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。     

端粒结合蛋白TRF2的研究进展

端粒DNA结合蛋白TRF2(TTAGGG repeat binding factor-2)以二聚体形式通过Myb结构域与端粒重复序列TTAGGG结合,并与TRF1、TIN2、Rap1、TINT1及POT1蛋白组成Shelterin蛋白复合物,协同在端粒动态平衡维持过程中起关键作用,进而影响整个基因组的稳定性。此外,TRF2在细胞DNA损伤应答过程中可能发挥重要作用。本文将对TRF2结构和功能研究的最新进展进行综述。     

骨肿瘤端粒长度变化与相关蛋白表达的关系

目的:研究骨肿瘤端粒长度变化与端粒结合蛋白即端粒重复结合因子1(TRF1)和端粒保护因子(POT1),端粒酶催化亚单位(hTERT),肿瘤相关基因P53、c-myc表达的关系,以了解骨肿瘤的分子特征。方法:采用免疫组织化学、端粒定量荧光原位杂交(Telo-FISH)和原位杂交检测了20例骨肉瘤、25例软骨肉瘤、14例骨的纤维结构不良中端粒长度、TRF1、POT1、hTERT、P53、c-myc的表达情况,并进行统计分析。结果:20例骨肉瘤平均长度为0.31,25例软骨肉瘤为0.41,14例骨的纤维结构不良为0.52。统计显示三者间端粒长度有显著差异(P<0.05)。骨肉瘤和软骨肉瘤TRF1、POT1阳性率均显著低于骨纤维结构不良(P<0.05)。而骨肉瘤和软骨肉瘤hTERT基因表达显著高于骨纤维结构不良(P<0.05)。骨肉瘤、软骨肉瘤P53、c-myc阳性率高于骨纤维结构不良(P<0.05)。统计分析骨肿瘤端粒长度变化与端粒结合蛋白TRF1、POT1的表达呈负相关性,与端粒酶hTERT基因表达、与P53蛋白核聚积,以及c-myc癌基因表达呈正相关性。结论:骨肿瘤端粒长度与恶性表型有关、端粒短缩与肿瘤基因突变相关。     

端粒-端粒酶系统与糖尿病和胰岛β细胞

端粒和端粒酶共同构成端粒-端粒酶系统,参与细胞衰老、增殖和凋亡的调控,在多种增龄性疾病中发挥重要作用。研究表明,端粒-端粒酶系统异常与糖尿病和胰岛β细胞功能异常有关,改变端粒酶的活性有希望作为糖尿病细胞治疗的措施之一。本文就端粒-端粒酶系统在糖尿病胰岛β细胞中的研究进展做一综述。     

端粒结合蛋白与端粒长度调控

真核细胞端粒DNA序列的丢失与细胞的衰老及凋亡有关.端粒酶的激活可维持端粒长度并使细胞获得无限增殖的能力.端粒结合蛋白则可能通过调节端粒酶或其他相关因子的行为参与对端粒长度的调控.近年有关端粒结合蛋白的研究取得了突破性进展并在此基础上建立了端粒长度调控模型.     

端粒、端粒酶结构功能研究进展

端粒是真核生物线性染色体末端由重复DNA序列和蛋白质结合形成的复合结构,其特殊的环形结构与多种结合蛋白形成了端粒的多重功能的基础。端粒的功能包括染色体末端的保护、引导减数分裂的同源染色体配对、参与DNA修复过程等;端粒酶具有逆转录酶特性和维持端粒长度的功能,其活性与恶性肿瘤的发生密切相关,调控因子错综复杂。     

端粒相关蛋白TIN2

TIN2（TRFI相互作用核蛋白2）是一重要的端粒相关蛋白。人TIN2蛋白包括N端,TRF1交互作用区（TRF1—Int）和C端3个结构域。它在TRF1复合物、TRF2复合物和Sheherin的功能中扮演关键角色,协同其它端粒蛋白维持端粒长度、结构和功能。TIN2与个体发育、细胞分化和肿瘤发生密切相关。     

