MCB Accepted Manuscript Posted Online 17 February 2015 Mol. Cell. Biol. doi:10.1128/MCB.01402-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Local mitochondrial-endolysosomal microfusion cleaves the
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voltage-dependent anion channel 1 to promote survival in hypoxia
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M. Christiane Brahimi-Horn1, Sandra Lacas-Gervais2, Ricardo Adaixo3, Karine Ilc1,
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Matthieu Rouleau4, Annick Notte5, Marc Dieu5, Carine Michels5, Thibault Voeltzel6,
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Véronique Maguer-Satta6, Joffrey Pelletier1, Marius Ilie1,7, Paul Hofman1,7, Bénédicte
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Manoury8, Alexander Schmidt3, Sebastian Hiller3, Jacques Pouysségur1,9 and Nathalie
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M. Mazure1
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University of Nice Sophia-Antipolis, Centre Antoine Lacassagne, 33 Avenue de Valombrose,
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06189 Nice, France. 2Centre Commun de Microscopie Appliquée, University of Nice Sophia-
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Antipolis, 28 Avenue Valombrose, 06103 Nice, France.
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Institute for Research on Cancer and Aging of Nice, CNRS-UMR 7284-Inserm U1081,
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Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland.
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Antipolis, Faculty of Medicine, 06107 Nice, France
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Léon Bérard
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28 rue Laennec, 69008 Lyon, France
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Louis Pasteur Hospital, Nice, France.
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Inserm U1013, Hôpital Necker, 149 rue de Sèvres, 75015 Paris, France
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Centre Scientifique de Monaco (CSM), Monaco
Laboratoire de PhysioMédecine Moléculaire, UMR 7370 CNRS, University of Nice Sophia-
URBC-NARILIS - University of Namur, Belgium Centre de Recherche en Cancérologie de Lyon, INSERM U1052, CNRS U5286, Centre
Human Tissue Biobank Unit/CRB and Laboratory of Clinical and Experimental Pathology,
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Corresponding author:
[email protected]
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Running title: Endolysosomal Modification of Hypoxic Mitochondria
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Keywords: apoptosis; asparaginyl endopeptidase; cancer metabolism; hypoxia; mitochondria;
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endolysosome; voltage-dependent anion channel.
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Abbreviations: AEP, asparaginyl endopeptidase; ATG, autophagy-related gene; BNIP3, Bcl-
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2/adenovirus
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phenylhydrazone; HIF-1, hypoxia-inducible factor-1; LC3, cytosolic light chain 3; Mfn1,
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mitofusin1; OXPHOS, oxidative phosphorylation; MEF, mouse embryonic fibroblasts;
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siRNA, small interfering RNA; STS, staurosporin; VDAC1, voltage-dependent anion channel
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1.
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Word count: Materials and Methods, 2 131 words; Combined Introduction, Results &
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Discussion, 3 240 words.
E1B
19-kDa
interacting
protein
3;
FCCP,
trifluorocarbonylcyanide
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2
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ABSTRCT
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The oxygen-limiting microenvironment (hypoxia) of tumors induces metabolic
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reprogramming and cell survival, but the underlying mechanisms implicating
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mitochondria remain poorly understood. We previously demonstrated that the hypoxia-
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inducible factor-1 mediated hyperfusion of mitochondria, by inducing Bcl-2/adenovirus
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E1B 19-kDa interacting protein 3, and post-translational truncation of the
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mitochondrial ATP transporter, the outer-membrane voltage-dependent anion channel
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1, in hypoxic cells. In addition, we showed that truncation was associated with increased
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resistance to drug-induced apoptosis and was indicative of increased patient
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chemoresistance. We now show that silencing of the tumor suppressor TP53 decreased
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truncation and increased drug-induced apoptosis. We also show that TP53 regulated
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truncation through induction of the mitochondrial protein Mieap. While we found that
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truncation was independent of mitophagy we observed local microfusion between
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mitochondria and endolysosomes in hypoxic cells in culture and in patient’s tumor
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tissue. Since we found that the endolysosomal asparagine endopeptidase was responsible
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for truncation, we propose that it is a readout of mitochondrial-endolysosomal
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microfusion in hypoxia. These novel findings provide the framework for a better
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understanding of hypoxic cell metabolism and cell survival through mitochondrial-
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endolysosomal microfusion regulated by the hypoxia-inducible factor-1 and TP53.
