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Cellular functions of the dual-targeted catalytic subunit of telomerase, telomerase reverse transcriptase — Potential role in senescence and aging
EXG-09362; No of Pages 5 Experimental Gerontology xxx (2014) xxx–xxx
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Review
Cellular functions of the dual-targeted catalytic subunit of telomerase, telomerase reverse transcriptase — Potential role in senescence and aging Niloofar Ale-Agha b,1, Nadine Dyballa-Rukes b,1, Sascha Jakob b, Joachim Altschmied b, Judith Haendeler a,b,⁎ a b
Central Institute of Clinical Chemistry and Laboratory Medicine, University of Duesseldorf, Germany IUF — Leibniz Research Institute for Environmental Medicine at the University of Duesseldorf gGmbH, 40225 Duesseldorf, Germany
a r t i c l e
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Article history: Received 15 December 2013 Received in revised form 17 February 2014 Accepted 20 February 2014 Available online xxxx Keywords: Aging Mitochondria Nucleus Senescence Telomerase reverse transcriptase
a b s t r a c t Over the last 40 years it has become clear that telomeres, the end of the chromosomes, and the enzyme telomerase reverse transcriptase (TERT), which is required to counteract their shortening, play a pivotal role in senescence and aging. However, over the last years several studies demonstrated that TERT belongs to the group of dual-targeted proteins. It contains a bipartite nuclear localization signal as well as a mitochondrial targeting sequence and, under physiological conditions, is found in both organelles in several cell types including terminally differentiated, post-mitotic cells. The canonical function of TERT is to prevent telomere erosion and thereby the development of replicative senescence and genetic instability. Besides telomere extension, TERT exhibits other non-telomeric activities such as cell cycle regulation, modulation of cellular signaling and gene expression, augmentation of proliferative lifespan as well as DNA damage responses. Mitochondrial TERT is able to reduce reactive oxygen species, mitochondrial DNA damage and apoptosis. Because of the localization of TERT in the nucleus and in the mitochondria, it must have different functions in the two organelles as mitochondrial DNA does not contain telomeric structures. However, the organelle-specific functions are not completely understood. Strikingly, the regulation by phosphorylation of TERT seems to reveal multiple parallels. This review will summarize the current knowledge about the cellular functions and post-translational regulation of the dual-targeted protein TERT. © 2014 Elsevier Inc. All rights reserved.
1. Introduction: telomeres and telomerase Already in the first half of the last century Hermann Muller and Barbara McClintock described the instability of broken chromosomes in species as distantly related as Drosophila and maize (McClintock, 1941; Muller, 1938). However, in their studies they never found structural changes involving the end of the chromosomes, which led Muller to the conclusion that this terminal region (in his words “terminal gene”) must have a special function in sealing the chromosome. To describe the uniqueness of these regions and to set them apart from the Abbreviations: TERT, telomerase reverse transcriptase; TERC, non-coding telomerase RNA component; ROS, reactive oxygen species; ETC, electron transport chain; mtDNA, mitochondrial DNA; NLS, nuclear localization signal; NES, nuclear export signal; MTS, mitochondrial targeting sequence. ⁎ Corresponding author at: Central Institute of Clinical Chemistry and Laboratory Medicine, University of Duesseldorf, and, IUF — Leibniz Research Institute for Environmental Medicine, Auf'm Hennekamp 50, 40225 Duesseldorf, Germany. Tel.: +49 211 3389 291; fax: +49 211 3389 331. E-mail addresses: [email protected] (N. Ale-Agha), [email protected] (N. Dyballa-Rukes), [email protected] (S. Jakob), [email protected] (J. Altschmied), [email protected] (J. Haendeler). 1 Both authors contributed equally to the work.
remainder of the chromosomes, he termed them telomeres combining the Greek words for end (telo) and part (meros). Almost 40 years later Joseph Gall and Elizabeth Blackburn described the presence of a tandem hexanucleotide repeat sequence CCCCAA at the end of extrachromosomal rDNA molecules in Tetrahymena (Blackburn and Gall, 1978). After it was shown by Elizabeth Blackburn and Jack Szostak that these were functionally equivalent to the ends of yeast chromosomes, they concluded that such repeated end sequences correspond to functional telomeres (Szostak and Blackburn, 1982). Nowadays, it is common knowledge that telomeres consist of tandem repeats of the hexanucleotide sequence TTAGGG (in mammals), adopt a higher order structure and are capped with a large number of single- and doublestrand DNA binding proteins (Blasco, 2005; de Lange, 2005; Pinto et al., 2011). A connection between telomeres, senescence and aging was derived from several independent lines of evidence. In 1965 Leonard Hayflick demonstrated that isolated fibroblasts show only a limited proliferative potential in culture and concluded that this finite lifetime of cells in vitro may be an expression of aging or senescence at the cellular level (Hayflick, 1965). A molecular basis and a link to telomeres were proposed by Alexey Olovnikov after he and James Watson had independently recognized the problem that during DNA replication, a small
http://dx.doi.org/10.1016/j.exger.2014.02.011 0531-5565/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Ale-Agha, N., et al., Cellular functions of the dual-targeted catalytic subunit of telomerase, telomerase reverse transcriptase — Potential role in senescence and aging, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.011
