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Depletion of oxaloacetate decarboxylase FAHD1 inhibits mitochondrial electron transport and induces cellular senescence in human endothelial cells
Experimental Gerontology 92 (2017) 7–12
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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Short report
Depletion of oxaloacetate decarboxylase FAHD1 inhibits mitochondrial electron transport and induces cellular senescence in human endothelial cells Michele Petit, Rafal Koziel, Solmaz Etemad, Haymo Pircher, Pidder Jansen-Dürr ⁎ Institute for Biomedical Aging Research, Universität Innsbruck, Rennweg 10, A-6020 Innsbruck, Austria
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Article history: Received 25 October 2016 Received in revised form 28 February 2017 Accepted 2 March 2017 Available online 08 March 2017 Keywords: Oxaloacetate decarboxylase FAHD1 Premature senescence Mitochondria Energy metabolism DNA damage
a b s t r a c t In this study we report the identification of FAH domain containing protein 1 (FAHD1), a recently described member of the fumarylacetoacetate hydrolase (FAH) superfamily of metabolic enzymes, as a novel player in the regulation of cellular senescence. FAHD1 was found in a proteomic screen searching for mitochondrial proteins, which are differentially regulated in mitochondria from young and senescent human endothelial cells, and subsequently identified as oxaloacetate decarboxylase. We report here that depletion of FAHD1 from human endothelial cells inhibited mitochondrial energy metabolism and subsequently induced premature senescence. Whereas senescence induced by FAHD1 depletion was not associated with DNA damage, we noted a reduction of mitochondrial ATP-coupled respiration associated with upregulation of the cdk inhibitor p21. These results indicate that FAHD1 is required for mitochondrial function in human cells and provide additional support to the growing evidence that mitochondrial dysfunction can induce cellular senescence by metabolic alterations independent of the DNA damage response pathway. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Cellular senescence is increasingly recognized as a physiological process involved in a variety of physiological and pathophysiological conditions in mammals. Thus, cellular senescence contributes to tumor suppression (Campisi, 2011), age-associated dysfunction (Campisi, 2013; van Deursen, 2014), and mammalian development (Burton and Krizhanovsky, 2014; Munoz-Espin et al., 2013). Senescent cells express a number of so-called senescence markers, including the cell cycle inhibitors p16INK4A and p21Cip1/Waf-1, as well as increased activity of senescence-associated β-galactosidase (Rodier and Campisi, 2011). Many senescent cells also secrete several cytokines, growth factors, and matrix metalloproteinases, collectively referred to as the senescence-associated secretory phenotype (SASP; (Coppe et al., 2008; Malaquin et al., 2016). In addition to these “classical” senescence markers, recent Abbreviations: BrdU, 5-bromo-2′deoxy-uridine; ETC, electron transport chain; FAHD1, fumarylacetoacetate hydrolase (FAH) domain-containing protein 1; FCCP, carbonyl cyanide 4(trifluoromethoxy) phenylhydrazone; HUVEC, human umbilical vein endothelial cells; JC-1, 5.5′,6,6′-tetrachloro-l,l′,3,Y-tetraethylbcnzimidazolocarbocyanine iodide; MMP, mitochondrial membrane potential; OAA, oxaloacetate; OCR, oxygen consumption rate; PI, propidium iodide; SA-β-Gal, senescence-associated β-galactosidase; SCR, scrambled; shRNA, short hairpin RNA; TCA cycle, tricarboxylic acid cycle. ⁎ Corresponding author at: Institute for Biomedical Aging Research, Rennweg 10, A6020 Innsbruck, Austria. E-mail address: [email protected] (P. Jansen-Dürr).
http://dx.doi.org/10.1016/j.exger.2017.03.004 0531-5565/© 2017 Elsevier Inc. All rights reserved.
evidence suggests that senescence-associated metabolic changes, including altered mitochondrial function, may be essential for the induction and maintenance of the senescent state (Nacarelli and Sell, 2016). Whereas replicative senescence and several forms of stress-induced senescence are characterized by DNA damage at telomeres and other genomic loci, in particular DNA double strand breaks, other types of senescence have been characterized that occur in the absence of DNA damage (Bhatia-Dey et al., 2016). Thus, developmental senescence (MunozEspin et al., 2013), senescence in response to a lowered ATP/AMP ratio (Zwerschke et al., 2003), and senescence induced by inhibition of the mitochondrial electron transport chain (ETC) (Stockl et al., 2006; Wiley et al., 2016) are not accompanied by detectable DNA damage. Besides human diploid fibroblasts which are still used in the vast majority of in vitro senescence studies, many other human cell types also display the senescence phenotype upon extended passaging. Thus, human umbilical vein endothelial cells (HUVEC) were shown to enter cellular senescence after roughly 60 population doublings (Wagner et al., 2001). As in fibroblasts, premature senescence can be induced in HUVEC by a variety of stimuli, including exposure to tertButyl-hydroperoxide (t-BHP) (Unterluggauer et al., 2003), which leads to induced oxidative stress. Of note, replicative senescence of HUVEC could be delayed by silencing the gene encoding NADPH oxidase NOX4 (Lener et al., 2009), indicating that H2O2 produced by endogenous NOX4, induces nuclear DNA damage and thereby accelerates senescence in this cell type. However, HUVEC senescence is also induced
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by the inhibition of glutaminase (Unterluggauer et al., 2008) which affects ATP production and other biosynthetic pathways based on glutaminolysis. However, the role of mitochondria in HUVEC senescence remained elusive. In an attempt to identify new mitochondrial regulators of cellular senescence, a proteomics approach was used to identify mitochondrial proteins, the expression of which is altered with cellular senescence, using HUVEC as model. Besides post-translationally modified isoforms of several mitochondrial ATPase subunits (Groebe et al., 2007), we also identified fumarylacetoacetate hydrolase (FAH) domain containing protein 1 (FAHD1) as a differentially expressed protein in mitochondria from young vs. senescent HUVEC. Subsequently, FAHD1 was functionally characterized as a mitochondrial metabolic enzyme with dual substrate specificity, which can both hydrolyze acylpyruvates (Pircher et al., 2011) and decarboxylase oxaloacetate (Pircher et al., 2015), a metabolite of the tricarboxylic acid (TCA) cycle. When the FAHD1 homolog fahd-1 was deleted in the nematode C. elegans, this resulted in a severe locomotion deficit which was accompanied by a significant reduction of both mitochondrial membrane potential and oxygen consumption (Taferner et al., 2015), suggesting that FAHD1 is required for mitochondrial function in nematodes. However, the role of FAHD1 in mammalian cells remained elusive. In the present communication, we have explored the effects of lentivirus mediated silencing of the FAHD1 gene in HUVEC.
