Abstract
Glutamine synthetase, encoded by the geneGLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion ofGlulin endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cellsGLULknockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced byGLULknockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation.
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Data availability
Figures1,4,5and Extended Data Figs.1,7, and8have associated raw data (uncropped blots and/or gel pictures) in Supplementary Fig. 1. Figures1,2and Extended Data Figs.1,4have associated raw data (Excel files) for all bar graphs representing data from experiments involving mouse models. For the molecular modelling of palmitoyl-CoA docking into GS, models and trajectories are available on Figshare (doi: 10.6084/m9.figshare.6575438). Any additional information required to interpret, replicate or build upon the Methods or findings reported in the manuscript is available from the corresponding author upon request.
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Acknowledgements
We acknowledge R. Levine for supplying purified bacterial GS; L. Van Den Bosch and W. Scheveneels for providing primary mouse astrocytes; S. Trenson, I. Crèvecoeur, S. Noppen, L. Van Berckelaer and R. Van Berwaer for technical assistance; S.-M. Fendt, D. Verdegem and C. Ulens for discussions and suggestions; W. Vermaelen, A. Acosta Sanchez, A. Brajic, A. Sobrino and M. Cockx for experimental assistance; L.-C. Conradi and A. Pircher for supplying materials; and E. Wauters, A. Wolthuis and J. Jaekers for providing tissues for EC isolations. HecBioSim and PRACE are acknowledged for allocation of computer time. J.G., A.R.C., C.D., C.L., J.K., F.M.-R., S.R. and S.Va. are supported by the FWO; A.Z. by LE&RN/FDRS; B.C. by IWT; U.B. by a Marie Curie-IEF Fellowship; H.H. by an EMBO Long-Term Fellowship; J.D.v.B. by a LSBR fellowship; R.Cu. by a British Heart Foundation Intermediate Clinical Fellowship; and X.W. by the American Cancer Society RSG, NIH/NCI and NIH/NIDDK. F.C., G.S. and F.L.G. are supported by the EPSRC; X.L. by the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center at Sun Yat-Sen University, and the National Natural Science Foundation of China (81330021 and 81670855). P.C. is supported by a Federal Government Belgium grant, long-term structural Methusalem funding by the Flemish Government, a Concerted Research Activities Belgium grant, grants from the FWO, Foundation against Cancer and ERC Advanced Research Grant. G.E. and M.Dew. received a Foundation against Cancer grant and G.E. received a FWO ‘Krediet aan navorsers’.
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Contributions
P.C. conceived the concept of the study and provided supervision. G.E., P.C., X.L., M.Dew. and L.S. contributed to the execution, support and analysis of experiments, and/or provided advice. G.E., C.D., A.R.C., J.G. and P.C. were involved in the experimental design. G.E., C.D., A.R.C., J.G., U.B., A.Z., H.H., S.Va., J.K., C.L., F.M.-R., B.C., L.R., S.Vi., K.B., S.W., J.S., L.S., S.L., R.Ch., R.Cu. and M.Dew. carried out molecular biology and in vivo experiments. B.G. performed mass spectrometry. J.v.R. and J.D.v.B. carried out RHO activity assays. M.DeR., G.J. and X.W. undertook palmitoylation experiments. G.S., F.C. and F.L.G. performed molecular dynamics simulations. S.R. and J.Ho. performed bimolecular fluorescence complementation and SPT experiments. G.E., C.D., A.R.C., J.G., R.Cu., U.B., C.L., S.R., L.T., B.C., M.Dew., J.Ho., S.L., B.G., F.L.G., J.D.v.B., X.W. and P.C. interpreted the data. W.H.L., Y.W. and J.Ha. provided necessary materials. G.E. and P.C. drafted the manuscript. All authors agreed on the final version of the manuscript.
