Cancer Letters, Journal Year: 2019, Volume and Issue: 471, P. 72 - 87
Published: Dec. 12, 2019
Language: Английский
Cell Reports, Journal Year: 2025, Volume and Issue: 44(1), P. 115171 - 115171
Published: Jan. 1, 2025
Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, arises in skeletal muscle and remains an undifferentiated state due to transcriptional post-transcriptional regulators. Among its subtypes, fusion-negative RMS (FN-RMS) accounts for majority of diagnoses population. MicroRNAs (miRNAs) are non-coding RNAs that modulate cell identity via regulation messenger (mRNAs). In this study, we identify miRNAs impacting FN-RMS identity, revealing miR-449a miR-340 as major regulators cycle p53 signaling. Through miR-eCLIP technology, demonstrate directly target transcripts involved glycolysis mitochondrial pyruvate transport, inhibiting carrier (MPC) complex. Pharmacological MPC inhibition induces a similar metabolic shift, reducing metastatic potential leading exit. Overall, miR-449 orchestrate positioning strategy shift cells toward non-tumorigenic, quiescent state.
Language: Английский
Citations
1
Cell Death Discovery, Journal Year: 2025, Volume and Issue: 11(1)
Published: Feb. 20, 2025
Recent progress in cancer metabolism research has identified lactylation as a critical post-translational modification influencing tumor development and progression. The process relies on lactate accumulation the activation of lactate-sensitive acyltransferases. Beyond its role epigenetic regulation, emerged significant factor evolution, offering fresh opportunities for developing targeted therapies that transcend traditional approaches. This review explores growing importance biology highlights potential advancing diagnostic tools therapeutic strategies.
Language: Английский
Citations
1
Cell Reports, Journal Year: 2019, Volume and Issue: 28(10), P. 2608 - 2619.e6
Published: Sept. 1, 2019
Hepatocellular carcinoma (HCC) is a devastating cancer increasingly caused by non-alcoholic fatty liver disease (NAFLD). Disrupting the Mitochondrial Pyruvate Carrier (MPC) in mice attenuates NAFLD. Thus, we considered whether MPC disruption also prevents HCC. Here, use N-nitrosodiethylamine plus carbon tetrachloride model of HCC development to test how liver-specific knock out affects hepatocellular tumorigenesis. Our data show that ablation markedly decreases tumorigenesis and MPC-deficient tumors transcriptomically downregulate glutathione metabolism. We observe depletion cultured hepatomas are synthetically lethal. Stable isotope tracing shows hepatocyte reroutes glutamine from synthesis into tricarboxylic acid (TCA) cycle. These results support where inducing metabolic competition for impairs limiting synthesis. findings raise possibility combining stress may be therapeutically useful additional cancers.
Language: Английский
Citations
72
The EMBO Journal, Journal Year: 2019, Volume and Issue: 38(10)
Published: April 12, 2019
Article12 April 2019Open Access Transparent process The yeast mitochondrial pyruvate carrier is a hetero-dimer in its functional state Sotiria Tavoulari Corresponding Author [email protected] orcid.org/0000-0002-4263-8905 Medical Research Council Mitochondrial Biology Unit, University of Cambridge, UK Search for more papers by this author Chancievan Thangaratnarajah Vasiliki Mavridou Michael E Harbour Jean-Claude Martinou Department Cell Biology, Geneva, Genève 4, Switzerland Edmund RS Kunji orcid.org/0000-0002-0610-4500 Information *,1, Thangaratnarajah1,3, Mavridou1, Harbour1, Martinou2 and *,1 1Medical 2Department 3Present address: Groningen Biomolecular Sciences Biotechnology Institute, Membrane Enzymology, Groningen, Netherlands *Corresponding author. Tel: +441223252850; Fax: +441223252875; E-mail: EMBO Journal (2019)38:e100785https://doi.org/10.15252/embj.2018100785 PDFDownload PDF article text main figures. Peer ReviewDownload summary the editorial decision including letters, reviewer comments responses to feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract (MPC) critical cellular homeostasis, as it required central metabolism transporting from cytosol into matrix. MPC has been implicated many diseases being investigated drug target. A few years ago, small membrane proteins, called MPC1 MPC2 mammals Mpc1, Mpc2 Mpc3 yeast, were proposed form large protein complexes responsible function. However, have never isolated their composition, oligomeric properties not defined. Here, we identify unit Saccharomyces cerevisiae. In contrast earlier hypotheses, demonstrate that hetero-dimer, multimeric complex. When engaged hetero-dimers, Mpc proteins can also homo-dimers are, however, inactive. We show described substrate transport inhibitor profiles are embodied hetero-dimer. This work provides foundation elucidating structure complex mechanism inhibition. Synopsis Yeast be containing different combinations three protomers. heterodimers Mpc1/Mpc3 