Journal of Molecular Biology
Volume 394, Issue 4, 11 December 2009, Pages 789-803
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Direct Binding of Glyceraldehyde 3-Phosphate Dehydrogenase to Telomeric DNA Protects Telomeres against Chemotherapy-Induced Rapid Degradation

https://doi.org/10.1016/j.jmb.2009.09.062Get rights and content

Abstract

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that displays several non-glycolytic activities, including the maintenance and/or protection of telomeres. In this study, we determined the molecular mechanism and biological role of the interaction between GAPDH and human telomeric DNA. Using gel-shift assays, we show that recombinant GAPDH binds directly with high affinity (Kd = 45 nM) to a single-stranded oligonucleotide comprising three telomeric DNA repeats, and that nucleotides T1, G5, and G6 of the TTAGGG repeat are essential for binding. The stoichiometry of the interaction is 2:1 (DNA:GAPDH), and GAPDH appears to form a high-molecular-weight complex when bound to the oligonucleotide. Mutation of Asp32 and Cys149, which are localized to the NAD-binding site and the active-site center of GAPDH, respectively, produced mutants that almost completely lost their telomere-binding functions both in vitro and in situ (in A549 human lung cancer cells). Treatment of A549 cells with the chemotherapeutic agents gemcitabine and doxorubicin resulted in increased nuclear localization of expressed wild-type GAPDH, where it protected telomeres against rapid degradation, concomitant with increased resistance to the growth-inhibitory effects of these drugs. The non-DNA-binding mutants of GAPDH also localized to the nucleus when expressed in A549 cells, but did not confer any significant protection of telomeres against chemotherapy-induced degradation or growth inhibition; this occurred without the involvement of caspase activation or apoptosis regulation. Overall, these data demonstrate that GAPDH binds telomeric DNA directly in vitro and may have a biological role in the protection of telomeres against rapid degradation in response to chemotherapeutic agents in A549 human lung cancer cells.

Introduction

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; E.C. 1.2.1.12) is a well-known and ubiquitous enzyme that functions in the glycolytic and gluconeogenic pathways of sugar metabolism, but has been linked to a diverse array of non-glycolytic activities. These include several transactions with nucleic acids (tRNA transport, DNA repair, transcription, and binding with viral RNAs), membrane fusion, vesicle transport, and apoptosis (for reviews, see Sirover1, 2, 3 and Berry and Boulton4). Its binding to the β-amyloid precursor protein5 and the pathogenic protein huntingtin6, 7 implicates GAPDH in the neurodegenerative disorders Alzheimer's disease and Huntington's disease, respectively. GAPDH also plays a role in cancer pathogenesis8, 9, 10 and translocates to the nucleus in response to several anti-cancer agents.11, 12, 13 Additionally, GAPDH exhibits pro-apoptotic activity,14, 15, 16, 17 which involves binding to the E3 ubiquitin ligase Siah118 and translocation of the enzyme to the nucleus, mostly in non-cancerous neuronal cells.12, 19, 20

In a recent study, Sundararaj et al. found that GAPDH binds telomeric DNA in vivo, and that its overexpression in A549 lung adenocarcinoma cells prevents shortening of telomeres following treatment with the anti-cancer agents gemcitabine (GMZ) and doxorubicin (DOX), suggesting a possible role for GAPDH in chemotherapeutic resistance.21 Telomeres are DNA–protein assemblies that protect the ends of chromosomes from being recognized by the DNA repair machinery as double-stranded (ds) breaks. Protection of the chromosome ends is essential for cell viability, while their shortening is associated with cell senescence (for a review, see Blackburn22). The finding that GAPDH may function to protect telomeres was intriguing, but raised many questions, including whether GAPDH can bind directly to telomeric DNA or requires other factors, whether it binds single-stranded (ss) or ds regions of telomeric DNA, whether it exhibits the high affinity and specificity expected of a telomere-binding protein, whether the DNA-binding site overlaps with the enzyme's catalytic site or is elsewhere, and, finally, whether the interaction serves to protect telomeres against rapid degradation in response to treatment with chemotherapeutic agents in human cancer cells and, if so, whether this protection occurs in a caspase-dependent or caspase-independent manner.

Here, we present data showing that recombinant GAPDH binds ss-telomeric DNA with high affinity, in the absence of any other factors, as a large-molecular-weight complex comprising at least two tetramers. We have also identified three bases of the hexameric telomere repeat that are essential for binding to GAPDH. NAD+ competition experiments, coupled with site-directed mutagenesis, show that the telomeric DNA-binding site comprises the NAD-binding site and the active site of the enzyme. It is also demonstrated here that the binding of GAPDH to telomeres is not unique to A549 cells because GAPDH also co-localizes with telomeres in non-cancerous murine lung epithelial (MLE-15) cells. In addition, we find that overexpressed wild-type (wt) GAPDH binds to telomeres in A549 cells and prevents the rapid degradation of telomeres in response to GMZ and DOX, whereas a similar expression of GAPDH mutants that do not bind telomeric DNA does not confer such protection. Importantly, we also show that ectopic expression of wt-GAPDH, but not of the non-DNA-binding mutants, was concomitant with the prevention of the anti-proliferative effects of GMZ and DOX in these cells in a caspase-independent manner. Overall, the data reveal a specific and direct interaction between GAPDH and telomeric DNA that is associated in vivo with the prevention of chemotherapy-induced rapid degradation of telomeres and growth inhibition of cells.