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part of DNA from the 3′-ends of linear chromosomes is unreplicated. Olovnikov explained Hayflick's theory of limited somatic cell division suggesting that this now called end-replication problem leads to loss of telomeric sequences, which he called “telogenes”, during successive mitoses finally resulting in cellular senescence and elimination of such cells (Olovnikov, 1973). However, up to now it is not clear whether telomere length could be used as a biomarker for aging because the findings in humans are contradictory as reviewed by Mather et al. (2011). Again Elizabeth Blackburn, together with her graduate student Carol Greider, discovered that telomere shortening is counteracted by the activity of a cellular ribonucleoprotein complex called telomerase, which they identified in Tetrahymena (Greider and Blackburn, 1985; Greider and Blackburn, 1987). The presence of a similar activity in human cells was demonstrated shortly thereafter establishing that telomerasemediated telomere maintenance is conserved throughout eukaryotes (Morin, 1989). The telomerase holoenzyme requires the catalytic proteinaceous subunit telomerase reverse transcriptase (TERT) and, besides multiple telomerase associated proteins, the non-coding telomerase RNA component (TERC), which serves as the template for the elongation of the telomere repeat sequences by TERT (Blasco, 2005). If telomerase is absent, telomeres shorten during each round of cell division until they reach a critically short-length threshold (Harley et al., 1990). This causes a DNA damage response and induces cell cycle arrest triggering senescence (Herbig et al., 2004). Along the same lines, the introduction of TERT into cells prolongs both their lifespan and their telomeres to lengths typical of young cells and reduces signs of senescence (Bodnar et al., 1998; Counter et al., 1998). These findings also suggest that permanent, high-level expression of TERT can lead to uncontrolled proliferation, which is reflected by the fact that many tumors exhibit reactivation of telomerase (Blasco, 2005). Therefore, telomerase activity must be tightly regulated, which on one hand is achieved by transcriptional regulation of the TERT gene, and on the other hand by controlling TERT activity on the posttranslational level (Aisner et al., 2002; Cong et al., 2002; Ducrest et al., 2002). It has been proposed for a long time that telomerase activity is absent from most human somatic cells and only present in tumor cells and adult stem cells of highly regenerative tissues, such as the immune system, skin and intestine (Forsyth et al., 2002). However, there is accumulating evidence that substantial telomerase activity is present in differentiated somatic cells, e.g. endothelial cells, smooth muscle cells and hepatocytes (Haendeler et al., 2004; Leri et al., 2000; Minamino and Kourembanas, 2001; Vasa et al., 2000; Werner et al., 2009, 2011; Yamaguchi et al., 1998).
executioner of apoptosis (Estaquier et al., 2012). Newer proteomic studies have revealed the presence of several hundred to more than 1000 proteins in these organelles, depending on the cell type and physiological situation (Balaban, 2010; Pagliarini et al., 2008) suggesting that mitochondria serve much broader functions than originally anticipated. When the functions of mitochondrial TERT were assessed, the first descriptions reported that it renders cells more susceptible to oxidative stress-induced mitochondrial DNA (mtDNA) damage, which can lead to apoptotic cell death (Santos et al., 2004, 2006). In contrast to these findings, we and others described a protective function for mitochondrial TERT by increasing the membrane potential, reducing reactive oxygen production, protecting mtDNA against damage and inhibiting apoptosis induction (Ahmed et al., 2008; Haendeler et al., 2009). Interestingly, TERT has also an impact on the ETC., as overexpression of TERT enhances overall respiratory chain activity in HEK 293 cells, with the most pronounced effect on complex I activity. This was corroborated in vivo by demonstrating significantly reduced respiratory chain activity in hearts of TERT-deficient mice in comparison to their wildtype littermates. Interestingly the respiration was unaltered in liver mitochondria of TERT-deficient mice as compared to their wildtype littermates. Thus, one could speculate that in regenerative organs, which are rich in mitochondria like the liver, mitochondrial TERT is not as important for respiration as in post-mitotic tissues, which depend on mitochondria like the heart. Further studies e.g. in liver-injured animals are needed to support this hypothesis. It was shown in cell culture that the effect of TERT on respiration requires the reverse transcriptase activity of the enzyme (Haendeler et al., 2009). Later on the group around Santos also showed that the absence of TERT has a negative impact on mitochondria supporting a positive role for this protein on mitochondrial functions. In addition, it was demonstrated that mitochondrial TERT works as a TERC-independent reverse transcriptase (Sharma et al., 2012). Furthermore, Indran et al. demonstrated in cancer cells that TERT overexpression reduces the basal cellular ROS levels, which was accompanied by a lower level of cytochrome C release to the cytosol and inhibition of endogenous ROS production in response to oxidative stress (Indran et al., 2011). In line with these findings was the study from Singhapol et al. demonstrating that mitochondrially localized TERT decreases mitochondrial ROS and thereby prevents nuclear DNA damage (Singhapol et al., 2013). The findings reviewed in this chapter support an anti-apoptotic function of nuclear as well as of mitochondrial TERT. These at first glance contradictory results could be explained by the different roles of TERT in the two compartments, namely regulation of gene expression in the nucleus and protection against increased ROS formation in the mitochondria.