端粒及端粒酶的研究进展

端粒是真核细胞染色体末端的特有结构,是由端粒结合蛋白和一段重复序列的端粒DNA组成的一个高度精密的复合体,在维持染色体末端稳定性,避免染色体被核酸酶降解等方面起着重要的作用。端粒的长度、结构及组织形式受多种端粒结合因子的调控。由于端粒的重要性,在哺乳动物细胞里,端粒的长度或端粒结构变化与癌症发生及细胞衰老有密切的关系。由于末端复制问题的存在,随着细胞分裂次数的增加,端粒不断缩短,细胞不可避免的走向衰老或凋亡。由于在细胞分裂过程中端粒长度的不断缩短与细胞分裂代数增加具有相关性,即端粒长度反应了细胞的分裂次数,因此有人将端粒形象的比喻为生物时钟。在90%的癌细胞中,端粒酶被重新激活,以此来维持端粒的长度,使细胞走向永生化。简要综述了端粒、端粒酶及端粒酶结合蛋白的最新研究进展。     

高等植物端粒和端粒酶的研究进展

端粒是构成真核生物线状染色体末端重要的DNA-蛋白质复合结构,DNA由简单的串联重复序列组成。它的合成由一个特殊的具有反转录活性的核糖核蛋白-端粒酶完成。端粒对染色体、整个生物基因组,甚至对细胞的稳定都具有重要意义。本文就植物端粒、端粒酶、端粒结合蛋白,以及端粒变化、端粒酶在植物生长发育中的调节作一概述。     

哺乳动物细胞衰老调节的新机制——端粒的表观遗传修饰

端粒（telomere）是位于真核生物染色体末端的保护性结构,在调节细胞衰老及细胞寿命等 方面具有重要意义.人们已在端粒结构中鉴定出了一系列的蛋白因子,如TRF1、TRF2、Pot1 ,Rap1、Tin2等,这些因子在保护端粒以及维持端粒合适长度的过程中具有重要作用.最近 人们发现,在端粒结构以及亚端粒区域中存在丰富的表观遗传修饰,该类修饰包括组蛋白的 三甲基化、组蛋白的乙酰基化以及DNA的甲基化等.并且发现这些修饰在端粒长度调节过程以 及端粒相关疾病的发生发展过程中具有重要意义.人们推测,该机制可能对哺乳动物的衰老过 程以及衰老相关的疾病等方面具有重要的调节作用.本文将对这些方面的最新研究进展作一 介绍.     

Telomerase inhibitor PinX1 provides a link between TRF1 and telomerase to prevent telomere elongation

Telomere maintenance is essential for protecting chromosome ends. Aberrations in telomere length have been implicated in cancer and aging. Telomere elongation by human telomerase is inhibited in cis by the telomeric protein TRF1 and its associated proteins. However, the link between TRF1 and inhibition of telomerase elongation of telomeres remains elusive because TRF1 has no direct effect on telomerase activity. We have previously identified one Pin2/TRF1-interacting protein, PinX1, that has the unique property of directly binding and inhibiting telomerase catalytic activity (Zhou, X. Z., and Lu, K. P. (2001) Cell 107, 347-359). However, nothing is known about the role of the PinX1-TRF1 interaction in the regulation of telomere maintenance. By identifying functional domains and key amino acid residues in PinX1 and TRF1 responsible for the PinX1-TRF1 interaction, we show that the TRF homology domain of TRF1 interacts with a minimal 20-amino acid sequence of PinX1 via hydrophilic and hydrophobic interactions. Significantly, either disrupting this interaction by mutating the critical Leu-291 residue in PinX1 or knocking down endogenous TRF1 by RNAi abolishes the ability of PinX1 to localize to telomeres and to inhibit telomere elongation in cells even though neither has any effect on telomerase activity per se. Thus, the telomerase inhibitor PinX1 is recruited to telomeres by TRF1 and provides a critical link between TRF1 and telomerase inhibition to prevent telomere elongation and help maintain telomere homeostasis.     

Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2

We investigated the control of telomere length by the human telomeric proteins TRF1 and TRF2. To this end, we established telomerase-positive cell lines in which the targeting of these telomeric proteins to specific telomeres could be induced. We demonstrate that their targeting leads to telomere shortening. This indicates that these proteins act in cis to repress telomere elongation. Inhibition of telomerase activity by a modified oligonucleotide did not further increase the pace of telomere erosion caused by TRF1 targeting, suggesting that telomerase itself is the target of TRF1 regulation. In contrast, TRF2 targeting and telomerase inhibition have additive effects. The possibility that TRF2 can activate a telomeric degradation pathway was directly tested in human primary cells that do not express telomerase. In these cells, overexpression of full-length TRF2 leads to an increased rate of telomere shortening.     