L-glutamine
(Sigma, Steinheim, Germany), and 1% penicillin streptomycin (Gibco, Eggenstein, Germany). 293FT cells were grown and maintained according to the supplier's user manual (Cat. nos. R70007, WFGE08S, Invitrogen). All cells were grown in an atmosphere of 5% CO2 at 37 °C and were subcultured by trypsinization with 0.05% trypSsin-EDTA (Sigma, Steinheim, Germany). 2.2. Metabolic flux analysis
Metabolic flux analysis was performed using Seahorse XFp Analyser (Seahorse Bioscience, North Billerica, MA). Shortly, 5 × 103 cells/well were seeded out one day before the experiment on the XFp cell culture miniplates (Seahorse Bioscience, North Billerica, MA) and analysed according to the protocols provided by the manufacturer. The Seahorse XFp Cell Mito Stress Test Kit (Seahorse Bioscience, North Billerica, MA, # 103010-100) was used. Obtained data were analysed using Wave 2.3.0 software (Agilent Technologies). 2.3. Determination of mitochondrial membrane potential
2. Material and methods
The electric potential of the inner mitochondrial membrane was determined in the cells pre-stained with the JC-1 fluorescent probe (Thermo Fisher Scientific, Vienna, Austria), as described previously (Koziel et al., 2013). Fluorescence was measured using the FACS Canto II flow cytometer (Becton Dickinson, Heidelberg, Germany).
2.1. Cell culture
2.4. Preparation of cellular extracts and western blot
HUVEC were isolated and maintained according to the methods described (Wagner et al., 2001). Cells were propagated in endothelial cell growth medium (EBM CC-3121 supplemented with CC-4133, Lonza, Walkersville, USA). U-2OS and HeLa cells purchased from ATCC (Manassas, VA) were propagated in Dulbecco's modified Eagle's medium (D5546, Sigma, Steinheim, Germany) supplemented with 10% heatinactivated fetal bovine serum (Sigma, Steinheim, Germany), 4 mM
Cellular protein lysates were prepared in RIPA buffer (50 mM Tris– HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 0.1% SDS) and subjected to standard immunoblotting, using primary antibodies against FAHD1 (rabbit polyclonal, dilution 1:1000, desribed in Pircher et al., 2011), α-tubulin (Cat.-No. T5168, dilution 1:20,000, Sigma, Vienna, Austria), p53 (Cat.-No. SC-126, dilution 1:000, Santa Cruz Biotechnology, Heidelberg, Germany), p21 (Cat.-No. 2947, dilution
Fig. 1. Downregulation of FAHD1 reduces cell proliferation in human endothelial cells. Early-passage HUVEC, were infected with 1 MOI of control (SCR) or FAHD1 knockdown lentivirus, as indicated, and FAHD1 levels determined by Western blot; α-tubulin served as loading control (A). Subsequently, cells were counted seven days after infection (B), and the rate of cell proliferation determined by BrdU incorporation assay (C). Cell death was assessed by co-staining with Annexin V and PI followed by flow cytometry (D).
M. Petit et al. / Experimental Gerontology 92 (2017) 7–12
1:000, Cell Signaling, Leiden, The Netherlands), γ-H2AX (Cat.-No. 2577, dilution 1:000, Cell Signaling, The Netherlands), p16 (Cat.-No. 554079, dilution 1:500, BD Biosciences, Vienna, Austria), lamin B1 (Cat.-No. ab16048, dilution 1:2000, Abcam, Cambridge, UK), HMGB1 (Cat.-No. ab18650, dilution 1:2000, Abcam, Cambridge, UK), Phosphop53 Ser15 (Cat.-No #9284S, dilution 1:1000, Cell Signaling, Leiden, Netherlands), and GAPDH (Cat.-No. SC-25778, dilution 1:2000, Santa Cruz Biotechnology, Heidelberg, Germany).
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with different lentivirus dilutions (0.01 μl, 0.1 μl, and 1 μl in 1 ml medium containing 8 μg/μl polybrene (Sigma, Steinheim, Germany) as transduction enhancer). 24 h after transduction, selection was started with medium containing 500 ng/ml puromycin (Merck Milipore, Darmstadt, Germany). After six days of selection, colonies were stained with crystal violet (1% in 10% ethanol) and counted. Titers were in the range of 8.5 × 105–1.2 × 106 transforming units (TU) per ml. 2.6. Lentiviral shRNA-mediated depletion of FAHD1 in HUVEC
2.5. Production of FAHD1 knockdown lentivirus Three different shRNA sequences were derived from the 3′ untranslated region of the FAHD1 mRNA (shRNA1: 5′-AGAUGAACCCU UCAAGAAA-3′, shRNA2: 5′-GAAACCCCGUCUCUACUAA-3′, shRNA3: 5′GCAUUCCAAGUGAUUCUUA-3′) and introduced into the pLKO.1 vector (Addgene). The pLKO.1- SCR control vector (Addgene, Cambridge, MA, plasmid 10,879) was used as a control. 293FT cells were cultivated in T75 flasks until they reached 90% confluency, followed by Metafectene (Biontex, Munich, Germany) transfection using 6 μg of the respective pLKO.1 plasmid together with 6 μg pMD2.G (Addgene, Cambridge, MA, plasmid 12,259) and 6 μg psPAX2 (Addgene, Cambridge, MA, plasmid 12,260) as packaging plasmids. The supernatant was replaced with 10 ml of 293FT growth medium on the next day. 72 h after transfection, the supernatant was collected, centrifuged at 400 ×g for 5 min, filtered through a 0.45 μm syringe filter, and stored at −80 °C. To determine the lentivirus titer, 50,000 U-2OS cells each were seeded on 6-well plates and cultivated overnight. On the next day, the medium was replaced
Young HUVEC (passage 5) were seeded on 6-well plates (40,000 cells per well) and cultivated in endothelial cell growth medium. On the next day, the medium was aspirated and replaced with 1 ml fresh endothelial cell growth medium containing 8 μg/μl polybrene and control (scrambled) or FAHD1 knockdown lentivirus (multiplicity of infection (MOI) = 1). On the next day, the medium was aspirated and replaced with 2 ml fresh endothelial cell growth medium. 500 ng/ml puromycin (Life technologies, A11138-03) was added on day 4. Seven days after infection, cells were trypsinized and counted (Casy, Schaerfe System, Reutlingen, Germany). 2.7. Cell proliferation and apoptosis assay Cell proliferation was assayed using the 5-bromo-2′deoxy-uridine (BrdU) labeling and detection kit I (Roche, Vienna, Austria). 6 × 105 cells were plated the day prior the assay. Next day, cells were labeled with BrdU for 1 h and analysed by flow cytometry (FACS Canto II, Becton Dickinson, Heidelberg, Germany).