2. Non-telomeric functions of TERT 3. TERT transport to nucleus and mitochondria Besides its pivotal role in ensuring telomere maintenance in the nucleus, a large number of studies provided evidence for non-telomeric functions of TERT. Amongst other things, TERT was shown to be involved in the regulation of gene expression (Geserick et al., 2006; Park et al., 2009; Smith et al., 2003). In addition, nuclear TERT protects against apoptosis independent of changes in telomere length (Haendeler et al., 2003a; Werner et al., 2011). Furthermore, it is essential for the beneficial effects of physical exercise (Werner et al., 2008, 2009). Surprisingly at that time, a number of years ago TERT was also detected in mitochondria independently by several groups (Ahmed et al., 2008; Haendeler et al., 2009; Santos et al., 2004, 2006). From the textbooks mitochondria are known to produce reduction equivalents in the citric acid cycle and ATP via oxidative phosphorylation. They are also a main source of reactive oxygen species (ROS) produced by complexes I, II and III of the electron transport chain (ETC.). While complexes I and II produce ROS only into the matrix, complex III generates ROS on both sides of the inner mitochondrial membrane (Murphy, 2009; Turrens, 2003). In addition, mitochondria play a crucial role in intrinsic apoptosis pathways and have thus been described as the central
The finding that TERT is present in two different cellular compartments raises the question of how TERT is distributed between these organelles. The passage of larger proteins across the nuclear pore complexes, which allow the passive diffusion of ions, metabolites and macromolecules up to a size of approximately 40 kDa, requires selective transport mechanisms. Therefore, proteins above this exclusion limit possess nuclear localization signals (NLS) of different kinds, which are recognized by specific import machineries (Marfori et al., 2011). Additionally, the traffic of proteins between the nuclear compartment and the cytosol can be controlled by nuclear export signals (NES) (Pemberton and Paschal, 2005). The nuclear-cytoplasmic shuttling of proteins can also be affected by phosphorylation, one of the most common post-translational modifications in the regulation and fine-tuning of many biological processes (Nardozzi et al., 2010). For TERT it has recently been shown that its nuclear import requires a so-called bipartite NLS and phosphorylation on a serine residue in this NLS by protein kinase B/Akt (Chung et al., 2012). This is in accordance with older studies demonstrating Akt phosphorylation of a synthetic peptide
Please cite this article as: Ale-Agha, N., et al., Cellular functions of the dual-targeted catalytic subunit of telomerase, telomerase reverse transcriptase — Potential role in senescence and aging, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.011
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encompassing this region and increased telomerase activity after phosphorylation, although no distinction between subcellular compartments was made in this study (Kang et al., 1999). Besides this NLS, TERT also contains a NES-like motif at its C-terminus, which can interact with the nuclear export receptor CRM1/exportin 1. The association with CRM1 and thus the nuclear export of TERT is prevented by binding of 14-3-3 proteins to a region in TERT downstream of its NES (Seimiya et al., 2000). This is in line with our findings that TERT interacts with CRM1/exportin 1 and that the export requires an active nuclear Ran GTPase (Haendeler et al., 2003a). Moreover, it has been reported that mutation of this NES-like motif led to the induction of premature senescence probably due to the inability of the protein to shuttle between the nucleus and the cytoplasm (Kovalenko et al., 2009). However, it is unclear whether this mutated TERT has lost other important properties independent of its catalytic activity. In summary, multiple control levels exist to regulate the nuclear availability of TERT. As the mitochondrial DNA codes for only 13 subunits of the ETC in humans, all other proteins present in the mitochondria must be encoded by nuclear genes and translated in the cytoplasm. Therefore, they have to be imported into these organelles, which, like the nuclear import require highly specialized transport systems that also determine to which part of the mitochondria a protein is delivered. Again, these transporters recognize specific motifs in their cargo molecules (Chacinska et al., 2002; Dudek et al., 2013). A genuine N-terminal mitochondrial targeting sequence (MTS) has been identified in TERT (Haendeler et al., 2009; Santos et al., 2004). Moreover, we have demonstrated that TERT to a large extent is found in the mitochondrial matrix and interacts with the mitochondrial translocases of outer membrane 20 and 40 and the translocase of inner membrane 23 thereby providing insights not only into its submitochondrial localization but also the transport mechanism (Haendeler et al., 2009). It is well documented for eukaryotic cells that proteins derived from a single gene can be localized to several organelles like the nucleus and mitochondria. This has been clearly demonstrated for a number of proteins from different functional classes including transcription factors and kinases and phosphatases as well as TERT. Interestingly, many proteins with dual localization in the nucleus and the mitochondria, including TERT, feature a new common denominator, regulation of respiratory chain activity (Büchner et al., 2010a). To achieve such a dual localization, different protein isoforms can be generated from alternative transcription initiation sites, the use of alternative initiation codons or differential splicing. However, there is increasing evidence, that a single translation product can be distributed between different organelles. The localization can then be defined by different relative affinities to the highly specialized transport complexes, steric configuration of the targeting signals, interaction with other proteins or post-translational modifications as well as redistribution via active export (Karniely and Pines, 2005; Yogev and Pines, 2011). For TERT, it is well described that phosphorylation can have an impact on its localization, however, the sequence of events is not fully uncovered yet. 4. Regulation of nuclear and mitochondrial TERT by phosphorylation As described above, TERT is regulated on the transcriptional level, but also by phosphorylation. The first report about this post-translational regulation demonstrated that phosphorylation of TERT by protein kinase C alpha represents an essential step in the generation of a functional telomerase in cancer cells (Li et al., 1998). Furthermore, it had been shown early on that Akt can phosphorylate TERT in melanoma cells thereby increasing its activity. Using synthetic peptides, the two serines in positions 227 and 823 were identified as the relevant target sites (Kang et al., 1999). The first observation of TERT phosphorylation in non-tumorous cells was made in vascular smooth muscle cells where this modification was linked to proliferation. Interestingly, treatment with the kinase inhibitor H7 reduced the accumulation of TERT as