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Extended data figures and tables
Extended Data Fig. 1GLULknockout impairs vessel sprouting.
a,GLULmRNA levels in HUVECs (n = 9 donors), lung ECs (n = 5), colon ECs (n = 4), liver ECs (n = 3), human umbilical artery ECs (HUAECs) (n = 2) and human blood outgrowth ECs (BOECs) (n = 2); (mean ± s.e.m.; *P< 0.05 versus HUVEC, Student’st-test) and in HEPG2 cells (mean ± s.e.m.;n = 3; *P< 0.05 versus HUVEC, Student’st-test).b,c, Western blot of GS protein levels in HUVECs and HEPG2 cells in medium containing 0.6 mM glutamine (+) or 0.025 mM glutamine (−) (b), and in isolated mouse liver ECs (mLiECs) and mouse astrocytes (c) (representative immunoblots of two independent experiments are shown).d,e, Genomic organization of theloxP-flankedGlulallele before and after Cre-mediated excision (d) and correct recombination of theloxallele (L) inGlulvECKOandGlulpECKOmice upon tamoxifen (Tam) treatment, as assessed by genomic DNA PCR (e; the PCR to amplify theloxP-flankedGlulallele (lox) or to amplify thecre-recombined allele (∆) were run in separate reactions but loaded in the same lane; the gel picture shown is representative of all control,GlulvECKOandGlulpECKOmice used in this study).f, Quantification of branchpoints at the rear of the plexus inGlulvECKOmice (mean ± s.e.m.;n = 10 mice forGlulvECKOand 11 for WT controls from 3 litters;*P< 0.05 versus WT littermates, mixed-models R statistics).g, Pericyte coverage of retinal microvessels in WT andGlulvECKOlittermates determined by NG2 staining and shown as the NG2+area as a percentage of the vessel area (mean ± s.e.m.;n = 4 mice for WT and 3 forGlulvECKOfrom 1 litter; NS,P > 0.05 versus WT, Student’st-test).h, Reduced complexity of the retinal vascular front in P5GlulvECKOcompared with WT mice, determined by the number of branches on distal sprouts (mean ± s.e.m.;n = 13 mice for WT and 21 forGlulvECKOfrom 5 litters;*P< 0.05 versus WT, Student’st-test).i, Quantification of EdU+ECs at the rear of the plexus (mean ± s.e.m.;n = 12 mice for WT and 22 forGlulvECKOfrom 4 litters; NS, P > 0.05 versus WT littermates, Student’st-test).j–m, IB4 staining of P5 retinal vascular plexuses from WT (j) andGlulpECKO(k) mice (representative pictures with magnification shown in the inset; A, artery; V, vein) and quantification of branch points at the front (l) and the rear (m) of the plexus (mean ± s.e.m.;n = 10 mice for WT and 18 forGlulpECKOfrom 4 litters;*P ≤0.05 versus WT littermates, Student’st-test).n–u, IB4 staining of the retinal microvasculature of three-week-old (P21) (n,o) and six-week-old (P42) (r,s) WT andGlulvECKOlittermates (A, artery; V, vein). The lower left insets display a higher magnification of the IB4-stained superficial plexus, whereas the lower right insets display a higher magnification of the deep plexus. The corresponding quantification of the vascular area (p,t) and the branch point density (q,u) in the superficial and the deep layer is also shown (mean ± s.e.m.;n = 8 mice for WT and 8 forGlulvECKOat P21, from two litters;n = 10 mice for WT and 14 forGlulvECKOat P42, from four litters; NS,P > 0.05 versus WT, Student’st-test).v–ag, Representative micrographs of heart (v,z), liver (w,aa) and kidney (x,ab) sections from WT andGlulvECKOlittermates immunostained for the EC marker endoglin and of lung (y,ac) sections immunostained for the EC marker CD34 and corresponding quantifications of endoglin+(ad, heart;ae, liver;af, kidney) or CD34+(ag, lung) vascular area (mean ± s.e.m.;n = 5 mice (4 for heart) for WT and 7 (6 for heart) forGlulvECKO, from two litters, NS,P > 0.05 versus WT, Student’st-test).ah–ai, Images of flat-mounted retinas from control (ah) and MSO-treated (ai) ROP mice (vaso-obliterated area in white). Images shown are representative of 7 (ah) and 6 (ai) mice. ExactPvalues: HUVEC versus lung ECs: 0.0278; HUVEC versus colon ECs: 0.1086; HUVEC versus liver ECs: 0.3334; HUVEC versus HEPG2: <0.0001 (a）;<0.0001 (f）;0.3491 (g）;<0.0001 (h）;0.8247 (i）;0.0012 (l）;0.050 (m）;superficial: 0.1218; deep: 0.1720 (p）;superficial: 0.9995; deep: 0.4289 (q）;superficial: 0.9792; deep: 0.6602 (t）;superficial: 0.7979; deep: 0.1275 (u）;0.9021 (ad）;0.2279 (ae）;0.7647 (af）;0.3614 (ag). Scale bars: 200 μm (j,k,n,o,r,s), 20 μm (v–ac), 1 mm (ah–ai). For gel source images, see Supplementary Fig.1.
