The Interaction Of Telomerase Catalytic Subunit HTERT With DDRGK1

Over the past few decades, telomere biology research showed that telomere length and telomerase activity are closely related to cell life and human diseases. Telomerase is one of the key telomere-binding proteins, which plays an important role in cellular senescence and tumor development by telomere-dependent or independent way.The activity of human telomerase is highly correlated to the expression of its catalytic subunit (human telomerase reverse transcriptase, hTERT), and the regulation of hTERT expression plays a pivotal role in regulating telomerase activity. However, the regulative mechanisms and biological functions of hTERT remains unclear.Human telomerase is a molecular weight of up to 2MD protein complex, but the molecular weight of hTERT-binding proteins which have been identified is much smaller, indicating that most components of telomerase have not been found. Therefore, the screening and identification of new hTERT-binding proteins are very important basis for the research on telomerase activity and biological function, and have an important scientific significance for elucidating the molecular mechanism of telomerase in the development of cancer and aging-related diseases.A yeast two-hybrid assay was performed to screen and identify new hTERT-binding proteins, taking the evolutionary conserved and telomerase-specific T-motif (547-594aa) as a "bait". It was found that DDRGK 1 (DDRGK domain-containing protein 1) which is one of the DDRGK family proteins can interact with hTERT. Exogenous hTERT and DDRGK1 were expressed in human breast cancer MCF-7 cell line, then reciprocal co-immunoprecipitation experiments were conducted and further confirmed DDRGK1 and hTERT may interact with each other. DDRGK1 and hTERT different mutants with Flag-tag were constructed and co-immunoprecipitation experiments were conducted to examine the region of the interaction, the results indicated that the fragment(567-594 amino acid residues) of hTERT is required for the interaction between DDRGK1 and hTERT.TRAP (telomeric repeat amplification protocol) experiments were performed to identify whether DDRGK1 can regulate activity of telomerase. Results indicated that the activity of telomerase was significantly inhibited when DDRGK1 were over-expressed in MCF-7 and HeLa cell lines. In contrast, gene silencing DDRGK 1 can promote its activity. Telomerase activity was highly correlated to hTERT expression, so that the regulation of hTERT expression played a key role in telomerase activity regulation, mainly included levels of transcription and post-transcription. Then it was found that overexpression or silencing DDRGK1 had no significant changes on hTERT messenger RNA (mRNA) levels in MCF-7 cell line through real-time quantitative PCR (Quantitative Real-time PCR, Q-PCR) experiments. While hTERT protein level was significantly decreased when DDRGK1 was over-expressed and its level highly increased when DDRGK1 was silenced, indicating that DDRGK1 regulates telomerase activity by affecting hTERT protein level.It has been reported that DDRGK1 played a crucial role in ubiquitination-like modification, named ufmylation (protein modification by UFM1). Moreover, DDRGK1、hTERT and UFM1 (Ubiquitin-fold modifier 1) showed highly relevant function on endoplasmic reticulum stress. Thence, the expression vectors of ufmylation system were constructed for the in-depth study of DDRGK 1 regulating hTERT activity as well as the biological function.