Role of the TRF2 Telomeric Protein in Cancer and Aging

Telomeres are the special heterochromatin that forms the ends of chromosomes, consisting of TTAGGG repeats and associated proteins. Telomeres protect the ends from degradation and recombination, and are essential for chromosomal stability. Both a minimal length of telomere repeats and the telomere-binding proteins are required for telomere protection. Telomerase is a DNA polymerase that specifically elongates telomeres, in this way regulating telomere length and function. A minimal telomere length is required to maintain tissue homeostasis. On one hand, critically short telomeres trigger loss of cell viability and premature death in mice deficient for telomerase activity. Furthermore, altered functioning of telomerase and telomere-interacting proteins is present in some human premature ageing syndromes and cancer. A new mouse model with critically short telomeres has been generated by over-expressing the TRF2 telomere-binding protein, K5-TRF2 mice. These mice show short telomeres in the presence of telomerase activity, leading to premature aging and increased cancer. Short telomeres in TRF2 mice can be rescued in the absence of the XPF nuclease, indicating that this enzyme rapidly degrades telomeres in the presence of increased TRF2 expression. K5-TRF2 mice represent a new tool to understand the consequences of critical telomere shortening a telomerase-proficient genetic background, more closely resembling human cancer and aging pathologies.     

Dissociation of Telomere Dynamics from Telomerase Activity in Human Thyroid Cancer Cells

Prevention of telomere erosion through acquisition of telomerase activity is thought to be an essential mechanism in most human cancer cells for avoidance of cellular senescence and crisis. It has been generally assumed that once telomerase has been activated, no further telomere shortening should ensue. We show here, however, that a much more complex pattern of telomere dynamics can exist in telomerase-positive immortal cancer cells. Using a panel of subclones derived from a human thyroid cancer cell line, K1E7, we found that some clones show persistent decline in mean telomere restriction fragment (TRF) length by up to 2 kb over 450 population doublings (pd), despite sustained high telomerase activity (as assessed by thein vitro“TRAP” assay). TRF length subsequently stabilized at around 5 kb, but with no corresponding increase in telomerase activity. One clone showed an even more unexpected biphasic time course, with the mean TRF length initially increasing by 1.5 kb over 90 pd, before “plateauing” and then returning over a similar period to its original value, again without any correlation to TRAP activity. Such dissociations between telomere dynamics and telomerase activity support the existence of additional controls on telomere length in the intact cell. Our observations are consistent with current negative-feedback models of telomere length regulation by telomere binding proteins and these cell lines should prove useful experimental tools for their further evaluation.     

Control of human telomere length by TRF1 and TRF2

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3' telomere terminus in TRF1- and TRF2-induced telomeric loops.     

Role of Pin2/TRF1 in telomere maintenance and cell cycle control

Telomeres are specialized structures found at the extreme ends of chromosomes, which have many functions, including preserving genomic stability, maintaining cell proliferative capacity, and blocking the activation of DNA-damage cell cycle checkpoints. Deregulation of telomere length has been implicated in cancer and ageing. Telomere maintenance is tightly regulated by telomerase and many other telomere-associated proteins and is also closely linked to cell cycle control, especially mitotic regulation. However, little is known about the identity and function of the signaling molecules connecting telomere maintenance and cell cycle control. Pin2/TRF1 was originally identified as a protein bound to telomeric DNA (TRF1) and as a protein involved in mitotic regulation (Pin2). Pin2/TRF1 negatively regulates telomere length and importantly, its function is tightly regulated during the cell cycle, acting as an important regulator of mitosis. Recent identification of many Pin2/TRF1 upstream regulators and downstream targets has provided important clues to understanding the dual roles of Pin2/TRF1 in telomere maintenance and cell cycle control. These results have led us to propose that Pin2/TRF1 functions as a key molecule in connecting telomere maintenance and cell cycle control.     