Mpc1/Mpc2 found constitute units respiratory fermentative conditions, respectively. cerevisiae purified functionally reconstituted liposomes. Reconstituted pH-gradient-dependent manner inhibited known inhibitors. Individual homodimers, which however functional. Introduction recent years, there an increasing understanding appreciation involved major human diseases, such cancer, neurodegeneration, cardiovascular metabolic disorders, obesity diabetes. key player fate cell (MPC), uptake matrix (Vanderperre et al, 2015), where enters tricarboxylic acid cycle other biosynthetic pathways. existence across inner had supported early on mitochondria, shown saturating pH-dependent (Papa 1971; Papa Paradies, 1974). addition, small-molecule inhibitors identified support notion (Halestrap Denton, 1974; Halestrap, 1975, 1976), but molecular identity remained unknown four decades. Major progress was made 2012, when activity discovered associated with two homologous (Bricker 2012; Herzig 2012). Drosophila, expression both necessary cerevisiae, expressed carbon source-dependent pattern, forming under conditions MPCFERM MPCOX complexes, respectively (Bender 2015; Compan 2015). As identification intensified efforts understand role cancer (Schell 2014, 2017; Yang 2014; Zhong Li 2016, Corbet 2018; Ohashi Bader 2019), diabetes (Colca 2013; Divakaruni Vigueira Gray McCommis 2015, 2016; Vadvalkar 2017) neurodegeneration (Ghosh Quansah 2018). Moreover, pathogenic mutations mpc1 gene rare severe syndromes, further enhancing clinical relevance transporter Additionally, number drugs, previously targets, now inhibit Du Ghosh Nancolas Nath Chen newly target first-generation insulin sensitisers, thiazolidinediones (TZDs; Colca 2013), originally exert action peroxisome proliferator-activated receptor gamma (PPARγ; Soccio Nanjan More recently, new generation TZD, bypassing PPARγ 2014) currently trials treatment Parkinson's disease, inhibiting 2016). Despite these advances, direct experimental system measure interactions drugs study inhibition available. Six after primary 2012), no report successful purification reconstitution hetero-complex. Consequently, composition protomer stoichiometry remain controversial Bender Nagampalli 2018) involvement questioned (Halestrap, hetero-complexes migrate at 150 kDa 2015) or even higher weights blue native gel electrophoresis, leading proposals might include additional, yet unidentified application chemical cross-linking produced bands corresponding monomers, dimers oligomers only published attempt purify hetero-complex, possible individual protomers (Nagampalli It high-order multi-species capable 2018), raising questions regarding carrier. first characterisation providing model future structural mechanistic studies. natural pyruvate. absence protomers, homo-dimers, they do Results Expression Although established still outstanding most straightforward way settle issues through components putative determine whether activity. used S. (Fig EV1) decided concentrate complex, principle oxidative phosphorylation (MPCOX; related MPCs organisms, whereas (MPCFERM), exists some fungi. principal approach hetero-complex tagging one Factor Xa cleavage site eight-histidine tag C-terminus 1A). To achieve co-expression pair, inducible bidirectional vector (Miller 1998) mitochondria mpc triple deletion strain SHY15 (Herzig C-terminally tagged Mpc1 individually mitochondria. Click here expand figure. Figure EV1. Topology Mpc3The alignment generated Clustal Omega manual curation (Sievers 2011). aligned residues coloured ZAPPO colour scheme aliphatic, polar, aromatic, positively charged, negatively Pro/Gly Cys pink, green, orange, blue, red, magenta yellow, asterisks indicate identical colon conserved substitutions. Also indicated transmembrane helices, loop regions N-terminal amphipathic helix. secondary elements assigned based PSIPRED (Buchan MEMSAT3 (Jones 1994) conservation analysis. Download figure PowerPoint 1. Purification stability analysis Strategy nickel-affinity chromatography. assessed SDS–PAGE immunoblot crude preparations. untagged (Mpc1), histidine-tagged (Mpc1his), (Mpc3his) (Mpc1/Mpc3his) detected antibodies raised against (left panel) (right dashed arrows. Five micrograms each affinity-purified analysed SDS–PAGE, visualised Coomassie Blue stain. Peptide mass finger printing (Table EV1). via thermal denaturation fluorescent CPM-adduct formation. (left) calculate derivative (right), apparent melting temperature, same coding. samples, panel (D), NanoDSF. changes 330 nm/350 nm ratio temperature (right). Colour coding (D). All well, presence partner 1B). notable though alone additional prominent band appeared, approximately weight SDS-resistant dimer, immunoblotting preparations 1B, left panel). expressing For reasons, chose co-express unmodified together With strategy, present 1:1 1C). highest yield (~1 mg per g mitochondria) achieved detergent lauryl