Fig. 2. Downregulation of FAHD1 induces premature senescence in human endothelial cells. Early-passage HUVEC, were infected with 1 MOI of control (SCR) or FAHD1 knockdown lentivirus, and stained for SA-β-Gal activity seven days after infection; the percentage of SA-β-Gal positive cells was determined (A). Senescence markers lamin B1 and HMGB1 were determined by Western blot; α-tubulin served as loading control (B).
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For detection of apoptosis, the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) was used. Cells were detached, washed with PBS and resuspended in Annexin V binding buffer at a concentration of 1 × 106 cells/ml. 5 μl Annexin V and 5 μl propidium iodide (PI) were added to 100 μl cells and incubated for 15 min at room temperature. After incubation, 400 μl of Annexin V buffer were added to the cell suspension and measured by flow cytometry (FACS Canto II, Becton Dickinson, Heidelberg, Germany).
2.8. Statistical analysis All experiments were repeated at least three times unless otherwise specified. Data represent the mean ± SEM of at least three independent experiments. Statistical comparisons were performed using unpaired Student's t-test (in all graphics: *p b 0.05, ** p b 0.01, *** p b 0.001).
3. Results and discussion To address the functional requirement of HUVEC for FAHD1, lentiviral vectors were used for knocking down endogenous FAHD1 protein in human umbilical vein endothelial cells (HUVEC) by shRNAs, using non-targeting shRNAs as control. Infection with lentiviral FAHD1 shRNA vectors significantly reduced FAHD1 levels in HUVEC (Fig. 1A), and the effect could be reproduced with two independent shRNA constructs (data not shown). shRNA-mediated depletion of FAHD1 led to significantly reduced cell numbers seven days after infection (Fig. 1B). When the rate of cell proliferation was assessed by BrdU incorporation experiments, we noted a significant drop in cell proliferation in response to FAHD1 inactivation (Fig. 1C) Staining of FAHD1-depleted cells with Annexin V and propidium iodide revealed that the rate of both apoptosis and necrosis was not changed by FAHD1 inactivation (Fig. 1D). Furthermore, depletion of FAHD1 induced premature
Fig. 3. Downregulation of FAHD1 in HUVEC reduces mitochondrial energy status. Mitochondrial membrane potential (MMP) in FAHD1-depleted and control (SCR) HUVEC was determined by JC-1 staining followed by flow cytometry. Cells treated with FCCP and oligomycin served as negative and positive controls, respectively (A). Mitochondrial function in FAHD1-depleted and control (SCR) HUVEC was determined by metabolic flux analysis using a Seahorse XFp Flux Analyser (B–D). Oxygen consumption rate (OCR) was continuously recorded in cells at baseline and after subsequent addition of oligomycin, FCCP, and rotenone/antimycin A for a total of 80 min (B); subsequently, data were used to calculate basal OCR and the spare respiratory capacity (C), as well as the proton leak and the OCR linked to ATP production (D). Aspartate (10 mM) was added to medium prior to shRNA-mediated depletion of FAHD1, and cell numbers determined after seven days (E).