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well as telomerase activity in the nucleus giving a first hint about phosphorylation-dependent distribution of TERT within cells (Minamino and Kourembanas, 2001). Similarly, TERT phosphorylation also occurred under hypoxic conditions in the same cell type and was correlated with prolonged cellular proliferation (Minamino et al., 2001). Shortly thereafter it was demonstrated that TERT interacts with the heat shock protein 90 (HSP90) and Akt in endothelial cells and it was suggested that this association in concert with the phosphorylation of TERT is necessary not only for maintaining telomerase activity, but also for apoptosis inhibition by TERT (Haendeler et al., 2003b). The short-term effects observed in this study suggest that this Aktdependent anti-apoptotic activity of TERT is not connected to telomere elongation. A protection against apoptosis by TERT independent of telomerase activity was later confirmed in lymphoma and colon carcinoma cells (Rahman et al., 2005). Loss of telomerase activity was also demonstrated in highly differentiated CD8(+)CD28(−)CD27(−) T cells. This loss in activation was not due to reduced TERT expression, but due to decreased Akt activity (Plunkett et al., 2007). In contrast to the protective effects of TERT modified by Akt, a serine/ threonine kinase, tyrosine phosphorylation of TERT leads to reduced telomerase activity in the nucleus. In HEK293 cells subjected to exogenous or endogenous oxidative stress for a few hours TERT is exported out of the nucleus. This export is preceded by phosphorylation on tyrosine 707 and prevented by the Src kinase family inhibitor PP2. Moreover, it was shown that inhibition of tyrosine phosphorylation at 707 increases the anti-apoptotic capacity of TERT, which led to the conclusion that this Src kinase family mediated tyrosine phosphorylation regulates TERT localization and thereby protection against apoptosis (Haendeler et al., 2003a). This nuclear export was not only observed under short-term oxidative stress, but also upon continuous cultivation of endothelial cells for more than 30 population doublings, which resulted in an increase in Src kinase activity and intracellular ROS levels accompanied by mtDNA damage, finally resulting in cellular senescence. This onset of cellular senescence could be delayed by incubation with antioxidants, which blocked the nuclear export of TERT suggesting that nuclear TERT plays a role in senescence induction (Haendeler et al., 2004). All these observations implicate that regulators of TERT phosphorylation are present in the nucleus. Nuclear localization of Akt has been demonstrated in various cellular systems (Martelli et al., 2012) including endothelial cells, where the aforementioned complex formation between Akt, HPS90 and TERT has been shown (Haendeler et al., 2003b). With respect to tyrosine phosphorylation-mediated nuclear export of TERT we have demonstrated the presence of Src family kinases in the nucleus (Jakob et al., 2008). A counterplayer of TERT export is the tyrosine phosphatase Shp-2. This phosphatase was also shown to be localized in the nucleus and is able to prevent phosphorylation of TERT on tyrosine 707 thereby retaining TERT in the nucleus (Jakob et al., 2008) (Fig. 1). After the surprising finding that TERT is present in mitochondria the analysis of its regulation in these organelles revealed striking parallels to the nucleus. Recently we could show that the levels of mitochondrial TERT in endothelial cells are decreased by oxidative stress, most likely by degradation, as so far no export mechanism for complete proteins from the mitochondria is known. This downregulation also critically depends on tyrosine 707 in TERT, as it was prevented by the substitution of this residue with phenylalanine. In the same study we demonstrated the presence of Akt and Src in the mitochondria and showed that Akt activation is decreased by oxidative stress, while Src activity is upregulated (Büchner et al., 2010b) (Fig. 2). In analogy, preliminary data from our laboratory suggest that Shp-2 is also present within the mitochondria (Jakob, unpublished results). This is not a specific feature of endothelial cells as Src, as well as Shp-2 have also been detected in rat brain mitochondria (Salvi et al., 2002, 2004). Interestingly, under oxidative stress, the levels of mitochondrial TERT in fibroblasts are increased with a concomitant nuclear exclusion of the enzyme (Ahmed et al., 2008). This could have two implications,
Please cite this article as: Ale-Agha, N., et al., Cellular functions of the dual-targeted catalytic subunit of telomerase, telomerase reverse transcriptase — Potential role in senescence and aging, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.011
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Fig. 1. Regulation of nuclear TERT by the Src kinase and the phosphatase Shp-2. Increased reactive oxygen species (ROS) levels activate Src kinase, which in turn phosphorylates TERT on tyrosine (Y) leading to nuclear export of TERT. The phosphatase Shp-2 counteracts this export either by dephosphorylating and thus inactivating Src or by directly dephosphorylating TERT.
either that the protein exported from the nucleus is imported into the mitochondria or that newly synthesized molecules are directed exclusively to mitochondria, possibly to protect them from damage. 5. Conclusions The role for TERT in telomere maintenance is a long known, welldescribed phenomenon. Interestingly, a large number of studies have shown that TERT clearly has non-telomeric functions. On one hand it can affect gene expression, which supposedly must be a nuclear effect. Furthermore, TERT protects cells against apoptosis in a telomere independent-fashion and this protective effect applies to nuclear as well as mitochondrial TERT. TERT in the mitochondria has additional functions, e.g. to increase the mitochondrial membrane potential,
reduce ROS production and to protect mtDNA. Currently, it is extremely difficult to clearly differentiate between overlapping functions of TERT in the nucleus and the mitochondria, because all previous studies were either done in completely TERT-deficient cells and mice or after expressing TERT, mutants of this protein or variants artificially targeted to one of the organelles in cells, which also express the endogenous protein. We have now set out to provide tools to solve this problem by creating mice ubiquitously expressing low levels of nuclear or mitochondrially targeted TERT. After backcrossing these animals onto a TERT-deficient background the offspring will contain TERT only in one of the two subcellular compartments throughout the body and therefore allow the dissection between nuclear and mitochondrial functions of TERT. Another open question concerns the distribution of TERT between the nucleus and the mitochondria. From the data available it seems that in terminally differentiated, post-mitotic cells nuclear TERT is less important than mitochondrial TERT and that therefore, under oxidative stress, TERT levels in the mitochondria are increased to protect these organelles from damage. Based on the concomitant decrease in nuclear TERT it is tempting to speculate that the protein found in the mitochondria was directly exported from the nucleus and imported into the mitochondria. However, under extreme stress, either too high or persistent, the mitochondrial pool of TERT is degraded leading to cell death. Since mitochondrial TERT seems to have an important role in various intracellular processes, the non-telomeric functions of TERT could be as relevant as the telomeric functions in aging and diseases. As these nontelomeric functions have only recently been unraveled, the sole focus on telomere length led to the proposal that telomerase loss and thus telomere shortening could serve as a biomarker of aging. However this view might change when the non-telomeric functions of TERT and their role in senescence and aging are characterized in more detail in the future. Conflict of interest The authors have no conflicts of interests. Acknowledgments This work was in part supported by the Deutsche Forschungsgemeinschaft to J.H. (HA2868/6-1 and HA2868/9-1) and to J.A. (AL288/2-1) and by the Leducq Transatlantic Network of Excellence 09 CVD_01 to J.H. The authors have nothing to disclose. References
Fig. 2. Regulation of TERT in the mitochondria. Increased reactive oxygen species (ROS) levels activate mitochondrial Src kinase and inactivate Akt. The Src-mediated phosphorylation of TERT induces its degradation. Shp-2 most likely prevents TERT phosphorylation analogously to the nucleus.