Extended Data Fig. 2 Effects of silencing and pharmacological inhibition of GS on EC viability and central metabolism.
a,GLULmRNA levels in control ECs and ECs transduced with two different non-overlapping shRNAs targetingGLUL(GLULKD1andGLULKD2;GLULKD1is used in the experiments in the text and is denotedGLULKD) or transfected with scrambled siRNA (SCR) or siRNA targetingGLUL(siGLUL). Data are expressed as a percentage of the respective control, denoted by the horizontal dotted line (mean ± s.e.m.;n = 28 independent experiments forGLULKD1,n = 3 independent experiments forGLULKD2andn = 9 independent experiments for siGLUL;*P< 0.05 versus the respective control; one-samplet-test).b,c, Quantification of number of sprouts (b) and total sprout length (c) for spheroid-sprouting assays withGLULKDECs和GLULKDECs expressing a shRNA-resistantGLULmutant (rGLULOE) (mean ± s.e.m.;n = 3 independent experiments;*P< 0.05 and NS,P > 0.05 versus control; one-way ANOVA with Dunnett’s multiple comparison versus control).d, Viability of control (Ctrl) andGLULKDECs as measured by lactate dehydrogenase (LDH) release assay (mean ± s.e.m.;n = 3 independent experiments; NS,P > 0.05 versus control, one-samplet-test).e, Intracellular levels of reactive oxygen species measured by CM-H2DCFDA staining (mean ± s.e.m.;n = 3 independent experiments; NS,P > 0.05 versus control, Student’st-test).f, Energy charge measurement (([ATP] + 1/2[ADP]) / ([ATP] + [ADP] + [AMP])) inGLULKDand control ECs (mean ± s.e.m.;n = 3 independent experiments; NS,P > 0.05 versus control, Student’st-test).g, Ratio of oxidized glutathione (GSSG) over total glutathione levels (GSSG/(GSH + GSSG)) inGLULKDand control ECs (mean ± s.e.m.;n = 4 independent experiments; NS,P > 0.05 versus control, Student’st-test).h, NADP/NADPH ratio inGLULKDand control ECs (mean ± s.e.m.;n = 5 independent experiments; NS,P > 0.05 versus control, one-samplet-test).i–k, Effect ofGLULknockdown on major metabolic fluxes including glycolysis (i), glucose oxidation (j) and glutamine oxidation (k) (mean ± s.e.m.;n = 3 independent experiments fori,n = 5 forjandn = 4 fork; NS,P > 0.05 versus control, one-samplet-test).l,m, Oxygen consumption rate (OCR) in control, MSO-treated andGLULKDECs in basal state and after injection of oligomycin, FCCP and antimycin A (l) (mean ± s.e.m.;n = 3 independent experiments), and calculation of OCRBAS, OCRATPand maximal respiration (m) (mean ± s.e.m.;n = 3 independent experiments). ExactPvalues:GLULKD1: <0.0001;GLULKD2: <0.0001; siGLUL: <0.0001 (a）;control versusGLULKD: 0.0147; control versusGLULKD+ rGLULOE: 0.9824 (b）;control versusGLULKD: 0.0083; control versusGLULKD+ rGLULOE: 0.6528 (c）;0.5717 (d）;0.8206 (e）;0.3715 (f）;0.4398 (g）;0.9291 (h）;0.4691 (i）;0.6643 (j）;0.6786 (k). CM-DCF, CM-H2DCFDA; OCRBAS, basal oxygen consumption rate; OCRATP, ATP-generating oxygen consumption rate; RFU, relative fluorescence units.
