端粒酶活性调节的分子机制

人端粒酶由RNA亚基、hTERT催化亚基和hTEP1调节蛋白等组成。端粒酶对端粒结构的稳定起着重要的作用,而端粒结构和端粒结合蛋白也影响着端粒酶活性。某些化疗药物通过破坏端粒结构下调端粒酶活性。端粒酶的激活需要hTERT基因的从头转录和各个蛋白亚基正确装配为端粒酶全酶。端粒酶活性调节的分子机制包括：（1）TERT基因的表达和转录是决定端粒酶活性的重要环节,受多种因素调控;（2）蛋白激酶Cα和蛋白激酶B磷酸化端粒酶蛋白而激活端粒酶,蛋白磷酸酯酶2A（PP2A）可逆转这一过程,下调端粒酶活性;（3）多种癌基因和抑癌基因及其编码的蛋白质也直接或间接与端粒蛋白、端粒酶蛋白反应,参与端粒酶活性的调控。     

Non-telomeric activities of telomerase

Recent results suggest that telomerase is involved in many more cellular processes than merely telomere elongation. These include telomere-independent anti-apoptotic, cytoprotective and pro-proliferative effects of telomerase or protection of mitochondrial DNA against oxidative stress. Telomerase also participates in DNA repair and its essential subunits, hTR and hTERT, are able to modulate independently the cell's response to DNA damage. Recent high throughput analyses of gene expression showed that hTERT expression modulates expression of about 300 genes, including genes involved in the regulation of cell cycle progression, proliferation and differentiation. Besides the well-known telomerase catalytic activity of RNA-dependent DNA polymerase, its RNA-dependent RNA polymerase activity was recently described in association with the RNA subunit of mitochondrial RNA processing endoribonuclease, thus suggesting involvement of telomerase in RNA interference processes. These recent discoveries open novel possibilities and entirely unexpected research perspectives, branching off from the mainstream telomere and telomerase research.     

C-terminal amino acids 290-328 of LPTS/PinX1 confer telomerase inhibition

LPTS/PinX1, a telomerase inhibitor composed of 328 amino acids, binds to the telomere associated protein Pin2/TRF1 and to the telomerase catalytic subunit hTERT. However, the mechanism by which LPTS/PinX1 regulates telomerase activity remains unclear. Here we show, for the first time, that LPTS/PinX1 uses different domains to interact with Pin2/TRF1 and hTERT. The LPTS/PinX1_254-289 fragment specifically binds to Pin2/TRF1, and LPTS/PinX1_290-328 can associate with hTERT. Compared with the full-length LPTS/PinX1 protein, LPTS/PinX1_290-328 shows stronger in vitro telomerase inhibitory activity. Moreover, the LPTS/PinX1 protein was recruited to telomeres for binding to Pin2/TRF1. Overexpression of LPTS/PinX1_290-328, which contains a nucleolus localization signal, in cells resulted in telomere shortening and progressive cell death. Conversely, telomere elongation was induced by expression of the dominant-negative LPTS/PinX1_1-289. Our results suggest that the C-terminal fragment of LPTS/PinX1 (LPTS/PinX1_290-328) contains a telomerase inhibitory domain that is required for the inhibition of telomere elongation and the induction of cell crisis. Our studies also provide evidence that LPTS/PinX1 interaction with Pin2/TRF1 may play a role in the stabilization of telomeres.     

Human Ku70/80 associates physically with telomerase through interaction with hTERT

Telomere length maintenance, an activity essential for chromosome stability and genome integrity, is regulated by telomerase- and telomere-associated factors. The DNA repair protein Ku (a heterodimer of Ku70 and Ku80 subunits) associates with mammalian telomeres and contributes to telomere maintenance. Here, we analyzed the physical association of Ku with human telomerase both in vivo and in vitro. Antibodies specific to human Ku proteins precipitated human telomerase in extracts from tumor cells, as well as from telomerase-immortalized normal cells, regardless of the presence of DNA-dependent protein kinase catalytic subunit. The same Ku antibodies also precipitated in vitro reconstituted telomerase, suggesting that this association does not require telomeric DNA. Moreover, Ku associated with the in vitro translated catalytic subunit of telomerase (hTERT) in the absence of telomerase RNA (hTR) or telomeric DNA. The results presented here are the first to report that Ku associates with hTERT, and this interaction may function to regulate the access of telomerase to telomeric DNA ends.     

Telomerase with mutated catalytic motifs has dominant negative effects on telomerase activity and inhibits cell growth

Telomerase catalytic subunit (TERT) seems a key factor controlling telomerase activity, telomere length, and cell growth. To further address this issue, we forced expression of a catalytically inactive mutant human TERT (hTERT) in hTERT-immortalised sheep fibroblasts to examine its effects. Expression of mutant hTERT compromised telomerase activity reconstituted by wild-type hTERT in a manner directly attributable to mutant hTERT expression level. High levels of mutant hTERT expression inhibited cell growth with a subset of cells entering replicative senescence. Furthermore, significant telomere attrition was evident in two of three clones with high levels of mutant hTERT expression. Our findings are consistent with the notion that hTERT homodimers are necessarily required to form a functional telomerase complex at the telomere substrate. We also highlight the requirement of a more thorough understanding of telomerase- and telomere-associated factors to understand fully the interplay that governs telomere homeostasis in vitro and in vivo.     