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Please cite this article as: Ale-Agha, N., et al., Cellular functions of the dual-targeted catalytic subunit of telomerase, telomerase reverse transcriptase — Potential role in senescence and aging, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.011
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Review
Cellular functions of the dual-targeted catalytic subunit of telomerase, telomerase reverse transcriptase — Potential role in senescence and aging Niloofar Ale-Agha b,1, Nadine Dyballa-Rukes b,1, Sascha Jakob b, Joachim Altschmied b, Judith Haendeler a,b,⁎ a b
Central Institute of Clinical Chemistry and Laboratory Medicine, University of Duesseldorf, Germany IUF — Leibniz Research Institute for Environmental Medicine at the University of Duesseldorf gGmbH, 40225 Duesseldorf, Germany
a r t i c l e
i n f o
Article history: Received 15 December 2013 Received in revised form 17 February 2014 Accepted 20 February 2014 Available online xxxx Keywords: Aging Mitochondria Nucleus Senescence Telomerase reverse transcriptase
a b s t r a c t Over the last 40 years it has become clear that telomeres, the end of the chromosomes, and the enzyme telomerase reverse transcriptase (TERT), which is required to counteract their shortening, play a pivotal role in senescence and aging. However, over the last years several studies demonstrated that TERT belongs to the group of dual-targeted proteins. It contains a bipartite nuclear localization signal as well as a mitochondrial targeting sequence and, under physiological conditions, is found in both organelles in several cell types including terminally differentiated, post-mitotic cells. The canonical function of TERT is to prevent telomere erosion and thereby the development of replicative senescence and genetic instability. Besides telomere extension, TERT exhibits other non-telomeric activities such as cell cycle regulation, modulation of cellular signaling and gene expression, augmentation of proliferative lifespan as well as DNA damage responses. Mitochondrial TERT is able to reduce reactive oxygen species, mitochondrial DNA damage and apoptosis. Because of the localization of TERT in the nucleus and in the mitochondria, it must have different functions in the two organelles as mitochondrial DNA does not contain telomeric structures. However, the organelle-specific functions are not completely understood. Strikingly, the regulation by phosphorylation of TERT seems to reveal multiple parallels. This review will summarize the current knowledge about the cellular functions and post-translational regulation of the dual-targeted protein TERT. © 2014 Elsevier Inc. All rights reserved.
1. Introduction: telomeres and telomerase Already in the first half of the last century Hermann Muller and Barbara McClintock described the instability of broken chromosomes in species as distantly related as Drosophila and maize (McClintock, 1941; Muller, 1938). However, in their studies they never found structural changes involving the end of the chromosomes, which led Muller to the conclusion that this terminal region (in his words “terminal gene”) must have a special function in sealing the chromosome. To describe the uniqueness of these regions and to set them apart from the Abbreviations: TERT, telomerase reverse transcriptase; TERC, non-coding telomerase RNA component; ROS, reactive oxygen species; ETC, electron transport chain; mtDNA, mitochondrial DNA; NLS, nuclear localization signal; NES, nuclear export signal; MTS, mitochondrial targeting sequence. ⁎ Corresponding author at: Central Institute of Clinical Chemistry and Laboratory Medicine, University of Duesseldorf, and, IUF — Leibniz Research Institute for Environmental Medicine, Auf'm Hennekamp 50, 40225 Duesseldorf, Germany. Tel.: +49 211 3389 291; fax: +49 211 3389 331. E-mail addresses: [email protected] (N. Ale-Agha), [email protected] (N. Dyballa-Rukes), [email protected] (S. Jakob), [email protected] (J. Altschmied), [email protected] (J. Haendeler). 1 Both authors contributed equally to the work.
remainder of the chromosomes, he termed them telomeres combining the Greek words for end (telo) and part (meros). Almost 40 years later Joseph Gall and Elizabeth Blackburn described the presence of a tandem hexanucleotide repeat sequence CCCCAA at the end of extrachromosomal rDNA molecules in Tetrahymena (Blackburn and Gall, 1978). After it was shown by Elizabeth Blackburn and Jack Szostak that these were functionally equivalent to the ends of yeast chromosomes, they concluded that such repeated end sequences correspond to functional telomeres (Szostak and Blackburn, 1982). Nowadays, it is common knowledge that telomeres consist of tandem repeats of the hexanucleotide sequence TTAGGG (in mammals), adopt a higher order structure and are capped with a large number of single- and doublestrand DNA binding proteins (Blasco, 2005; de Lange, 2005; Pinto et al., 2011). A connection between telomeres, senescence and aging was derived from several independent lines of evidence. In 1965 Leonard Hayflick demonstrated that isolated fibroblasts show only a limited proliferative potential in culture and concluded that this finite lifetime of cells in vitro may be an expression of aging or senescence at the cellular level (Hayflick, 1965). A molecular basis and a link to telomeres were proposed by Alexey Olovnikov after he and James Watson had independently recognized the problem that during DNA replication, a small
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Please cite this article as: Ale-Agha, N., et al., Cellular functions of the dual-targeted catalytic subunit of telomerase, telomerase reverse transcriptase — Potential role in senescence and aging, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.011
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part of DNA from the 3′-ends of linear chromosomes is unreplicated. Olovnikov explained Hayflick's theory of limited somatic cell division suggesting that this now called end-replication problem leads to loss of telomeric sequences, which he called “telogenes”, during successive mitoses finally resulting in cellular senescence and elimination of such cells (Olovnikov, 1973). However, up to now it is not clear whether telomere length could be used as a biomarker for aging because the findings in humans are contradictory as reviewed by Mather et al. (2011). Again Elizabeth Blackburn, together with her graduate student Carol Greider, discovered that telomere shortening is counteracted by the activity of a cellular ribonucleoprotein complex called telomerase, which they identified in Tetrahymena (Greider and Blackburn, 1985; Greider and Blackburn, 1987). The presence of a similar activity in human cells was demonstrated shortly thereafter establishing that telomerasemediated telomere maintenance is conserved throughout eukaryotes (Morin, 1989). The telomerase holoenzyme requires the catalytic proteinaceous subunit telomerase reverse transcriptase (TERT) and, besides multiple telomerase associated proteins, the non-coding telomerase RNA component (TERC), which serves as the template for the elongation of the telomere repeat sequences by TERT (Blasco, 2005). If telomerase is absent, telomeres shorten during each round of cell division until they reach a critically short-length threshold (Harley et al., 1990). This causes a DNA damage response and induces cell cycle arrest triggering senescence (Herbig et al., 2004). Along the same lines, the introduction of TERT into cells prolongs both their lifespan and their telomeres to lengths typical of young cells and reduces signs of senescence (Bodnar et al., 1998; Counter et al., 1998). These findings also suggest that permanent, high-level expression of TERT can lead to uncontrolled proliferation, which is reflected by the fact that many tumors exhibit reactivation of telomerase (Blasco, 2005). Therefore, telomerase activity must be tightly regulated, which on one hand is achieved by transcriptional regulation of the TERT gene, and on the other hand by controlling TERT activity on the posttranslational level (Aisner et al., 2002; Cong et al., 2002; Ducrest et al., 2002). It has been proposed for a long time that telomerase activity is absent from most human somatic cells and only present in tumor cells and adult stem cells of highly regenerative tissues, such as the immune system, skin and intestine (Forsyth et al., 2002). However, there is accumulating evidence that substantial telomerase activity is present in differentiated somatic cells, e.g. endothelial cells, smooth muscle cells and hepatocytes (Haendeler et al., 2004; Leri et al., 2000; Minamino and Kourembanas, 2001; Vasa et al., 2000; Werner et al., 2009, 2011; Yamaguchi et al., 1998).
