (ADP-ribose) to TRF1 to diminish its ability to bind to telomeric DNA in vitro and open the telomeric complex, allowing access to telomerase. Long-term overexpression of tankyrsae in telomerase-positive cells results in a gradual and progressive elongation of telomeres and tankyrase acts as a positive regulator of telomere elongation in vivo. Thus, the regulating of tankyrase PARP activity and the interaction between tankyrase and TRF1 are the main causes of altered telomere homeostasis.There is a known NLS (nuclear localization signal) in TRF1 which is a nuclear protein. Tankyrase can locate in cytoplasm and nuclear in a TRF1 -dependent manner. How they finely regulate telomere length according to cell response? Are there some other molecules involved in modifying and regulating them in these processes? If the protein level of tankyrase is inhibited and its nuclear function is blocked, can the negative regulating ability of TRF1 on telomere length be strengthened? On the other hand, if the protein level of TRF1 is inhibited, can tankyrase's positive role on telomereenhanced or be weaked because of decreasing into nuclear? What will happen on cell growth, telomere length and senscence on these conditions?To challenge these questions, we founded the cell model to block tankyrase into nuclear, screened the siRNA sequences to interfere tankyrsae and TRF1, investigated the relationship between PKA and TRF1, tested whether PKA could phosphorylate TRF1 in vitro directly. The preliminary results as follow:1. TRF1 cDNA was cloned from HeLa cells and fused to the His6 protein. The fusion protein were purified by affinity chromagraphy and was used to prepare antiserum.The antiserum of TRF1 could be used in Western-blot and Immunofluorescence cell staining to analysis the endogenous TRF1 from different mammalian cell lines.2. The cell model of blocking tankyrase into nuclear was founded. By dominant negative inhibit, overexpressed of 68 amino acids of NH4- terminal of TRF1 can inhibite the protein level of tankyrase in nuclear and cell proliferation.3. Two siRNA sequences that could weak protein level of tankyrase and three siRNA sequences on TRF1 were screened.4. By in vitro phosphorylation assay, HiS6-TRF1 was phosphorylated by ATM which was actived by 10Gy γ-irradiation one hour before, and this phosphorylation could be inhibited by Wortmannin. Immunoblot analysis of the anti-PKA immunoprecipitate with myc antibody revealed that PKA and overexpressed myc-TRF1 could form immunoprecipitative complex. The in vitro phosphorylation of PKA showed that PKA could phosphorylate His6-TRF1, which could be inhibited PKA inhibitor H89.In short, our cell model and siRNA sequences focus on tankyrase and TRF1 might shed new lights on tankyrase function and its interaction between TRF1 on cell growth, scensence and cancer.