Mechanism of dominant-negative telomerase function

Human telomerase uses its integral core components, hTR and hTERT, to maintain telomeres in many cell types. Expression of a dominant-negative mutant of the catalytic subunit of telomerase, DN-hTERT, has been shown to cause telomere shortening and ultimately cell death in a number of tumor-derived cell lines. However, the mechanism of dominant-negative hTERT function and its fate inside the cell are still unknown. In order to understand the effect of the dominant-negative on wild-type hTERT, each was fused with GFP and expressed in telomerase-positive cells. GFP-DN-hTERT expression resulted in cytoplasmic exportation and degradation via ubiquitination. Co-expression of wild-type GFP-hTERT with an untagged DN-hTERT resulted in decreased wild-type hTERT levels, export to the cytoplasm, and increased ubiquitination, suggesting that DN-hTERT complexes with wild-type hTERT to induce cytoplasmic localization. Based on the cytoplasmic degradation, we propose two new mechanisms of dominant-negative hTERT, employing the theory of interactive dimerization. First, the heterodimer of DN-hTERT with wild-type hTERT is exported to the cytoplasm for ubiquitin-mediated protein degradation, and second, the heterodimer may be degraded at a faster rate than the wild-type hTERT homodimer. Understanding mechanisms of telomerase degradation will guide future drug design to target sites on telomerase important for catalytic activity and protein stability.     

Catalytically active human telomerase mutants with allele-specific biological properties

Expression of the catalytic subunit of human telomerase, hTERT, extends human primary fibroblast life span. Such life span extension has generally been reported to be accompanied by net telomere lengthening, which led to the hypothesis that it is the telomere lengthening that causes the life span extension. Here we show that hTERT+C and hTERT-FlagC, mutant telomerase proteins with either 10 additional residues or a FLAG epitope added to the hTERT C-terminus, confer significant but limited life span extension to IMR90 human primary lung fibroblasts. However, as the cells continue to grow for >100 population doublings past their normal senescence point, bulk telomere length continues to erode to lengths much shorter than those seen at the senescence of control telomerase-negative cells. Expression of hTERT+C immortalized IMR90 cells transformed by three different oncogenes. Again, bulk telomeres became much shorter than those of the control cells at crisis. Additional hTERT mutants were constructed and analyzed similarly. Enzymatically active hTERT-N125A+T126A, like other previously reported conserved GQ domain mutants and C-terminally HA-tagged hTERT, failed to extend life span. Another GQ domain mutant, hTERT-E79A, was indistinguishable from wild-type hTERT in its cell growth effects, but there was no net telomere lengthening. These results uncover further hTERT allele-specific phenotypes that uncouple telomerase activity, net telomere lengthening and life span extension.     

Transient expression of human telomerase extends the life span of normal human fibroblasts

We utilized the Cre/lox recombination system to transiently express the catalytic subunit of telomerase (hTERT) in normal diploid foreskin fibroblasts (BJ cells). A retroviral construct containing an hTERT cDNA, flanked by loxP-sites was introduced into near senescent BJ cells (population doubling 85). At population doubling (PD) 92, which exceeds the typical life span of these cells, we excised the gene via Cre-mediated recombination. All clones lost telomerase activity and showed telomere shortening over an additional 50 PDs. Interestingly, the average telomere length in these cells became shorter than in untreated BJ cells at senescence. This may be due to hTERT preferentially elongating the shortest telomeres, leading to greater length uniformity. In summary, transient telomerase expression and only a very small average telomere elongation by hTERT resulted in a 50% increase in life span of human fibroblasts. This suggests a potentially safe use of hTERT in tissue engineering.     

Human MCRS2, a cell-cycle-dependent protein, associates with LPTS/PinX1 and reduces the telomere length

Human LPTS/PinX1 is a telomerase-inhibitory protein, which binds to the telomere protein Pin2/TRF1 and the catalytic subunit hTERT of telomerase. To explore the proteins that might be involved in the telomerase pathway, we performed a yeast two-hybrid screening with LPTS/PinX1 as the bait. A novel gene, MCRS2, encoding for an isoform of MCRS1/p78 and MSP58 was isolated. The expression of MCRS2 protein is cell-cycle dependent, accumulating in the very early S phase. MCRS2 interacts with LPTS/PinX1 in vitro, in vivo and colocalizes with LPTS/PinX1 in cells. MCRS2 and its amino terminus inhibit telomerase activity in vitro and long-term overexpression of MCRS2 in SMMC-7721 cells results in a gradual and progressive shortening of telomeres. Our findings suggest that MCRS2 might be a linker between telomere maintenance and cell-cycle regulation.     

A high-throughput assay for a human telomerase protein-human telomerase RNA interaction

The rapid rate at which cancer cells divide necessitates a mechanism for telomere maintenance, and in approximately 90% of all cancer types the enzyme telomerase is used to maintain the length of telomeric DNA. Telomerase is a multi-subunit enzyme that minimally contains a catalytic protein subunit, hTERT, and an RNA subunit, hTR. Proper assembly of telomerase is critical for its enzymatic activity and therefore is a requirement for the proliferation of most cancer cells. We have developed the first high-throughput screen capable of identifying small molecules that specifically perturb human telomerase assemblage. The screen uses a scintillation proximity assay to identify compounds that prevent a specific and required interaction between hTR and hTERT. Rather than attempting to disrupt all of the individual hTR-hTERT interactions, we focused the screen on the interaction of the CR4-CR5 domain of hTR with hTERT. The screen employs a biotin-labeled derivative of the CR4-CR5 domain of hTR that independently binds [(35)S]hTERT in a functionally relevant manner. The complex between hTERT and biotin-labeled RNA can be captured on streptavidin-coated scintillation proximity beads. Use of 96-well filter plates and a vacuum manifold enables rapid purification of the beads. After optimization, statistical evaluation of the screen generated a Z' factor of 0.6, demonstrating the high precision of the assay.