Joachim Altschmied

Mitochondrial Telomerase Reverse Transcriptase Binds to and Protects Mitochondrial DNA and Function From Damage

Objective—The enzyme telomerase and its catalytic subunit the telomerase reverse transcriptase (TERT) are important for maintenance of telomere length in the nucleus. Recent studies provided evidence for a mitochondrial localization of TERT. Therefore, we investigated the exact localization of TERT within the mitochondria and its function. Methods and Results—Here, we demonstrate that TERT is localized in the matrix of the mitochondria. TERT binds to mitochondrial DNA at the coding regions for ND1 and ND2. Binding of TERT to mitochondrial DNA protects against ethidium bromide–induced damage. TERT increases overall respiratory chain activity, which is most pronounced at complex I and dependent on the reverse transcriptase activity of the enzyme. Moreover, mitochondrial reactive oxygen species are increased after genetic ablation of TERT by shRNA. Mitochondrially targeted TERT and not wild-type TERT revealed the most prominent protective effect on H2O2-induced apoptosis. Lung fibroblasts from 6-month-old TERT−/− mice (F2 generation) showed increased sensitivity toward UVB radiation and heart mitochondria exhibited significantly reduced respiratory chain activity already under basal conditions, demonstrating the protective function of TERT in vivo. Conclusion—Mitochondrial TERT exerts a novel protective function by binding to mitochondrial DNA, increasing respiratory chain activity and protecting against oxidative stress–induced damage.

Nuclear Redox-Signaling Is Essential for Apoptosis Inhibition in Endothelial Cells—Important Role for Nuclear Thioredoxin-1

Objective—The redox regulator thioredoxin-1 (Trx) is a potent antioxidative enzyme and exerts important cellular functions. Physiological concentrations of reactive oxygen species (ROS) and of nitric oxide (NO) act as second messengers. Previously, we demonstrated that ROS and NO reduced apoptosis in a Trx-dependent manner. The aim of this study was to determine the underlying mechanisms. Methods and Results—First, we investigated the localization of Trx after H2O2 and NO. Both induced nuclear import of Trx, which required karyopherin-&agr;. siRNA against karyopherin-&agr; inhibited nuclear import of Trx. Analysis of the Trx amino acid sequence and subsequent immunoprecipitation studies revealed that Trx(K81/82E) is not imported into the nucleus under H2O2 treatment and Trx(K81/82/85E) was retained in the cytosol and induced cell death. Trx(K81/82E) abolished the antiapoptotic capacity of H2O2. Glutathione S-transferase P1 (GST-P1) was identified as one major target regulated by H2O2. siRNA against GST-P1 abolished the antiapoptotic effect of H2O2. Cysteine 69, but not cysteines 32 and 35, which are all required for the complete antiapoptotic function of Trx, is not imported into the nucleus. Conclusion—H2O2-induced nuclear import of Trx depends on karyopherin-&agr; and NO. Trx-dependent induction of GST-P1 expression is required for apoptosis inhibition in endothelial cells.

Nuclear Protein Tyrosine Phosphatase Shp-2 Is One Important Negative Regulator of Nuclear Export of Telomerase Reverse Transcriptase

Aging is one major risk factor for numerous diseases. The enzyme telomerase reverse transcriptase (TERT) plays an important role for aging and apoptosis. Previously, we demonstrated that inhibition of oxidative stress-induced Src kinase family-dependent nuclear export of TERT results in delayed replicative senescence and reduced apoptosis sensitivity. Therefore, the aim of this study was to investigate mechanisms inhibiting nuclear export of TERT. First, we demonstrated that H2O2-induced nuclear export of TERT was abolished in Src, Fyn, and Yes-deficient embryonic fibroblasts. Next, we wanted to identify one potential negative regulator of this export process. One candidate is the protein tyrosine phosphatase Shp-2 (Shp-2), which can counteract activities of the Src kinase family. Indeed, Shp-2 was evenly distributed between the nucleus and cytosol. Nuclear Shp-2 associates with TERT in endothelial cells and dissociates from TERT prior to its nuclear export. Overexpression of Shp-2 wt inhibited H2O2-induced export of TERT. Overexpression of the catalytically inactive, dominant negative Shp-2 mutant (Shp-2(C459S)) reduced endogenous as well as overexpressed nuclear TERT protein and telomerase activity, whereas it had no influence on TERT(Y707F). Binding of TERT(Y707F) to Shp-2 is reduced compared with TERTwt. Ablation of Shp-2 expression led only to an increased tyrosine phosphorylation of TERTwt, but not of TERT(Y707F). Moreover, reduced Shp-2 expression decreased nuclear telomerase activity, whereas nuclear telomerase activity was increased in Shp-2-overexpressing endothelial cells. In conclusion, Shp-2 retains TERT in the nucleus by regulating tyrosine 707 phosphorylation.