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senescence in HUVEC, as shown by an increased percentage of cells staining positive for senescence-associated β-galactosidase (SA-β-Gal) (Fig. 2A). As additional senescence markers, we determined by Western blot the levels of lamin B1 and nuclear HMGB1, which are significantly reduced in senescent cells, including cells that are driven into senescence by mitochondrial impairment (Wiley et al., 2016). We found that the levels of both senescence marker proteins were significantly down-regulated in FAHD1-depleted cells (Fig. 2B). These findings suggest that FAHD1 is required for cell proliferation in human endothelial cells and its inactivation induces premature senescence. To address the effect of FAHD1 depletion on mitochondrial function, mitochondrial membrane potential (MMP) was determined in HUVEC infected with lentiviral vectors carrying non-targeting or FAHD1 specific shRNA (Fig. 3). Cells were stained with JC-1 and the ratio of cells with high vs. low MMP was determined by flow cytometry. Cells with reduced MMP, obtained by FCCP treatment, and cells with increased MMP, obtained by short-term pre-treatment with oligomycin, served as negative and positive controls, respectively. We found that depletion of FAHD1 significantly reduced mitochondrial membrane potential in HUVEC (Fig. 3A). To further explore effects of FAHD1 depletion on mitochondrial function, mitochondrial function was analysed by Seahorse metabolic flux analyser (Fig. 3B). We found that both basal oxygen consumption and spare respiratory capacity were reduced in FAHD1-depleted cells (Fig. 3B, C); respiration coupled to ATP production was also reduced (Fig. 3D). It was shown by others that inhibition of mitochondrial respiration leads to a depletion of metabolites required for nucleotide biosynthesis, such as aspartate, and that the proliferation defect of cells with compromised electron transport chain (ETC) activity can be overcome by the addition of exogenous aspartate to cell culture media (Birsoy et al., 2015). When aspartate was added to FAHD1-depleted HUVEC, cell proliferation was partly rescued (Fig. 3E), supporting the idea that inhibition of mitochondrial function by FAHD1 KD limits the availability of metabolites for nucleotide biosynthesis and this effect contributes to decreased cell proliferation. We next assessed molecular mechanisms underlying premature senescence observed in FAHD1-depleted HUVEC (Fig. 4A). Cellular extracts were prepared and assessed by Western blot using antibodies to
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p53. Of note, we observed slightly increased p53 protein levels in FAHD1-depleted cells (Fig. 4B), which were accompanied by a significant increase in the level of the cdk inhibitor p21Cip1/Waf-1 (Fig. 4C), which is known to contribute to the senescence-associated proliferation arrest. To determine if premature senescence induced by FAHD1 depletion in HUVEC was due to DNA damage, cellular extracts were probed with an antibody to phosphorylated γ-H2AX, a well-established marker for DNA damage, in particular DNA double strand breaks. As positive control, DNA damage was induced in human diploid fibroblasts by the genotoxic agent cisplatin which is known to induce DNA damage (Spitkovsky et al., 1997). As expected, control cells did not display positive staining for γ-H2AX in Western blot; likewise, γ-H2AX staining was undetectable in FAHD1-depleted cells (Fig. 4D). Control and FAHD1-depleted cells were also stained for γ-H2AX by indirect immunofluorescence, again using cisplatin-treated cells as positive control. The proportion of DNA damage foci per cell was strongly increased by cisplatin treatment but not significantly changed by FAHD1 inactivation (Fig. S1). Together, these observations indicate that depletion of FAHD1 does not increase DNA damage. As we also observed no significant changes in p53 phosphorylation in FAHD1-depleted cells (data not shown), p53-independent activation of p21 gene expression, for which ample precedent exists in the literature (Warfel and El-Deiry, 2013), cannot be ruled out. We also analysed expression of the cdk inhibitor p16INK4A, which is known to contribute to senescence – associated growth arrest. HeLa cells, a cervical carcinoma cell line with constitutive expression of p16INK4A (Pauck et al., 2014), was used as positive control. We found that depletion of FAHD1 did not result in upregulation of p16INK4A expression in HUVEC (Fig. 4E). Whereas p16INK4A is considered an important player driving senescence in many cases, recent reports from several laboratories suggest that cellular senescence without increased p16INK4A expression can be observed in several instances, such as senescence during embryonic development (Burton and Krizhanovsky, 2014) or mitochondrial dysfunction-associated senescence (Wiley et al., 2016). The data reported here suggest that shRNA-mediated depletion of the oxaloacetate decarboxylase FAHD1 from human endothelial cells reduces oxygen consumption and inhibits the function of the
Fig. 4. Senescence markers in FAHD1-depleted HUVEC. Extracts were prepared from FAHD1-depleted and control (SCR) cells, and the expression of FAHD1 (A), and the senescence markers p53 (B), p21 (C), γH2AX (D), and p16INK4A (E) was analysed by Western blot. Cisplatin treated human fibroblasts served as positive control for γH2AX Western blot, and HeLa cells as positive control for p16 Western blot. α-tubulin and GAPDH served as loading controls.
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mitochondrial electron transport chain, characterized by reduced mitochondrial membrane potential and a reduced respiration coupled to ATP production. This finding extends our previous observation, that germline ablation of the nematode homolog of FAHD1 impairs mitochondrial function in C. elegans, to mammalian cells. The data reported here provide additional insight into FAHD1 function in the mitochondria, in particular suggesting that the function of the mitochondrial electron transport chain depends on FAHD1. Flux through the ETC is inevitably coupled to a functional TCA cycle, and it was shown that succinate dehydrogenase (SDH) in ETC complex II is inhibited by OAA (Kotlyar and Vinogradov, 1984). Using a very sensitive assay based on 14 C labeled acetyl-CoA, we reported previously that germline deletion of FAHD1 in the mouse led to a twofold increase of OAA concentration in mouse liver and kidney (Pircher et al., 2015). Although we were unable to determine the OAA concentration in HUVEC by this assay due to technical limitations, we consider it likely that depletion of FAHD1 leads to increased concentration of its substrate OAA in mitochondria, which may in turn inhibit SDH activity and thereby reduce electron flux in the ETC. There is precedence that inhibition of the mitochondrial ETC can induce premature senescence in human cells. First, it was shown that chronic exposure to antimycin A and/or oligomycin induces premature senescence in human foreskin fibroblasts and this effect is independent of mitochondrial ROS production (Stockl et al., 2006); moreover, Wiley et al. recently reported that depletion of mitochondrial sirtuin SIRT3 induced premature senescence, referred to as mitochondrial dysfunction induced senescence (MiDas), in human diploid lung fibroblasts and this effect could be reproduced by ETC inhibitors rotenone and antimycin A (Wiley et al., 2016). Similar to our findings with FAHD1-depleted HUVEC, Wiley et al. observed a moderate increase in p53 concentration in combination with a robust upregulation of p21 in the absence of DNA damage in Sirt3-depleted fibroblasts, providing further evidence to suggest the involvement of the mitochondrial ETC in FAHD1-depleted HUVEC. In summary, the data reported here indicate that human FAHD1 shares with the nematode protein the ability to protect the function of the mitochondrial electron transport chain and that its downregulation in human endothelial cells induces cellular senescence in the absence of DNA damage. Conflict of interest The authors declare no conflict of interest. Acknowledgements Work in PJD's laboratory was supported by the European Commission (H2020 Project FRAILOMIC). We are grateful to Michael Neuhaus, Brigitte Jenewein, and Annabella Pittl for expert technical assistance. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.exger.2017.03.004. References Bhatia-Dey, N., Kanherkar, R.R., Stair, S.E., Makarev, E.O., Csoka, A.B., 2016. Cellular senescence as the causal nexus of aging. Front. Genet. 7 (13). Birsoy, K., Wang, T., Chen, W.W., Freinkman, E., Abu-Remaileh, M., Sabatini, D.M., 2015. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis. Cell 162 (3), 540–551 (Jul 30).