Extended Data Fig. 3GLULknockdown reduces EC motility.
a, Wound closure in control andGLULKD2EC monolayer scratch assays with or without pretreatment with mitomycin C (mean ± s.e.m.;n = 7 and 5 independent experiments with and without mitomycin C, respectively;*P< 0.05 versus corresponding control; Student’st-test).b, Quantification of lamellipodial area (as a percentage of total cellular area) in control andGLULKD2ECs (mean ± s.e.m.;n = 3 independent experiments;*P< 0.05 versus control; Student’st-test).c, Wound closure in monolayer scratch assays with SCR- and siGLUL-transfected ECs (mean ± s.e.m.;n = 5 independent experiments;*P< 0.05 versus SCR; Student’st-test).d, Quantification of lamellipodial area (as a percentage of total cellular area) in SCR- and siGLUL-transfected ECs (mean ± s.e.m.;n = 5 independent experiments;*P< 0.05 versus SCR; Student’st-test).e, Proliferation of SCR- and siGLUL-transfected ECs, as measured by [3H]thymidine incorporation into DNA (mean ± s.e.m.;n = 3 independent experiments; NS,P > 0.05 versus SCR; Student’st-test). ExactPvalues: control versusGLULKD2: 0.0290; control versusGLULKD2+ MitoC: 0.0223 (a）;0.0088 (b）;0.0407 (c）;0.0083 (d）;0.4335 (e).
Extended Data Fig. 4 Effects ofGLULsilencing on cytoskeleton and barrier function.
a–h, Images of control (a,c,e,g) andGLULKD(b,d,f,h) ECs after staining for α-tubulin (a,b), F-actin (c,d) and nuclear staining (e,f）;gandhshow merged images. The images shown are representative of 3 independent experiments.i–k, Representative images of phalloidin (F-actin) + Hoechst-stained liver ECs 6 h after isolation from control (i) and MSO-treated (j) mice, and corresponding quantification of F-actin levels (k) (mean ± s.e.m.;n = 5 mice per group;*P< 0.05 versus control, Student’st-test).l–n, Representative images of phalloidin-stained confluent monolayer control (l) andGLULKD(m) ECs aligning a scratch wound, and quantification of F-actin levels (n) (mean ± s.e.m.;n = 5 independent experiments;*P< 0.05 versus control, Student’st-test).o, Quantification of the length of discontinuous and continuous VE-cadherin-stained junctions in control andGLULKDECs (mean ± s.e.m.;n = 4 independent experiments;*P< 0.05 versus control, Student’st-test).p, Quantification of VE-cadherin gap size index in control andGLULKDEC monolayers (mean ± s.e.m.;n = 4 independent experiments;*P< 0.05 versus control, Student’st-test).q–v, Corresponding representative images of monolayer control (q,s,u) andGLULKD(r,t,v) ECs stained for VE-cadherin (q,r,u,v) and F-actin (s,t,u,v). Yellow arrows inrpoint to discontinuous VE-cadherin junctions and yellow asterisks indicate intracellular gaps.w, Quantification of transendothelial electrical resistance in control andGLULKDEC monolayers (mean ± s.e.m.;n = 4 independent experiments;*P< 0.05 versus control, Student’st-test at each time point).x–z, Quantification (x) of Evans blue dye extracted from the ears of control and MSO-treated mice, induced by topical application of mustard oil (n = 4 mice for each condition,*P< 0.05; Student’st-test), and representative pictures of the leakage of Evans blue dye into the ear tissue in control (y) and MSO-treated (z) mice. ExactPvalues: 0.0030 (k）;0.0036 (n）;continuous control versusGLULKD: 0.0005; discontinuous control versusGLULKD: 0.0005 (o）;0.0356 (p）;0.0181 (w）;0.0002 (x). Scale bars: 20 μm (a–h,l,m), 10 μm (i,j,q–v). AU, arbitrary units.
Extended Data Fig. 5 Enzymatic activity of GS and its role in EC migration.