Increased shelterin mRNA expression in peripheral blood mononuclear cells and skeletal muscle following an ultra-long-distance running event

Located at the end of chromosomes, telomeres are progressively shortened with each replication of DNA during aging. Integral to the regulation of telomere length is a group of proteins making up the shelterin complex, whose tissue-specific function during physiological stress is not well understood. In this study, we examine the mRNA and protein levels of proteins within and associated with the shelterin complex in subjects (n = 8, mean age = 44 yr) who completed a physiological stress of seven marathons in 7 days. Twenty-two to 24 h after the last marathon, subjects had increased mRNA levels of DNA repair enzymes Ku70 and Ku80 (P < 0.05) in both skeletal muscle and peripheral blood mononuclear cells (PBMCs). Additionally, the PBMCs displayed an increment in three shelterin protein mRNA levels (TRF1, TRF2, and Pot-1, P < 0.05) following the event. Seven days of ultrarunning did not result in changes in mean telomere length, telomerase activity, hTert mRNA, or hterc mRNAs found in PBMCs. Higher protein concentrations of TRF2 were found in skeletal muscle vs. PBMCs at rest. Mean telomere length in skeletal muscle did not change and did not contain detectable levels of htert mRNA or telomerase activity. Furthermore, changes in the PBMCs could not be attributed to changes in the proportion of subtypes of CD4(+) or CD8(+) cells. We have provided the first evidence that, in humans, proteins within and associated with the shelterin complex increase at the mRNA level in response to a physiological stress differentially in PBMCs and skeletal muscle.     

Telomere instability in a human tumor cell line expressing a dominant-negative WRN protein

Werner Syndrome (WS) is an autosomal recessive disease characterized by premature aging and chromosome instability. The protein involved in WS, WRN, is a RecQ-type helicase that also has exonuclease activity. WRN has been demonstrated to bind to a variety of other proteins, including RPA, DNA-PKcs, and TRF2, suggesting that WRN is involved in DNA replication, repair, recombination, and telomere maintenance. In culture, WS cells show premature senescence, which can be overcome by transfection with an expression vector containing the gene for the catalytic subunit of telomerase. However, telomerase expression does not eliminate chromosome instability in WS cells, which led to the proposal that telomere loss is not the cause of the high rate of chromosome rearrangements in WS cells. In the present study, we have investigated how a WRN protein containing a dominant-negative mutation (K577M-WRN) influences the stability of telomeres in a human tumor cell line expressing telomerase. The results demonstrate an increased rate of telomere loss and chromosome fusion in cells expressing K577M-WRN. Expression of K577M-WRN results in reduced levels of telomerase activity, however, the absence of detectable changes in average telomere length demonstrates that WRN-associated telomere loss results from stochastic events involving complete telomere loss or loss of telomere capping function. Thus, telomere loss can contribute to chromosome instability in cells deficient in WRN regardless of the expression of telomerase activity.     

Generation and characterization of telomere length maintenance in tankyrase 2-deficient mice

Telomere length and function are crucial factors that determine the capacity for cell proliferation and survival, mediate cellular senescence, and play a role in malignant transformation in eukaryotic systems. The telomere length of a specific mammalian species is maintained within a given range by the action of telomerase and telomere-associated proteins. TRF1 is a telomere-associated protein that inhibits telomere elongation by its binding to telomere repeats, preventing access to telomerase. Human TRF1 interacts with tankyrase 1 and tankyrase 2 proteins, two related members of the tankyrase family shown to have poly(ADP-ribose) polymerase activity. Human tankyrase 1 is reported to ADP-ribosylate TRF1 and to down-regulate the telomeric repeat binding activity of TRF1, resulting in telomerase-dependent telomere elongation. Human tankyrase 2 is proposed to have activity similar to that of tankyrase 1, although tankyrase 2 function has been less extensively characterized. In the present study, we have assessed the in vivo function of mouse tankyrase 2 by germ line gene inactivation and show that inactivation of tankyrase 2 does not result in detectable alteration in telomere length when monitored through multiple generations of breeding. This finding suggests that either mouse tankyrases 1 and 2 have redundant functions in telomere length maintenance or that mouse tankyrase 2 differs from human tankyrase 2 in its role in telomere length maintenance. Tankyrase 2 deficiency did result in a significant decrease in body weight sustained through at least the first year of life, most marked in male mice, suggesting that tankyrase 2 functions in potentially telomerase-independent pathways to affect overall development and/or metabolism.