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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Short report
Depletion of oxaloacetate decarboxylase FAHD1 inhibits mitochondrial electron transport and induces cellular senescence in human endothelial cells Michele Petit, Rafal Koziel, Solmaz Etemad, Haymo Pircher, Pidder Jansen-Dürr ⁎ Institute for Biomedical Aging Research, Universität Innsbruck, Rennweg 10, A-6020 Innsbruck, Austria
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Article history: Received 25 October 2016 Received in revised form 28 February 2017 Accepted 2 March 2017 Available online 08 March 2017 Keywords: Oxaloacetate decarboxylase FAHD1 Premature senescence Mitochondria Energy metabolism DNA damage
a b s t r a c t In this study we report the identification of FAH domain containing protein 1 (FAHD1), a recently described member of the fumarylacetoacetate hydrolase (FAH) superfamily of metabolic enzymes, as a novel player in the regulation of cellular senescence. FAHD1 was found in a proteomic screen searching for mitochondrial proteins, which are differentially regulated in mitochondria from young and senescent human endothelial cells, and subsequently identified as oxaloacetate decarboxylase. We report here that depletion of FAHD1 from human endothelial cells inhibited mitochondrial energy metabolism and subsequently induced premature senescence. Whereas senescence induced by FAHD1 depletion was not associated with DNA damage, we noted a reduction of mitochondrial ATP-coupled respiration associated with upregulation of the cdk inhibitor p21. These results indicate that FAHD1 is required for mitochondrial function in human cells and provide additional support to the growing evidence that mitochondrial dysfunction can induce cellular senescence by metabolic alterations independent of the DNA damage response pathway. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Cellular senescence is increasingly recognized as a physiological process involved in a variety of physiological and pathophysiological conditions in mammals. Thus, cellular senescence contributes to tumor suppression (Campisi, 2011), age-associated dysfunction (Campisi, 2013; van Deursen, 2014), and mammalian development (Burton and Krizhanovsky, 2014; Munoz-Espin et al., 2013). Senescent cells express a number of so-called senescence markers, including the cell cycle inhibitors p16INK4A and p21Cip1/Waf-1, as well as increased activity of senescence-associated β-galactosidase (Rodier and Campisi, 2011). Many senescent cells also secrete several cytokines, growth factors, and matrix metalloproteinases, collectively referred to as the senescence-associated secretory phenotype (SASP; (Coppe et al., 2008; Malaquin et al., 2016). In addition to these “classical” senescence markers, recent Abbreviations: BrdU, 5-bromo-2′deoxy-uridine; ETC, electron transport chain; FAHD1, fumarylacetoacetate hydrolase (FAH) domain-containing protein 1; FCCP, carbonyl cyanide 4(trifluoromethoxy) phenylhydrazone; HUVEC, human umbilical vein endothelial cells; JC-1, 5.5′,6,6′-tetrachloro-l,l′,3,Y-tetraethylbcnzimidazolocarbocyanine iodide; MMP, mitochondrial membrane potential; OAA, oxaloacetate; OCR, oxygen consumption rate; PI, propidium iodide; SA-β-Gal, senescence-associated β-galactosidase; SCR, scrambled; shRNA, short hairpin RNA; TCA cycle, tricarboxylic acid cycle. ⁎ Corresponding author at: Institute for Biomedical Aging Research, Rennweg 10, A6020 Innsbruck, Austria. E-mail address: [email protected] (P. Jansen-Dürr).
http://dx.doi.org/10.1016/j.exger.2017.03.004 0531-5565/© 2017 Elsevier Inc. All rights reserved.
evidence suggests that senescence-associated metabolic changes, including altered mitochondrial function, may be essential for the induction and maintenance of the senescent state (Nacarelli and Sell, 2016). Whereas replicative senescence and several forms of stress-induced senescence are characterized by DNA damage at telomeres and other genomic loci, in particular DNA double strand breaks, other types of senescence have been characterized that occur in the absence of DNA damage (Bhatia-Dey et al., 2016). Thus, developmental senescence (MunozEspin et al., 2013), senescence in response to a lowered ATP/AMP ratio (Zwerschke et al., 2003), and senescence induced by inhibition of the mitochondrial electron transport chain (ETC) (Stockl et al., 2006; Wiley et al., 2016) are not accompanied by detectable DNA damage. Besides human diploid fibroblasts which are still used in the vast majority of in vitro senescence studies, many other human cell types also display the senescence phenotype upon extended passaging. Thus, human umbilical vein endothelial cells (HUVEC) were shown to enter cellular senescence after roughly 60 population doublings (Wagner et al., 2001). As in fibroblasts, premature senescence can be induced in HUVEC by a variety of stimuli, including exposure to tertButyl-hydroperoxide (t-BHP) (Unterluggauer et al., 2003), which leads to induced oxidative stress. Of note, replicative senescence of HUVEC could be delayed by silencing the gene encoding NADPH oxidase NOX4 (Lener et al., 2009), indicating that H2O2 produced by endogenous NOX4, induces nuclear DNA damage and thereby accelerates senescence in this cell type. However, HUVEC senescence is also induced
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by the inhibition of glutaminase (Unterluggauer et al., 2008) which affects ATP production and other biosynthetic pathways based on glutaminolysis. However, the role of mitochondria in HUVEC senescence remained elusive. In an attempt to identify new mitochondrial regulators of cellular senescence, a proteomics approach was used to identify mitochondrial proteins, the expression of which is altered with cellular senescence, using HUVEC as model. Besides post-translationally modified isoforms of several mitochondrial ATPase subunits (Groebe et al., 2007), we also identified fumarylacetoacetate hydrolase (FAH) domain containing protein 1 (FAHD1) as a differentially expressed protein in mitochondria from young vs. senescent HUVEC. Subsequently, FAHD1 was functionally characterized as a mitochondrial metabolic enzyme with dual substrate specificity, which can both hydrolyze acylpyruvates (Pircher et al., 2011) and decarboxylase oxaloacetate (Pircher et al., 2015), a metabolite of the tricarboxylic acid (TCA) cycle. When the FAHD1 homolog fahd-1 was deleted in the nematode C. elegans, this resulted in a severe locomotion deficit which was accompanied by a significant reduction of both mitochondrial membrane potential and oxygen consumption (Taferner et al., 2015), suggesting that FAHD1 is required for mitochondrial function in nematodes. However, the role of FAHD1 in mammalian cells remained elusive. In the present communication, we have explored the effects of lentivirus mediated silencing of the FAHD1 gene in HUVEC.