Nuclear Redox Signaling

Reactive oxygen species have been described to modulate proteins within the cell, a process called redox regulation. However, the importance of compartment-specific redox regulation has been neglected for a long time. In the early 1980s and 1990s, many in vitro studies introduced the possibility that nuclear redox signaling exists. However, the functional relevance for that has been greatly disregarded. Recently, it has become evident that nuclear redox signaling is indeed one important signaling mechanism regulating a variety of cellular functions. Transcription factors, and even kinases and phosphatases, have been described to be redox regulated in the nucleus. This review describes several of these proteins in closer detail and explains their functions resulting from nuclear localization and redox regulation. Moreover, the redox state of the nucleus and several important nuclear redox regulators [Thioredoxin-1 (Trx-1), Glutaredoxins (Grxs), Peroxiredoxins (Prxs), and APEX nuclease (multifunctional DNA-repair enzyme) 1 (APEX1)] are introduced more precisely, and their necessity for regulation of transcription factors is emphasized.

Activation of the Xmrk proto-oncogene of Xiphophorus by overexpression and mutational alterations

Xmrk is a receptor tyrosine kinase closely related to the human EGF receptor. In the teleost fish Xiphophorus two versions of the Xmrk gene exist, an oncogene (ONC) and a proto-oncogene (INV). While ONC-Xmrk is the melanoma-inducing gene, INV-Xmrk appears not to be involved in transformation of pigment cells. To elucidate the mechanism that converts the proto-oncogene into a transforming oncogene a comparative analysis of the structure, expression and function of both versions of the gene was performed. In contrast to ONC-Xmrk which is expressed at high levels in melanoma cells, the proto-oncogene INV-Xmrk is ubiquitously expressed at very low levels indicating overexpression as one possible reason for tumorigenicity by ONC-Xmrk. As sequence comparison of the proto-oncogene and the oncogene revealed a number of amino acid changes, a possible effect of these mutations on the activation of the ONC-Xmrk receptor was determined. A constitutive activation of the oncogenic receptor was found and ectopic expression of INV-Xmrk after microinjection into medakafish embryos did not lead to the high tumour rate in transgenic fish as observed for the oncogene. Our data therefore suggest that overexpression of the receptor alone is not sufficient for melanoma induction, but that in addition activating mutations in ONC-Xmrk are responsible for its full tumorigenic potential.

Downregulation of mitochondrial telomerase reverse transcriptase induced by H2O2 is Src kinase dependent

Telomerase with its catalytic subunit telomerase reverse transcriptase (TERT) prevents telomere erosion in the nucleus. In addition, telomerase has also telomere-independent functions in protection from apoptosis. Unexpectedly, TERT was found in the mitochondria. However, its regulation in this organelle is completely unknown. Here, we demonstrate that mitochondrial TERT is downregulated by exposure to H(2)O(2) in primary human endothelial cells. This depletion is dependent on the Src phosphorylation site within TERT, tyrosine 707. In accordance with this finding, we also detected Src in the mitochondria and demonstrated that Src is activated upon H(2)O(2) treatment. This regulation of mitochondrial TERT is reminiscent of the situation in the nucleus from where TERT is exported under conditions of oxidative stress in a Src kinase dependent manner. In addition, Akt1 was also found in the mitochondria and H(2)O(2) treatment led to reduced active Akt1 in these organelles, suggesting that similar regulatory mechanisms operate in mitochondria and the nucleus.