executioner of apoptosis (Estaquier et al., 2012). Newer proteomic studies have revealed the presence of several hundred to more than 1000 proteins in these organelles, depending on the cell type and physiological situation (Balaban, 2010; Pagliarini et al., 2008) suggesting that mitochondria serve much broader functions than originally anticipated. When the functions of mitochondrial TERT were assessed, the first descriptions reported that it renders cells more susceptible to oxidative stress-induced mitochondrial DNA (mtDNA) damage, which can lead to apoptotic cell death (Santos et al., 2004, 2006). In contrast to these findings, we and others described a protective function for mitochondrial TERT by increasing the membrane potential, reducing reactive oxygen production, protecting mtDNA against damage and inhibiting apoptosis induction (Ahmed et al., 2008; Haendeler et al., 2009). Interestingly, TERT has also an impact on the ETC., as overexpression of TERT enhances overall respiratory chain activity in HEK 293 cells, with the most pronounced effect on complex I activity. This was corroborated in vivo by demonstrating significantly reduced respiratory chain activity in hearts of TERT-deficient mice in comparison to their wildtype littermates. Interestingly the respiration was unaltered in liver mitochondria of TERT-deficient mice as compared to their wildtype littermates. Thus, one could speculate that in regenerative organs, which are rich in mitochondria like the liver, mitochondrial TERT is not as important for respiration as in post-mitotic tissues, which depend on mitochondria like the heart. Further studies e.g. in liver-injured animals are needed to support this hypothesis. It was shown in cell culture that the effect of TERT on respiration requires the reverse transcriptase activity of the enzyme (Haendeler et al., 2009). Later on the group around Santos also showed that the absence of TERT has a negative impact on mitochondria supporting a positive role for this protein on mitochondrial functions. In addition, it was demonstrated that mitochondrial TERT works as a TERC-independent reverse transcriptase (Sharma et al., 2012). Furthermore, Indran et al. demonstrated in cancer cells that TERT overexpression reduces the basal cellular ROS levels, which was accompanied by a lower level of cytochrome C release to the cytosol and inhibition of endogenous ROS production in response to oxidative stress (Indran et al., 2011). In line with these findings was the study from Singhapol et al. demonstrating that mitochondrially localized TERT decreases mitochondrial ROS and thereby prevents nuclear DNA damage (Singhapol et al., 2013). The findings reviewed in this chapter support an anti-apoptotic function of nuclear as well as of mitochondrial TERT. These at first glance contradictory results could be explained by the different roles of TERT in the two compartments, namely regulation of gene expression in the nucleus and protection against increased ROS formation in the mitochondria.