L-glutamine
(Sigma, Steinheim, Germany), and 1% penicillin streptomycin (Gibco, Eggenstein, Germany). 293FT cells were grown and maintained according to the supplier's user manual (Cat. nos. R70007, WFGE08S, Invitrogen). All cells were grown in an atmosphere of 5% CO2 at 37 °C and were subcultured by trypsinization with 0.05% trypSsin-EDTA (Sigma, Steinheim, Germany). 2.2. Metabolic flux analysis
Metabolic flux analysis was performed using Seahorse XFp Analyser (Seahorse Bioscience, North Billerica, MA). Shortly, 5 × 103 cells/well were seeded out one day before the experiment on the XFp cell culture miniplates (Seahorse Bioscience, North Billerica, MA) and analysed according to the protocols provided by the manufacturer. The Seahorse XFp Cell Mito Stress Test Kit (Seahorse Bioscience, North Billerica, MA, # 103010-100) was used. Obtained data were analysed using Wave 2.3.0 software (Agilent Technologies). 2.3. Determination of mitochondrial membrane potential
2. Material and methods
The electric potential of the inner mitochondrial membrane was determined in the cells pre-stained with the JC-1 fluorescent probe (Thermo Fisher Scientific, Vienna, Austria), as described previously (Koziel et al., 2013). Fluorescence was measured using the FACS Canto II flow cytometer (Becton Dickinson, Heidelberg, Germany).
2.1. Cell culture
2.4. Preparation of cellular extracts and western blot
HUVEC were isolated and maintained according to the methods described (Wagner et al., 2001). Cells were propagated in endothelial cell growth medium (EBM CC-3121 supplemented with CC-4133, Lonza, Walkersville, USA). U-2OS and HeLa cells purchased from ATCC (Manassas, VA) were propagated in Dulbecco's modified Eagle's medium (D5546, Sigma, Steinheim, Germany) supplemented with 10% heatinactivated fetal bovine serum (Sigma, Steinheim, Germany), 4 mM
Cellular protein lysates were prepared in RIPA buffer (50 mM Tris– HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 0.1% SDS) and subjected to standard immunoblotting, using primary antibodies against FAHD1 (rabbit polyclonal, dilution 1:1000, desribed in Pircher et al., 2011), α-tubulin (Cat.-No. T5168, dilution 1:20,000, Sigma, Vienna, Austria), p53 (Cat.-No. SC-126, dilution 1:000, Santa Cruz Biotechnology, Heidelberg, Germany), p21 (Cat.-No. 2947, dilution
Fig. 1. Downregulation of FAHD1 reduces cell proliferation in human endothelial cells. Early-passage HUVEC, were infected with 1 MOI of control (SCR) or FAHD1 knockdown lentivirus, as indicated, and FAHD1 levels determined by Western blot; α-tubulin served as loading control (A). Subsequently, cells were counted seven days after infection (B), and the rate of cell proliferation determined by BrdU incorporation assay (C). Cell death was assessed by co-staining with Annexin V and PI followed by flow cytometry (D).
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1:000, Cell Signaling, Leiden, The Netherlands), γ-H2AX (Cat.-No. 2577, dilution 1:000, Cell Signaling, The Netherlands), p16 (Cat.-No. 554079, dilution 1:500, BD Biosciences, Vienna, Austria), lamin B1 (Cat.-No. ab16048, dilution 1:2000, Abcam, Cambridge, UK), HMGB1 (Cat.-No. ab18650, dilution 1:2000, Abcam, Cambridge, UK), Phosphop53 Ser15 (Cat.-No #9284S, dilution 1:1000, Cell Signaling, Leiden, Netherlands), and GAPDH (Cat.-No. SC-25778, dilution 1:2000, Santa Cruz Biotechnology, Heidelberg, Germany).
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with different lentivirus dilutions (0.01 μl, 0.1 μl, and 1 μl in 1 ml medium containing 8 μg/μl polybrene (Sigma, Steinheim, Germany) as transduction enhancer). 24 h after transduction, selection was started with medium containing 500 ng/ml puromycin (Merck Milipore, Darmstadt, Germany). After six days of selection, colonies were stained with crystal violet (1% in 10% ethanol) and counted. Titers were in the range of 8.5 × 105–1.2 × 106 transforming units (TU) per ml. 2.6. Lentiviral shRNA-mediated depletion of FAHD1 in HUVEC
2.5. Production of FAHD1 knockdown lentivirus Three different shRNA sequences were derived from the 3′ untranslated region of the FAHD1 mRNA (shRNA1: 5′-AGAUGAACCCU UCAAGAAA-3′, shRNA2: 5′-GAAACCCCGUCUCUACUAA-3′, shRNA3: 5′GCAUUCCAAGUGAUUCUUA-3′) and introduced into the pLKO.1 vector (Addgene). The pLKO.1- SCR control vector (Addgene, Cambridge, MA, plasmid 10,879) was used as a control. 293FT cells were cultivated in T75 flasks until they reached 90% confluency, followed by Metafectene (Biontex, Munich, Germany) transfection using 6 μg of the respective pLKO.1 plasmid together with 6 μg pMD2.G (Addgene, Cambridge, MA, plasmid 12,259) and 6 μg psPAX2 (Addgene, Cambridge, MA, plasmid 12,260) as packaging plasmids. The supernatant was replaced with 10 ml of 293FT growth medium on the next day. 72 h after transfection, the supernatant was collected, centrifuged at 400 ×g for 5 min, filtered through a 0.45 μm syringe filter, and stored at −80 °C. To determine the lentivirus titer, 50,000 U-2OS cells each were seeded on 6-well plates and cultivated overnight. On the next day, the medium was replaced
Young HUVEC (passage 5) were seeded on 6-well plates (40,000 cells per well) and cultivated in endothelial cell growth medium. On the next day, the medium was aspirated and replaced with 1 ml fresh endothelial cell growth medium containing 8 μg/μl polybrene and control (scrambled) or FAHD1 knockdown lentivirus (multiplicity of infection (MOI) = 1). On the next day, the medium was aspirated and replaced with 2 ml fresh endothelial cell growth medium. 500 ng/ml puromycin (Life technologies, A11138-03) was added on day 4. Seven days after infection, cells were trypsinized and counted (Casy, Schaerfe System, Reutlingen, Germany). 2.7. Cell proliferation and apoptosis assay Cell proliferation was assayed using the 5-bromo-2′deoxy-uridine (BrdU) labeling and detection kit I (Roche, Vienna, Austria). 6 × 105 cells were plated the day prior the assay. Next day, cells were labeled with BrdU for 1 h and analysed by flow cytometry (FACS Canto II, Becton Dickinson, Heidelberg, Germany).