Section snippets

GAPDH binds directly to telomeric DNA

Previous in vivo studies suggested that GAPDH binds to telomeric DNA and protects the ends of telomeres from degradative enzymes.21 In vitro binding between GAPDH and telomeric DNA, however, was only observed in a gel-binding assay that used UV radiation to cross-link the complexes.21 Hence, it was not known whether GAPDH could bind directly to telomeric DNA or whether other proteins were required in vivo. To address this, we developed an electrophoretic mobility shift assay (EMSA) and used

Discussion

In the present study, we have determined that GAPDH interacts with ss-telomeric DNA with high affinity and specificity, and that binding requires Asp32 and Cys149, which are located in the active-site region of the enzyme. We also show that the binding of GAPDH to telomeres in vivo results in the protection of telomere length, which correlates with the protection of A549 cells from the growth-inhibitory effects of the chemotherapeutic agents GMZ and DOX.

Oligonucleotides

The oligonucleotides used in this study are as follows:

  • [32P]5′-(TTAGGG)3-3′

  • 5′-(ATAGGG)3-3′

  • 5′-(TTAGGG)3-3′

  • 5′-(TTTGGG)3-3′5′-(TTACGG)3-3′5′-(TTAGCG)3-3′

  • 5′-(TTAGGC)3-3′

  • 5′-(CCCTAA)3-3′

  • [32P]5′-(AGGGTT)3-3′

  • 5′-(AGATGTAGCAATAGTAGT)-3′

The oligonucleotides were purchased from Integrated DNA Technologies, Inc. (Coralville, IA) and gel purified with denaturing polyacrylamide gel electrophoresis. For EMSA, 2–4 pmol of ss-5′-(TTAGGG)3-3′ or ss-5′-(AGGGTT)3-3′ oligonucleotides were labeled with 10 μCi of [γ-32

Acknowledgements

The authors wish to thank Dr. S. Lemon (University of Texas) for providing cDNA of human liver GAPDH and Dr. R. Nicholas (UNC Chapel Hill) for the pT7-HTb vector, both of which were used in this study. We also thank Adam Smolka, Sivakumar Ramalingam, and Can Emre Senkal for technical advice. This work was supported by National Institutes of Health grant CA88932 (to B.O.).

References (49)

  • AzamS. et al.

    Human glyceraldehyde-3-phosphate dehydrogenase plays a direct role in reactivating oxidized forms of the DNA repair enzyme APE1

    J. Biol. Chem.

    (2008)
  • BrownV.M. et al.

    A novel CRM1-mediated nuclear export signal governs nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase following genotoxic stress

    J. Biol. Chem.

    (2004)
  • MaruyamaW. et al.

    Glyceraldehyde-3-phospate dehydrogenase is translocated into nuclei through Golgi apparatus during apoptosis induced by 6-hydroxydopamine in human dopaminergic SH-SY5Y cells

    Neurosci. Lett.

    (2002)
  • ColellA. et al.

    GAPDH and autophagy preserve survival after apoptotic cytochrome c release in the absence of caspase activation

    Cell

    (2007)
  • OgretmenB. et al.

    Role of ceramide in mediating the inhibition of telomerase activity in A549 human lung adenocarcinoma cells

    J. Biol. Chem.

    (2001)
  • SiroverM.A.

    Role of the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase, in normal cell function and in cell pathology

    J. Cell. Biochem.

    (1997)
  • SiroverM.A.

    New nuclear functions of the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase, in mammalian cells

    J. Cell. Biochem.

    (2005)
  • BerryM.D. et al.

    Glyceraldehyde-3-phosphate dehydrogenase and apoptosis

    J. Neurosci. Res.

    (2000)
  • SchulzeH. et al.

    Rat brain glyceraldehyde-3-phosphate dehydrogenase interacts with the recombinant cytoplasmic domain of Alzheimer's beta-amyloid precursor protein

    J. Neurochem.

    (1993)
  • BurkeJ.R. et al.

    Huntingtin and DRPLA proteins selectively interact with the enzyme GAPDH

    Nat. Med.

    (1996)
  • BaeB.I. et al.

    Mutant huntingtin: nuclear translocation and cytotoxicity mediated by GAPDH

    Proc. Natl Acad. Sci. USA

    (2006)
  • DuZ.X. et al.

    Involvement of glyceraldehyde-3-phosphate dehydrogenase in tumor necrosis factor-related apoptosis-inducing ligand-mediated death of thyroid cancer cells

    Endocrinology

    (2007)
  • KimS. et al.

    Regulation of oncogenic transcription factor hTAF(II)68-TEC activity by human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

    Biochem. J.

    (2007)
  • XingC. et al.

    Identification of GAPDH as a protein target of the saframycin antiproliferative agents

    Proc. Natl Acad. Sci. USA

    (2004)
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    Present address for E. Apohan: Department of Biology, Faculty of Education, Inonu University, Malatya 44060, Turkey.

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