Oxidative Stress–Induced Degradation of Thioredoxin-1 and Apoptosis Is Inhibited by Thioredoxin-1–Actin Interaction in Endothelial Cells

Objective—Thioredoxin-1 (Trx-1), one important antioxidative enzyme in endothelial cells, is required for apoptosis inhibition. Apoptosis induction is dependent on cytoskeletal changes, which depend on actin rearrangements. Therefore, we wanted to elucidate whether a physical interaction exists between Trx-1 and actin and what the functional consequences are. Methods and Results—Combined immunoprecipitation/mass spectrometry identified actin as a new binding partner for Trx-1. A separate pool of Trx-1 forms a complex with apoptosis signaling kinase 1. Actin is required for stress fiber formation; thus, the interaction of actin with Trx-1 might interfere with this process. Stress fiber formation, which is directly linked to the phosphorylation of focal adhesion kinase (FAK), occurs as early as 1 hour after H2O2 treatment. It is inhibited by Trx-1 overexpression, treatment with exogenous Trx-1, or inhibition of FAK. Prolonged incubation with H2O2 induced stress fiber formation, reduced Trx-1 protein levels, and increased apoptosis. All these processes were inhibited by preincubation with the FAK inhibitor PF573228. On the contrary, incubation with PF573228 1 hour after H2O2 treatment did not block stress fiber formation, degradation of Trx-1, or apoptosis. Conclusion—These data demonstrate that the actin–Trx-1 complex protects Trx-1 from degradation and, thus, endothelial cells from apoptosis. Reciprocally, Trx-1 prevents stress fiber formation.

Interacting with Thioredoxin-1—Disease or No Disease?

SIGNIFICANCE Many cardiovascular disorders are accompanied by a deregulated cellular redox balance resulting in elevated levels of intracellular reactive oxygen species (ROS). One major antioxidative cellular molecule is thioredoxin-1 (Trx-1). Its indispensability is demonstrated by the embryonic lethality of Trx-1 deficient mice. Trx-1 is ubiquitously expressed in cells and has numerous, diverse functions. It not only reduces oxidized proteins or, together with peroxiredoxins, detoxifies H(2)O(2), but also binds to several proteins and thereby regulates their functions. The interaction partners of Trx-1 differ depending on its localization in the cytosol or in the nucleus. RECENT ADVANCES/CRITICAL ISSUES Over the past decade it has become clear that Trx-1 is not only critical for tumor functions, which has resulted in therapeutic approaches targeting this protein, but also essential for proper functions of the vasculature and the heart. Changes in post-translational modifications of Trx-1 or in its interactions with other proteins can lead to a switch from a physiologic state of cells and organs to diverse pathologies. This review provides insights into the role of Trx-1 in different physiological situations and cardiac hypertrophy, ischemia reperfusion injury, heart failure, atherosclerosis, and diabetes mellitus type 2, underscoring the central role of Trx-1 in cardiovascular health and disease. FUTURE DIRECTIONS Thus, the manipulation of Trx-1 activity in the heart and/or vasculature, for example, by small molecules, seems to be a promising therapeutic option in cardiovascular diseases, as general anti-oxidant treatments would not take into account interactions of Trx-1 with other proteins and also eliminate vital ROS.

A New Kid on the Block: PKD1: A Promising Target for Antiangiogenic Therapy?

The formation of blood vessels through the process of angiogenesis is critical in normal vascular development and numerous vascular disorders. The most prominent stimulus for angiogenic processes in endothelial cells is vascular endothelial growth factor (VEGF), which regulates their migration, proliferation, and survival.1 The crucial role for this factor is documented by the lethal phenotype resulting from disruption of a single allele in mice.2,3 Binding of VEGF to its receptors activates several intracellular signaling molecules, among them phospholipase Cγ (PLCγ), protein kinase C (PKC), protein kinase D (PKD), and phosphatidyl-insitol-3 kinase (PI3K).1 The VEF-induced signaling events finally culminate in gene expression changes in the nucleus. See accompanying article on page 1782 A key regulator of gene expression is chromatin structure, which is largely determined by the acetylation status of histones. In general, acetylation of these nucleosomal proteins by histone acetyl transferases (HATs) stimulates transcription, whereas deacetylation by histone deacetylases (HDACs) leads to transcriptional repression. The HDAC family comprises 18 members in humans, which are classified based on their homologies to yeast proteins. …

Cellular functions of the dual-targeted catalytic subunit of telomerase, telomerase reverse transcriptase — Potential role in senescence and aging

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.