Fig. 2. Downregulation of FAHD1 induces premature senescence in human endothelial cells. Early-passage HUVEC, were infected with 1 MOI of control (SCR) or FAHD1 knockdown lentivirus, and stained for SA-β-Gal activity seven days after infection; the percentage of SA-β-Gal positive cells was determined (A). Senescence markers lamin B1 and HMGB1 were determined by Western blot; α-tubulin served as loading control (B).
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For detection of apoptosis, the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) was used. Cells were detached, washed with PBS and resuspended in Annexin V binding buffer at a concentration of 1 × 106 cells/ml. 5 μl Annexin V and 5 μl propidium iodide (PI) were added to 100 μl cells and incubated for 15 min at room temperature. After incubation, 400 μl of Annexin V buffer were added to the cell suspension and measured by flow cytometry (FACS Canto II, Becton Dickinson, Heidelberg, Germany).
2.8. Statistical analysis All experiments were repeated at least three times unless otherwise specified. Data represent the mean ± SEM of at least three independent experiments. Statistical comparisons were performed using unpaired Student's t-test (in all graphics: *p b 0.05, ** p b 0.01, *** p b 0.001).
3. Results and discussion To address the functional requirement of HUVEC for FAHD1, lentiviral vectors were used for knocking down endogenous FAHD1 protein in human umbilical vein endothelial cells (HUVEC) by shRNAs, using non-targeting shRNAs as control. Infection with lentiviral FAHD1 shRNA vectors significantly reduced FAHD1 levels in HUVEC (Fig. 1A), and the effect could be reproduced with two independent shRNA constructs (data not shown). shRNA-mediated depletion of FAHD1 led to significantly reduced cell numbers seven days after infection (Fig. 1B). When the rate of cell proliferation was assessed by BrdU incorporation experiments, we noted a significant drop in cell proliferation in response to FAHD1 inactivation (Fig. 1C) Staining of FAHD1-depleted cells with Annexin V and propidium iodide revealed that the rate of both apoptosis and necrosis was not changed by FAHD1 inactivation (Fig. 1D). Furthermore, depletion of FAHD1 induced premature
Fig. 3. Downregulation of FAHD1 in HUVEC reduces mitochondrial energy status. Mitochondrial membrane potential (MMP) in FAHD1-depleted and control (SCR) HUVEC was determined by JC-1 staining followed by flow cytometry. Cells treated with FCCP and oligomycin served as negative and positive controls, respectively (A). Mitochondrial function in FAHD1-depleted and control (SCR) HUVEC was determined by metabolic flux analysis using a Seahorse XFp Flux Analyser (B–D). Oxygen consumption rate (OCR) was continuously recorded in cells at baseline and after subsequent addition of oligomycin, FCCP, and rotenone/antimycin A for a total of 80 min (B); subsequently, data were used to calculate basal OCR and the spare respiratory capacity (C), as well as the proton leak and the OCR linked to ATP production (D). Aspartate (10 mM) was added to medium prior to shRNA-mediated depletion of FAHD1, and cell numbers determined after seven days (E).
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senescence in HUVEC, as shown by an increased percentage of cells staining positive for senescence-associated β-galactosidase (SA-β-Gal) (Fig. 2A). As additional senescence markers, we determined by Western blot the levels of lamin B1 and nuclear HMGB1, which are significantly reduced in senescent cells, including cells that are driven into senescence by mitochondrial impairment (Wiley et al., 2016). We found that the levels of both senescence marker proteins were significantly down-regulated in FAHD1-depleted cells (Fig. 2B). These findings suggest that FAHD1 is required for cell proliferation in human endothelial cells and its inactivation induces premature senescence. To address the effect of FAHD1 depletion on mitochondrial function, mitochondrial membrane potential (MMP) was determined in HUVEC infected with lentiviral vectors carrying non-targeting or FAHD1 specific shRNA (Fig. 3). Cells were stained with JC-1 and the ratio of cells with high vs. low MMP was determined by flow cytometry. Cells with reduced MMP, obtained by FCCP treatment, and cells with increased MMP, obtained by short-term pre-treatment with oligomycin, served as negative and positive controls, respectively. We found that depletion of FAHD1 significantly reduced mitochondrial membrane potential in HUVEC (Fig. 3A). To further explore effects of FAHD1 depletion on mitochondrial function, mitochondrial function was analysed by Seahorse metabolic flux analyser (Fig. 3B). We found that both basal oxygen consumption and spare respiratory capacity were reduced in FAHD1-depleted cells (Fig. 3B, C); respiration coupled to ATP production was also reduced (Fig. 3D). It was shown by others that inhibition of mitochondrial respiration leads to a depletion of metabolites required for nucleotide biosynthesis, such as aspartate, and that the proliferation defect of cells with compromised electron transport chain (ETC) activity can be overcome by the addition of exogenous aspartate to cell culture media (Birsoy et al., 2015). When aspartate was added to FAHD1-depleted HUVEC, cell proliferation was partly rescued (Fig. 3E), supporting the idea that inhibition of mitochondrial function by FAHD1 KD limits the availability of metabolites for nucleotide biosynthesis and this effect contributes to decreased cell proliferation. We next assessed molecular mechanisms underlying premature senescence observed in FAHD1-depleted HUVEC (Fig. 4A). Cellular extracts were prepared and assessed by Western blot using antibodies to