2. Non-telomeric functions of TERT 3. TERT transport to nucleus and mitochondria Besides its pivotal role in ensuring telomere maintenance in the nucleus, a large number of studies provided evidence for non-telomeric functions of TERT. Amongst other things, TERT was shown to be involved in the regulation of gene expression (Geserick et al., 2006; Park et al., 2009; Smith et al., 2003). In addition, nuclear TERT protects against apoptosis independent of changes in telomere length (Haendeler et al., 2003a; Werner et al., 2011). Furthermore, it is essential for the beneficial effects of physical exercise (Werner et al., 2008, 2009). Surprisingly at that time, a number of years ago TERT was also detected in mitochondria independently by several groups (Ahmed et al., 2008; Haendeler et al., 2009; Santos et al., 2004, 2006). From the textbooks mitochondria are known to produce reduction equivalents in the citric acid cycle and ATP via oxidative phosphorylation. They are also a main source of reactive oxygen species (ROS) produced by complexes I, II and III of the electron transport chain (ETC.). While complexes I and II produce ROS only into the matrix, complex III generates ROS on both sides of the inner mitochondrial membrane (Murphy, 2009; Turrens, 2003). In addition, mitochondria play a crucial role in intrinsic apoptosis pathways and have thus been described as the central
The finding that TERT is present in two different cellular compartments raises the question of how TERT is distributed between these organelles. The passage of larger proteins across the nuclear pore complexes, which allow the passive diffusion of ions, metabolites and macromolecules up to a size of approximately 40 kDa, requires selective transport mechanisms. Therefore, proteins above this exclusion limit possess nuclear localization signals (NLS) of different kinds, which are recognized by specific import machineries (Marfori et al., 2011). Additionally, the traffic of proteins between the nuclear compartment and the cytosol can be controlled by nuclear export signals (NES) (Pemberton and Paschal, 2005). The nuclear-cytoplasmic shuttling of proteins can also be affected by phosphorylation, one of the most common post-translational modifications in the regulation and fine-tuning of many biological processes (Nardozzi et al., 2010). For TERT it has recently been shown that its nuclear import requires a so-called bipartite NLS and phosphorylation on a serine residue in this NLS by protein kinase B/Akt (Chung et al., 2012). This is in accordance with older studies demonstrating Akt phosphorylation of a synthetic peptide
Please cite this article as: Ale-Agha, N., et al., Cellular functions of the dual-targeted catalytic subunit of telomerase, telomerase reverse transcriptase — Potential role in senescence and aging, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.011
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encompassing this region and increased telomerase activity after phosphorylation, although no distinction between subcellular compartments was made in this study (Kang et al., 1999). Besides this NLS, TERT also contains a NES-like motif at its C-terminus, which can interact with the nuclear export receptor CRM1/exportin 1. The association with CRM1 and thus the nuclear export of TERT is prevented by binding of 14-3-3 proteins to a region in TERT downstream of its NES (Seimiya et al., 2000). This is in line with our findings that TERT interacts with CRM1/exportin 1 and that the export requires an active nuclear Ran GTPase (Haendeler et al., 2003a). Moreover, it has been reported that mutation of this NES-like motif led to the induction of premature senescence probably due to the inability of the protein to shuttle between the nucleus and the cytoplasm (Kovalenko et al., 2009). However, it is unclear whether this mutated TERT has lost other important properties independent of its catalytic activity. In summary, multiple control levels exist to regulate the nuclear availability of TERT. As the mitochondrial DNA codes for only 13 subunits of the ETC in humans, all other proteins present in the mitochondria must be encoded by nuclear genes and translated in the cytoplasm. Therefore, they have to be imported into these organelles, which, like the nuclear import require highly specialized transport systems that also determine to which part of the mitochondria a protein is delivered. Again, these transporters recognize specific motifs in their cargo molecules (Chacinska et al., 2002; Dudek et al., 2013). A genuine N-terminal mitochondrial targeting sequence (MTS) has been identified in TERT (Haendeler et al., 2009; Santos et al., 2004). Moreover, we have demonstrated that TERT to a large extent is found in the mitochondrial matrix and interacts with the mitochondrial translocases of outer membrane 20 and 40 and the translocase of inner membrane 23 thereby providing insights not only into its submitochondrial localization but also the transport mechanism (Haendeler et al., 2009). It is well documented for eukaryotic cells that proteins derived from a single gene can be localized to several organelles like the nucleus and mitochondria. This has been clearly demonstrated for a number of proteins from different functional classes including transcription factors and kinases and phosphatases as well as TERT. Interestingly, many proteins with dual localization in the nucleus and the mitochondria, including TERT, feature a new common denominator, regulation of respiratory chain activity (Büchner et al., 2010a). To achieve such a dual localization, different protein isoforms can be generated from alternative transcription initiation sites, the use of alternative initiation codons or differential splicing. However, there is increasing evidence, that a single translation product can be distributed between different organelles. The localization can then be defined by different relative affinities to the highly specialized transport complexes, steric configuration of the targeting signals, interaction with other proteins or post-translational modifications as well as redistribution via active export (Karniely and Pines, 2005; Yogev and Pines, 2011). For TERT, it is well described that phosphorylation can have an impact on its localization, however, the sequence of events is not fully uncovered yet. 4. Regulation of nuclear and mitochondrial TERT by phosphorylation As described above, TERT is regulated on the transcriptional level, but also by phosphorylation. The first report about this post-translational regulation demonstrated that phosphorylation of TERT by protein kinase C alpha represents an essential step in the generation of a functional telomerase in cancer cells (Li et al., 1998). Furthermore, it had been shown early on that Akt can phosphorylate TERT in melanoma cells thereby increasing its activity. Using synthetic peptides, the two serines in positions 227 and 823 were identified as the relevant target sites (Kang et al., 1999). The first observation of TERT phosphorylation in non-tumorous cells was made in vascular smooth muscle cells where this modification was linked to proliferation. Interestingly, treatment with the kinase inhibitor H7 reduced the accumulation of TERT as