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INTRODUCTION
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Hypoxia is a natural occurring stress that results in compensatory changes in metabolism and
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cell survival during embryonic development and tumor growth. Hypoxia stabilizes and
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activates the transcription factor hypoxia-inducible factor (HIF) through inhibition of oxygen-
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dependent hydroxylases that earmark the alpha subunit of HIF for proteasomal degradation
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(1). HIF induces or represses the expression of genes implicated in a myriad of functions
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including those regulating metabolism and resistance to drug-induced cell death. Genes
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coding for the enzymes of the glycolytic pathway, including hexokinase, are highly induced
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by HIF-1 and this is in part responsible for the switch in metabolism from mitochondrial
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respiration to glycolysis in cancer cells. Considerable studies have pointed to the Warburg
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effect, also termed aerobic glycolysis, as the major adaptive response of cancer cells, but
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mitochondrial metabolism and mitochondrial dynamics are also starting to be recognized as
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important adaptive strategies of cancer cells (2). Mitochondria are critical organelles that
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regulate both metabolism and cell death. They are dynamic organelles that continuously
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undergo fission and fusion during cell growth (3, 4). Under stress conditions, such as nutrient
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depletion or hypoxia, mitochondria either fragment or are degraded by HIF-dependent
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mitophagy (mitochondrial removal by autophagy) (5), or hyperfuse together to form
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elongated or rounded structures that optimize ATP production and promote cell survival (6-
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11).
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We reported previously that certain cell lines exposed to hypoxia contained enlarged
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mitochondria (6). We found that the mechanism was HIF-1- and Bcl-2/adenovirus E1B 19-
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kDa interacting protein 3 (BNIP3/BNIP3L)-dependent but that it was independent of
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mitophagy. In addition, the hypoxic cells were more resistant to stimulated cell death than
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normoxic cells (12). Furthermore, we reported that the mitochondrial outer membrane protein,
4
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the voltage-dependent anion channel 1 (VDAC1) was post-translationally C-terminal cleaved
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in these cells, in a HIF-1-dependent manner, and in human lung adenocarcinoma tissue (12).
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VDAC mediates transport of ions and small metabolites such as ADP/ATP, from and into
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mitochondria (13). Three mammalian isoforms of VDAC exist in eukaryotic cells. VDACs
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bind hexokinase, the first enzyme of the glycolytic pathway, and in so doing provide ATP for
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conversion of glucose to glucose-6-P. VDACs also play a key role in apoptosis through Ca2+
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regulation of VDAC1 expression and binding of anti-apoptotic proteins of the Bcl-2 family
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(14, 15).
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The TP53 transcription factor plays an important role in the response to and regulation of
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metabolic stress in cancer (16, 17). It is known that a TP53-inducible protein Mieap (also
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referred to as Spata18) (18) controls mitochondrial quality through interaction with the HIF-1-
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inducible protein BNIP3 (19). In addition, Mieap has been proposed to induce accumulation
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of lysosomal proteins within mitochondria by way of repairing damaged mitochondria (20).
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In the present study, we investigated further the mechanism behind the hypoxic regulation
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of the truncation of VDAC1. We proposed that enlarged hypoxic mitochondria make fusional
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contact with late endolysosomes through TP53-induced Mieap, in promoting cell survival.
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Furthermore, we report that VDAC1 is cleaved at loop 14 by the endolysosomal protease
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asparagine endopeptidase (also termed legumain). Intimate contact between mitochondria and
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vacuoles has only been described in yeast (21, 22) and in erythroid cells (23). This crosstalk
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between organelles was found to regulate lipid transport, cellular metabolism and iron
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transport. We now show that spatial and functional inter-organellar connection also exists in
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eukaryotes, both in cells in culture and in lung adenocarcinomas of patients. We propose
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finally that VDAC post-translational modification marks mitochondria for protection from
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mitophagy and reflects a survival strategy of hypoxic cancer cells in vitro and in vivo in
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patients.
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MATERIALS AND METHODS
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Cells and hypoxic conditions. Cells were grown in Dulbecco's Modified Eagle's Medium
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(DMEM) (Gibco-BRL) supplemented with 5 or 10% inactivated fetal bovine serum as
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appropriate. Dr. van de Wetering provided the LS174 cells. MEF p53+/+ and -/- were
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provided by Dr P Roux. An INVIVO2 200 anaerobic workstation (Ruskinn Technology
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Biotrace International Plc) set at 1% O2, 94% N2 and 5% CO2 was used for hypoxic
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incubation. HeLa cells were incubated for 48h in hypoxia while the others, unless otherwise
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indicated, were incubated for 72h.