maltose neopentyl glycol (LMNG), supplemented tetraoleoyl cardiolipin. stoichiometric could Triton X-100, decyl (DMNG) n-dodecyl β-D-maltoside (DDM), cardiolipin, albeit lower yields EV2). LMNG eluted single peak size-exclusion chromatography, indicating monodisperse EV3). fractions showed during purification, consistent stable EV3A, inset). own cardiolipin 1C), least times lower, despite similar levels 1B), issues. After histidine-tag cleavage, contained again dimer peptide fingerprinting EV2. detergentsThe Mpc1/Mpc3p X-100. solubilisation performed Materials Methods buffer 2% (w/v) DDM DMNG 1% affinity nickel Sepharose columns washed wash buffers 0.1% DDM, X-100 0.1 mg/ml (TOCL). samples solubilisate (Sol), remaining resin (B) flow-through (FT). Asterisks proteins. EV3. Size-exclusion chromatography 500 μg nickel-affinity-purified A280 profiles, symmetrical peak. Mpc3. Mpc1/Mpc3. Data information: Insets (A B) collected Next, thermostability evaluate folding components. purpose, monitored unfolding populations ramp 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM), reacts cysteine becoming exposed due 1D). residue very temperatures (52.0 51.7°C, respectively), clearly showing folded solution. own, did display profile, high fluorescence throughout ramp. result means either unfolded already exposed. discriminate between possibilities, nano differential scanning fluorimetry (nanoDSF; Fig 1E), relies environment endogenous tyrosine tryptophan residues. Again, temperatures, 48.5 48.0°C, respectively, those obtained CPM. 64.5°C, correspond possibly aggregation artefact. ability demonstrated (MPCFERM) using histidine EV4A B). compared Mpc2, EV4). equally well EV4A), sufficient quantities EV4B Table Interestingly though, together, successfully formed 42°C EV4C). EV4. (Mpc2his) (Mpc1/Mpc2his) knock-out low, visible stain, CPM method. Thermal (upper data (lower Time course homo-exchange liposomes comparison empty ΔpH 1.6 (n = 2). hetero-complexes, demonstrating interactions. Of yields, 10°C thermostable was, therefore, selected state. molar does resolve issue overall Previous kDa, electrophoretic mobility electrophoresis 2012) largely accepted literature. gels depends lipids extraction, detergent-lipid micelle (DL) bind anomalous migration (Crichton 2013). Therefore, appropriate technique sizing Another involves 7 15 Å long cross-linkers. cross-linked appeared multiple cannot excluded states non-specific events. determined mass, subunit linked multi-angle laser light scattering (SEC-MALLS; 2A protein–detergent–lipid (PDL) itself (Slotboom 2008; ter Beek average contributing 30.8 ± 1.4 163.3 5.1 EV2 2B). corresponded sum theoretical masses (15 kDa) (17.1 kDa), demonstrates Similar results step 2. dimeric Nickel-affinity-purified SEC-MALLS analysis, trace black line. (PDL, green), detergent–lipid (DL, blue) (P, red) indicated. Protein staining (B, Mpc3, designation (B). (D, inset) (B D) represent characteristic experiment repeated independently five biological repeats summarised exclude stoichiometries, internal consistency (Wen 1996), calculated experimentally mass. hetero-dimeric smallest difference 1). since co-purify equimolar amounts 2B, inset), possibility corresponds separate eliminated. Stoichiometry Absorbance (=1 g/l) silico Mw (kDa) Difference Monomer 1 0 1.628 32.92 17.92 1.868 17.12 28.69 11.57 Homo-dimer 2 1.633 29.97 32.9 2.93 1.869 34.21 28.68 −5.53 Hetero-dimer 1.757 32.1 30.83 −1.27 Homo-trimer 3 1.629 44.95 −12.05 51.31 29.6 −21.71 Hetero-trimer 1.716 47.07 31.21 −15.86 1.796 49.19 30.12 −19.07 Since (Figs 1C EV3) solution, 2C D). 182.9 7.9 34.8 0.7 2D), twice kDa). Thus, forms Mpc1. active investigate transport, protocol. prepared proteoliposomes loaded 5 mM unlabelled pH 8.0 3) 7.4 EV5), initiated addition radiolabelled (50 μM) outside. dependence our assays external EV5). 3. transports 8) 6) 1.6. Kinetic 3). assayed initial rates concentration range 25–600 μM. KM 299 μM 318 409 repeats. physiological 4) 4). ΔpH, time Inhibition [14C]-pyruvate UK5099 (1–100 μM), Zaprinast (1–1,000 lonidamine (10–10,000 7ACC2 (5–500 μM). points mean technical replicates typical experiment. IC50 measurements replicated, UK5099, (average IC50: 9 7, 18 8 27 13 μM, respectively) 118 24 TZDs, pioglitazone rosiglitazone 6). replicated: (A) repeats, (C, (E, F) Zaprinast, TZDs. error bars standard deviation D F). EV5. Effect Mpc1/Mpc3-containing diffusion liposomesPyruvate tested conditions. At 1.0, 6.4 2.0, 5.4 observed diffusion, accumulation liposomes, acidic 5.4, reached than proteoliposomes. previous studies suggesting cross "absorption", depending protonation (Klingenberg, 1970; Zahlten 1972; Bakker van Dam, selection critical. Our finalised protocol achieving maximal minimal. 6.4, yieldi
Language: Английский
Citations
60
Cancer Letters, Journal Year: 2019, Volume and Issue: 471, P. 72 - 87
Published: Dec. 12, 2019
Language: Английский
Citations
58