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p53. Of note, we observed slightly increased p53 protein levels in FAHD1-depleted cells (Fig. 4B), which were accompanied by a significant increase in the level of the cdk inhibitor p21Cip1/Waf-1 (Fig. 4C), which is known to contribute to the senescence-associated proliferation arrest. To determine if premature senescence induced by FAHD1 depletion in HUVEC was due to DNA damage, cellular extracts were probed with an antibody to phosphorylated γ-H2AX, a well-established marker for DNA damage, in particular DNA double strand breaks. As positive control, DNA damage was induced in human diploid fibroblasts by the genotoxic agent cisplatin which is known to induce DNA damage (Spitkovsky et al., 1997). As expected, control cells did not display positive staining for γ-H2AX in Western blot; likewise, γ-H2AX staining was undetectable in FAHD1-depleted cells (Fig. 4D). Control and FAHD1-depleted cells were also stained for γ-H2AX by indirect immunofluorescence, again using cisplatin-treated cells as positive control. The proportion of DNA damage foci per cell was strongly increased by cisplatin treatment but not significantly changed by FAHD1 inactivation (Fig. S1). Together, these observations indicate that depletion of FAHD1 does not increase DNA damage. As we also observed no significant changes in p53 phosphorylation in FAHD1-depleted cells (data not shown), p53-independent activation of p21 gene expression, for which ample precedent exists in the literature (Warfel and El-Deiry, 2013), cannot be ruled out. We also analysed expression of the cdk inhibitor p16INK4A, which is known to contribute to senescence – associated growth arrest. HeLa cells, a cervical carcinoma cell line with constitutive expression of p16INK4A (Pauck et al., 2014), was used as positive control. We found that depletion of FAHD1 did not result in upregulation of p16INK4A expression in HUVEC (Fig. 4E). Whereas p16INK4A is considered an important player driving senescence in many cases, recent reports from several laboratories suggest that cellular senescence without increased p16INK4A expression can be observed in several instances, such as senescence during embryonic development (Burton and Krizhanovsky, 2014) or mitochondrial dysfunction-associated senescence (Wiley et al., 2016). The data reported here suggest that shRNA-mediated depletion of the oxaloacetate decarboxylase FAHD1 from human endothelial cells reduces oxygen consumption and inhibits the function of the
Fig. 4. Senescence markers in FAHD1-depleted HUVEC. Extracts were prepared from FAHD1-depleted and control (SCR) cells, and the expression of FAHD1 (A), and the senescence markers p53 (B), p21 (C), γH2AX (D), and p16INK4A (E) was analysed by Western blot. Cisplatin treated human fibroblasts served as positive control for γH2AX Western blot, and HeLa cells as positive control for p16 Western blot. α-tubulin and GAPDH served as loading controls.
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mitochondrial electron transport chain, characterized by reduced mitochondrial membrane potential and a reduced respiration coupled to ATP production. This finding extends our previous observation, that germline ablation of the nematode homolog of FAHD1 impairs mitochondrial function in C. elegans, to mammalian cells. The data reported here provide additional insight into FAHD1 function in the mitochondria, in particular suggesting that the function of the mitochondrial electron transport chain depends on FAHD1. Flux through the ETC is inevitably coupled to a functional TCA cycle, and it was shown that succinate dehydrogenase (SDH) in ETC complex II is inhibited by OAA (Kotlyar and Vinogradov, 1984). Using a very sensitive assay based on 14 C labeled acetyl-CoA, we reported previously that germline deletion of FAHD1 in the mouse led to a twofold increase of OAA concentration in mouse liver and kidney (Pircher et al., 2015). Although we were unable to determine the OAA concentration in HUVEC by this assay due to technical limitations, we consider it likely that depletion of FAHD1 leads to increased concentration of its substrate OAA in mitochondria, which may in turn inhibit SDH activity and thereby reduce electron flux in the ETC. There is precedence that inhibition of the mitochondrial ETC can induce premature senescence in human cells. First, it was shown that chronic exposure to antimycin A and/or oligomycin induces premature senescence in human foreskin fibroblasts and this effect is independent of mitochondrial ROS production (Stockl et al., 2006); moreover, Wiley et al. recently reported that depletion of mitochondrial sirtuin SIRT3 induced premature senescence, referred to as mitochondrial dysfunction induced senescence (MiDas), in human diploid lung fibroblasts and this effect could be reproduced by ETC inhibitors rotenone and antimycin A (Wiley et al., 2016). Similar to our findings with FAHD1-depleted HUVEC, Wiley et al. observed a moderate increase in p53 concentration in combination with a robust upregulation of p21 in the absence of DNA damage in Sirt3-depleted fibroblasts, providing further evidence to suggest the involvement of the mitochondrial ETC in FAHD1-depleted HUVEC. In summary, the data reported here indicate that human FAHD1 shares with the nematode protein the ability to protect the function of the mitochondrial electron transport chain and that its downregulation in human endothelial cells induces cellular senescence in the absence of DNA damage. Conflict of interest The authors declare no conflict of interest. Acknowledgements Work in PJD's laboratory was supported by the European Commission (H2020 Project FRAILOMIC). We are grateful to Michael Neuhaus, Brigitte Jenewein, and Annabella Pittl for expert technical assistance. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.exger.2017.03.004. References Bhatia-Dey, N., Kanherkar, R.R., Stair, S.E., Makarev, E.O., Csoka, A.B., 2016. Cellular senescence as the causal nexus of aging. Front. Genet. 7 (13). Birsoy, K., Wang, T., Chen, W.W., Freinkman, E., Abu-Remaileh, M., Sabatini, D.M., 2015. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis. Cell 162 (3), 540–551 (Jul 30).
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