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Pharmacological inhibitors and chemicals. Bafilomycin A1 was purchased from
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Calbiochem. Brefeldin A, chloroquine diphosphate, LY-294002, 3-methyladenine, rapamycin,
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valinomycin,
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trifluorocarbonylcyanide phenylhydrazone were from Sigma.
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Transfection of plasmids and RNA interference. The plasmid YFP-Parkin was purchased
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from Addgene (24). The 21-nucleotide RNAs were synthesized by Eurogentec or OriGene.
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HeLa or LS174 cells were transfected with 40 or 100nM of siRNA twice at an interval of 24h
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prior to normoxia or hypoxia, as described (6).
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The
pepstatin
siRNA
A,
sequences
E64d,
were
rotenone
as
antimycin,
follows: VDAC1
oligomycin
VDAC1 3’UTR
(forward) (forward)
and
5’5’-
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GAUACACUCAGACUCUAAA
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CUCCAGGUUAAAGUUGAUUCA-3’,
133
GAAGAUUUGGCCUUAAUAU-3’,
siRNA
TP53#1
(forward)
5’-
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GACUCCAGUGGUAAUCUAC-3’,
siRNA
TP53#3
(forward)
5’-
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UAUGGCGGGAGGUAGACUG_3’,
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previously (25), siRNA ATG4B (forward) 5’-CUGAAGAUGACUUCAAUGA-3’, siRNA
-3’,
VDAC2
(forward)
siATG5/ATG6/ATG7/Beclin
were
5’-
described
6
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LAMP2 (forward) 5’-GAAAAUGCCACUUGCCUUU-3’, siRNA Mieap#1 (forward) 5’-
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GUAGCAGUGACUUAAGGCUAAG-3’,
siRNA
Mieap#2
(forward)
5’-
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GGUGCAGGGACAACUCUUUGGG-3’,
siRNA
Mieap#3
(forward)
5’-
140
GAGAUAUGUUGCAUUGCCUUUGC-5’,
siRNA
Mieap#4
(forward)
5’-
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GCACUAUCUGCCUAGGUAACUGC-5’,
siRNA
Lamp1A
(forward)
5’-
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AGAAAUGCAACACGUUA-5’,
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GCUGUGCGGUCUUAUGCAU-5’, siBNIP3 et siB3L were described previously (25),
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siMfn1 were previously described (12), Three siRNA to asparagine endopeptidase and a
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scrambled siRNA were purchased from Origene; the sequences were not disclosed.
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Caspase activation and cell death induced by staurosporin. Quantification of the caspase
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3/7 activity was done using a luciferin/luciferase based assay (Caspase-Glo 3/7 kit, Promega)
148
according to the manufacturer’s instructions. Each condition was performed 8 times and the
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entire experiment was performed three times. Significant differences are based on the
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Student’s t test * p < 0.005. Staurosporin (1 µM) was added 4h prior to assay for caspase 3/7
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activity. Cell death was also determined by trypan blue exclusion and confirmed with an
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ADAM cell counter.
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Transmission electron microscopy. Cells were fixed in situ with 1.6% glutaraldehyde in
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0.1M phosphate buffer at room temperature for at least 1h and then conserved at 4°C.
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Samples were rinsed in the same buffer and then postfixed with 1% osmium tetroxide and 1%
156
potassium ferrocyanide in 0.1M cacodylate buffer for 1h at room temperature to enhance the
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staining of cytoplasmic membranes. Cells were rinsed with distilled water, dehydrated in
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alcohol and embedded in epoxy resin. Embedded samples were then processed for ultrathin
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sectioning and observed with a JEM1400 transmission electron microscope (Jeol, Tokyo,
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Japan) equipped with a Morada CCD camera (Olympus SIS, Rungis, France).