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well as telomerase activity in the nucleus giving a first hint about phosphorylation-dependent distribution of TERT within cells (Minamino and Kourembanas, 2001). Similarly, TERT phosphorylation also occurred under hypoxic conditions in the same cell type and was correlated with prolonged cellular proliferation (Minamino et al., 2001). Shortly thereafter it was demonstrated that TERT interacts with the heat shock protein 90 (HSP90) and Akt in endothelial cells and it was suggested that this association in concert with the phosphorylation of TERT is necessary not only for maintaining telomerase activity, but also for apoptosis inhibition by TERT (Haendeler et al., 2003b). The short-term effects observed in this study suggest that this Aktdependent anti-apoptotic activity of TERT is not connected to telomere elongation. A protection against apoptosis by TERT independent of telomerase activity was later confirmed in lymphoma and colon carcinoma cells (Rahman et al., 2005). Loss of telomerase activity was also demonstrated in highly differentiated CD8(+)CD28(−)CD27(−) T cells. This loss in activation was not due to reduced TERT expression, but due to decreased Akt activity (Plunkett et al., 2007). In contrast to the protective effects of TERT modified by Akt, a serine/ threonine kinase, tyrosine phosphorylation of TERT leads to reduced telomerase activity in the nucleus. In HEK293 cells subjected to exogenous or endogenous oxidative stress for a few hours TERT is exported out of the nucleus. This export is preceded by phosphorylation on tyrosine 707 and prevented by the Src kinase family inhibitor PP2. Moreover, it was shown that inhibition of tyrosine phosphorylation at 707 increases the anti-apoptotic capacity of TERT, which led to the conclusion that this Src kinase family mediated tyrosine phosphorylation regulates TERT localization and thereby protection against apoptosis (Haendeler et al., 2003a). This nuclear export was not only observed under short-term oxidative stress, but also upon continuous cultivation of endothelial cells for more than 30 population doublings, which resulted in an increase in Src kinase activity and intracellular ROS levels accompanied by mtDNA damage, finally resulting in cellular senescence. This onset of cellular senescence could be delayed by incubation with antioxidants, which blocked the nuclear export of TERT suggesting that nuclear TERT plays a role in senescence induction (Haendeler et al., 2004). All these observations implicate that regulators of TERT phosphorylation are present in the nucleus. Nuclear localization of Akt has been demonstrated in various cellular systems (Martelli et al., 2012) including endothelial cells, where the aforementioned complex formation between Akt, HPS90 and TERT has been shown (Haendeler et al., 2003b). With respect to tyrosine phosphorylation-mediated nuclear export of TERT we have demonstrated the presence of Src family kinases in the nucleus (Jakob et al., 2008). A counterplayer of TERT export is the tyrosine phosphatase Shp-2. This phosphatase was also shown to be localized in the nucleus and is able to prevent phosphorylation of TERT on tyrosine 707 thereby retaining TERT in the nucleus (Jakob et al., 2008) (Fig. 1). After the surprising finding that TERT is present in mitochondria the analysis of its regulation in these organelles revealed striking parallels to the nucleus. Recently we could show that the levels of mitochondrial TERT in endothelial cells are decreased by oxidative stress, most likely by degradation, as so far no export mechanism for complete proteins from the mitochondria is known. This downregulation also critically depends on tyrosine 707 in TERT, as it was prevented by the substitution of this residue with phenylalanine. In the same study we demonstrated the presence of Akt and Src in the mitochondria and showed that Akt activation is decreased by oxidative stress, while Src activity is upregulated (Büchner et al., 2010b) (Fig. 2). In analogy, preliminary data from our laboratory suggest that Shp-2 is also present within the mitochondria (Jakob, unpublished results). This is not a specific feature of endothelial cells as Src, as well as Shp-2 have also been detected in rat brain mitochondria (Salvi et al., 2002, 2004). Interestingly, under oxidative stress, the levels of mitochondrial TERT in fibroblasts are increased with a concomitant nuclear exclusion of the enzyme (Ahmed et al., 2008). This could have two implications,
Please cite this article as: Ale-Agha, N., et al., Cellular functions of the dual-targeted catalytic subunit of telomerase, telomerase reverse transcriptase — Potential role in senescence and aging, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.011
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Fig. 1. Regulation of nuclear TERT by the Src kinase and the phosphatase Shp-2. Increased reactive oxygen species (ROS) levels activate Src kinase, which in turn phosphorylates TERT on tyrosine (Y) leading to nuclear export of TERT. The phosphatase Shp-2 counteracts this export either by dephosphorylating and thus inactivating Src or by directly dephosphorylating TERT.
either that the protein exported from the nucleus is imported into the mitochondria or that newly synthesized molecules are directed exclusively to mitochondria, possibly to protect them from damage. 5. Conclusions The role for TERT in telomere maintenance is a long known, welldescribed phenomenon. Interestingly, a large number of studies have shown that TERT clearly has non-telomeric functions. On one hand it can affect gene expression, which supposedly must be a nuclear effect. Furthermore, TERT protects cells against apoptosis in a telomere independent-fashion and this protective effect applies to nuclear as well as mitochondrial TERT. TERT in the mitochondria has additional functions, e.g. to increase the mitochondrial membrane potential,
reduce ROS production and to protect mtDNA. Currently, it is extremely difficult to clearly differentiate between overlapping functions of TERT in the nucleus and the mitochondria, because all previous studies were either done in completely TERT-deficient cells and mice or after expressing TERT, mutants of this protein or variants artificially targeted to one of the organelles in cells, which also express the endogenous protein. We have now set out to provide tools to solve this problem by creating mice ubiquitously expressing low levels of nuclear or mitochondrially targeted TERT. After backcrossing these animals onto a TERT-deficient background the offspring will contain TERT only in one of the two subcellular compartments throughout the body and therefore allow the dissection between nuclear and mitochondrial functions of TERT. Another open question concerns the distribution of TERT between the nucleus and the mitochondria. From the data available it seems that in terminally differentiated, post-mitotic cells nuclear TERT is less important than mitochondrial TERT and that therefore, under oxidative stress, TERT levels in the mitochondria are increased to protect these organelles from damage. Based on the concomitant decrease in nuclear TERT it is tempting to speculate that the protein found in the mitochondria was directly exported from the nucleus and imported into the mitochondria. However, under extreme stress, either too high or persistent, the mitochondrial pool of TERT is degraded leading to cell death. Since mitochondrial TERT seems to have an important role in various intracellular processes, the non-telomeric functions of TERT could be as relevant as the telomeric functions in aging and diseases. As these nontelomeric functions have only recently been unraveled, the sole focus on telomere length led to the proposal that telomerase loss and thus telomere shortening could serve as a biomarker of aging. However this view might change when the non-telomeric functions of TERT and their role in senescence and aging are characterized in more detail in the future. Conflict of interest The authors have no conflicts of interests. Acknowledgments This work was in part supported by the Deutsche Forschungsgemeinschaft to J.H. (HA2868/6-1 and HA2868/9-1) and to J.A. (AL288/2-1) and by the Leducq Transatlantic Network of Excellence 09 CVD_01 to J.H. The authors have nothing to disclose. References
Fig. 2. Regulation of TERT in the mitochondria. Increased reactive oxygen species (ROS) levels activate mitochondrial Src kinase and inactivate Akt. The Src-mediated phosphorylation of TERT induces its degradation. Shp-2 most likely prevents TERT phosphorylation analogously to the nucleus.
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Please cite this article as: Ale-Agha, N., et al., Cellular functions of the dual-targeted catalytic subunit of telomerase, telomerase reverse transcriptase — Potential role in senescence and aging, Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.02.011