siRNA
Lamp2A
(forward)
5’-
7
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For immunogold staining, cells were fixed with 4% paraformaldehyde, 0.1%
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glutaraldehyde in 0.1M phosphate buffer (PB) (pH 7.4) for 2h and were processed for
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ultracryomicrotomy according to a slightly modified Tokuyasu method. In brief, a cell
164
suspension was spun down in 10% gelatin. After immersion in 2.3M sucrose (in pH 7.4, 0.1M
165
PB) overnight at 4°C, the samples were rapidly frozen in liquid nitrogen. Ultrathin (70nm
166
thick) cryosections were prepared with an ultracryomicrotome (Leica EMFCS, Austria) and
167
mounted on formvar-coated nickel grids (Electron Microscopy Sciences, Fort Washington,
168
PA, USA). Immunostaining was performed with an automated immunogold labeling system
169
Leica EM IGL as following: the grids were incubated successively in PBS containing 50mM
170
NH4Cl (2x 5min), PBS containing 1% BSA (2x 5min.), PBS containing the relevant primary
171
antibody (Abcam: VDAC1, ab15895; Lamp1, ab25630. Lamp2, ab25631) in 1% BSA for 1h,
172
PBS containing 0.1% BSA (3x 5min), PBS containing 1% BSA and 15nm colloidal gold
173
conjugated protein AG (CMC, University Medical Center, Utrecht, The Netherlands), PBS
174
containing 0.1% BSA for 5min, PBS for 5min twice. Lastly, the samples were fixed for
175
10min with 1% glutaraldehyde, rinsed in distilled water and were contrasted with a mixture of
176
methylcellulose/sucrose and 0.3% uranyl acetate on ice. After drying in air, sections were
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examined under a JEOL 1400 transmission electron microscope. For lamp1 and lamp2
178
staining with mouse primary antibodies, an additional step was added before the incubation
179
with the colloidal gold protein AG, the grids were incubated with a secondary rabbit antibody
180
raised against mouse IgG (DAKO Z0259).
181
Respirometry and extracellular acidification. The cellular oxygen consumption rate (OCR)
182
and extracellular acidification rate (ECAR) were obtained using a Seahorse XF96
183
extracellular flux analyzer from Seahorse Bioscience (North Billerica, MA, USA). The final
184
concentrations of the agents are given in the legends. Experiments were performed according
185
to the manufacturer’s instructions. Protein standardization was performed after each
8
186
experiment, with no noticeable differences in protein concentration and cell phenotype.
187
Immunoblotting. For immunoblotting cells were lysed in 1.5x SDS buffer and the protein
188
concentration determined using the BCA assay. 40µg of protein of whole cell extracts was
189
resolved by SDS-PAGE and transferred onto a PVDF membrane (Millipore). Membranes
190
were blocked in 5% non-fat milk in TN buffer (50mM Tris-HCl pH7.4, 150mM NaCl) and
191
incubated in the presence of the primary and then secondary antibodies in 5% non-fat milk in
192
TN buffer. The rabbit polyclonal antibody to central regions of VDAC1 was purchased from
193
Abcam (ab15895). Rabbit polyclonal anti-HIF-1
194
and characterized in our laboratory (26). Anti-phospho-TP53 (Ser15) (Cell Signaling
195
Technology (Boston, MA, USA), anti-TA TP73 (IMG-246, Imgenex), anti-Mieap (Novus
196
Biologicals, Cambridge, UK), anti-p62 (Sigma-Aldrich (St Louis, MO, USA), anti-TP63,
197
anti-beclin 1 (Novus Biological, Cambridge, UK). Mouse anti--tubulin and -actin were
198
from Sigma, Anti-asparaginyl endopeptidase (Sigma Prestige) was from Sigma. Anti-BNIP3
199
and BNIP3L were described previously (25). After washing in TN buffer containing 1%
200
Triton-X100 and then in TN buffer, immunoreactive bands were visualized with the ECL
201
system (Amersham Biosciences). The ECL signal for -tubulin or -actin were used as a
202
loading control.
203
Pull-down with BNIP3 and mass spectrometry. HepG2 cells with stable expression of the
204
Tet repressor (pcDNA6/TR, Life Technologies) were transiently transfected with the pcDNA
205
4/TO plasmid expressing HaloTag-BNIP3 fusion protein using lipofectamine. 24h after the
206
transfection, cells were then incubated in the presence of 1µg/ml tetracyclin to induce
207
HaloTag-Bnip3 expression and incubated without or with etoposide 50µM in hypoxia (1%
208
O2) for 16h.
209
Protein pull-down was performed according to the HaloTag® Mammalian Pull-Down
210
System protocol (Promega). EV protease cleavage (30 units of PROTEV proteases in 50 l of
9
211
protev cleavage buffer for 1h at 25°C) was used to isolate the entire complex including the
212
bait protein BNIP3 fused to the HaloTag. After elution, proteins were boiled 5min with PPS
213
(3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate)
214
Discovery; 0.8% final concentration), reduced for 30min at 50°C with DTT (5mM) and then
215
alkylated for 30min in dark using iodoacetamide (15mM). Samples were then digested
216
overnight at 37°C with trypsin (Trypsin Gold, Mass Spectrometry Grade; Promega). Prior to
217
MS, samples were acidified with 1µl of 12N HCl and PPS detergent was hydrolysed after 45-
218
min incubation at 37°C followed by centrifugation (10min, 16 000 X g) at 4°C. Peptides were
219
analyzed using a nano-LC-ESI-MS/MS maXis Impact UHR-TOF (Bruker, Bremen,
220
Germany) coupled with a nanoLC UltiMate 3000 (Thermo). The digests were separated by
221
reverse-phase liquid chromatography using a 75µm X 250mm reverse phase Thermo column
222
(Acclaim PepMap 100 C18) in an Ultimate 3000 liquid chromatography system. The mobile
223
phase A was 95 % water/5% acetonitrile, 0.1% formic acid. Mobile phase B was 20%
224
water/80 % acetonitrile, 0.1% formic acid. The digest (8 µl) was injected, and the organic
225
content of the mobile phase was increased linearly from 5%B to 40% in 85min and from 40%
226
B to 100% B in 10min. The column effluent was connected to a Captive Spray (Bruker). In
227
survey scan, MS spectra were acquired for 0.5 s in the m/z range between 50 and 2200. The
228
15 most intense peptides ions 2+ or 3+ were sequenced. The collision-induced dissociation
229
(CID) energy was automatically set according to mass to charge (m/z) ratio and charge state
230
of the precursor ion. MaXis and Thermo systems were piloted by Compass HyStar 3.2
231
(Bruker).
Silent
Surfactant
(Protein
232
Peak lists were created using DataAnalysis 4.0 (Bruker) and saved as a MGF file for use
233
with ProteinScape 3.1 (Bruker) with Mascot 2.4 as search engine (Matrix Science). Enzyme
234
specificity was set to trypsin and the maximum number of missed cleavages per peptide was
235
set to one. Carbamidomethylation, oxidation of methionine and Gln–pyro-Glu were allowed
10
236
as variable modification. Mass tolerance for monoisotopic peptide window was 7 ppm and the
237
MS/MS tolerance window was set to 0.05Da. The peak lists were searched against the
238
mammalian database (02122011, 2075986 entries) with an automatic decoy database search.
239
Scaffold 4.3 (Proteome Software) was used to validate MS/MS based peptide and protein
240
identification. Peptide identifications were accepted if they could be established at greater
241
than 95% probability by the peptide prophet algorithm (27) with Scaffold delta-identified
242
peptides. Protein identification was accepted if they could be established at greater than 5.0%
243
probability to achieve an FDR less than 1.0% and contained at least 2 identified peptides.
244
Protein probabilities were assigned with the Protein Prophet algorithm (28).
245
Mass spectrometry of VDAC1 and VDAC1-C. Imperial blue SDS-PAGE stained excised
246
bands corresponding to full length VDAC1 from normoxic and hypoxic cell mitochondria and
247
VDAC1-C from hypoxic HeLa cell mitochondria were digested in gel with trypsin as
248
recently described (29). For LC-MS analysis, peptides were separated on a RP-LC column
249
(75 μm x 20 cm) packed in-house with C18 resin (Magic C18 AQ 3 μ m; Michrom
250
BioResources, Auburn, CA, USA) using a linear gradient from 95% solvent A (98% water,
251
2% acetonitrile, 0.15% formic acid) and 5% solvent B (98% acetonitrile, 2% water, 0.15%
252
formic acid) to 30% solvent B over 40 minutes at a flow rate of 0.2 μl/min. Each survey scan
253
acquired in the Orbitrap at 60,000 FWHM was followed by 20 MS/MS scans of the most
254
intense precursor ions in the linear ion trap with enabled dynamic exclusion for 20 sec.
255
Charge state screening was employed to select for ions with at least two charges and rejecting
256
ions with undetermined charge state. The normalized collision energy was set to 32% and one
257
microscan was acquired for each spectrum. The ion accumulation time was set to 300ms (MS)
258
and 50ms (MS/MS).
259
For peptide identification, raw files were converted to mgf-format using the MM-
260
conversion tool (version 3.9, www.massmatrix.org) and searched against a decoy (consisting
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261
of forward and reverse protein sequences) human SwissProt database (download date
262
16/05/2012) containing VDAC and known contaminants resulting in a total of 41,251 protein
263
sequences using Mascot (Matrix Science, version 2.4). The search parameters were set as
264
follows; semi tryptic specificity was required, up to two missed cleavages were allowed,
265
carbamidomethyl C was set as fixed and oxidation (M) as a variable modification, 10 ppm
266
precursor mass tolerance and 0.6 Da fragment mass tolerance for CID tandem mass spectra.
267
After importing the data to the Scaffold software (http://www.proteomesoftware.com, version
268
4.2.1) the FDR rate was set to