a, Scheme for the15NH4+labelling of glutamate and glutamine with unlabelled carbons (blue) and labelled nitrogens (red).b,15N incorporation into glutamine (measured as the percentage of isotope enrichment in glutamine either asM+ 1 (singly labelled) orM+ 2 (doubly labelled), 30 min after adding15NH4+) in medium with dialysed serum and different glutamine concentrations (mean ± s.e.m.;n = 3 independent experiments; one-way ANOVA with Dunnett’s multiple comparisons versus 4 mM; *P< 0.05).c,15N incorporation into glutamate (measured as the percentage of isotope enrichment inM+ 1) and glutamine (measured as the percentage of isotope enrichment inM+ 1 (singly labelled) andM+ 2 (doubly labelled)), 30 min after adding increasing concentrations of15NH4Cl (mean ± s.e.m.;n = 3 independent experiments).d, Scheme of glutamine labelling from [U-13C]glutamate with unlabelled nitrogens (blue) and labelled carbons (red).e, Contribution of labelled [U-13C]glutamate to intracellular glutamine at various glutamine concentrations (percentage of isotope enrichment inM+ 5 glutamine and glutamate, 30 min after adding the tracer) (mean ± s.e.m.;n = 3 independent experiments; one-way ANOVA with Dunnett’s multiple comparisons versus 4 mM; *P< 0.05).f, Scheme for the contribution of carbons from [U-13C]glucose to glutamine, with labelled carbons (red) and unlabelled carbons (blue). Incorporation is shown after one turn of the tricarboxylic acid (TCA) cycle.g, Total contribution of carbons from [U-13C]glucose to α-ketoglutarate, glutamate and glutamine in ECs in medium with or without glutamine, 48 h after adding the tracer (mean ± s.e.m.;n = 3 independent experiments;*P< 0.05 versus total contribution in glutamine at 0.6 mM external glutamine, one-way ANOVA with Dunnett’s multiple comparisons).h, Incorporation of15N into glutamine (measured as the percentage of isotope enrichment inM+ 1 (singly labelled) andM+ 2 (doubly labelled), 30 min after adding15NH4+) in ECs and HEPG2 cells (mean ± s.e.m.;n = 4 independent experiments. ND, not detected).i,13C-glutamine uptake kinetics in control, MSO-treated andGLULKDECs和subsequent conversion to glutamate. SeeMethodsfor explanation of the different time points. Data are for theM+ 5 isotopomer, as a percentage of the total intracellular glutamine or glutamate pool (mean ± s.e.m.;n = 3 independent experiments, except for 30 min for whichn = 1 experiment; no statistical differences between control, MSO-treated andGLULKDECs were observed for glutamine or for glutamate; one-way ANOVA with Dunnett’s multiple comparison versus control at each time point; no statistical analysis was performed at 30 min).j,14C-glutamine uptake in control andGLULKDECs (mean ± s.e.m.;n = 5 independent experiments; NS,P > 0.05 versus control, one-samplet-test).k, Ratio of intracellular glutamine and glutamate levels in control andGLULKDECs (mean ± s.e.m.;n = 3 independent experiments; NS,P > 0.05 versus control, Student’st-test).l, Velocity measurement of control andGLULKDECs at different glutamine concentrations (mean ± s.e.m.;n = 4 independent experiments;*P< 0.05 versus corresponding control, mixed-models R statistics).m,n, Effect of glutamine concentration on sprout number (m) and total sprout length (n) in control andGLULKDspheroids (mean ± s.e.m.;n = 3 independent experiments; *P< 0.05 versus corresponding control, mixed-models R statistics).o,p, Number of sprouts per spheroid (o) and total sprout length (p) in control and MSO-treated EC spheroids (mean ± s.e.m.;n = 3 independent experiments;*P< 0.05 versus control, paired Student’st-test).q–s, Effect of MSO-treatment on EC motility parameters: wound closure of mitomycin C-treated ECs (q) (mean ± s.e.m.;n = 11 independent experiments; *P< 0.05 versus control, Student’st-test), lamellipodial area (r) (mean ± s.e.m.;n = 10 independent experiments; *P< 0.05 versus control, paired Student’st-test) and F-actin levels, 1 h after latrunculin wash-out (s) (mean ± s.e.m.;n = 4 independent experiments; *P< 0.05 versus control, one-samplet-test).t, [3在控制和MSO-treat H]胸腺嘧啶核苷掺入ed ECs (mean ± s.e.m.;n = 3 independent experiments; NS,P > 0.05 versus control, one-samplet-test). ExactPvalues:M+ 1 0.025 mM versusM+ 1 4 mM: 0.0096;M+ 1 0.6 mM versusM+ 1 4 mM: 0.1206;M+ 2 0.025 mM versusM+ 2 4 mM: 0.0839;M+ 2 0.6 mM versusM+ 2 4 mM: 0.9921 (b）;GluM+ 5 0.6 mM versus GluM+ 5 4 mM: 0.9372; GluM+ 5 0.025 mM + MSO versus GluM+ 5 4 mM: 0.0034; GluM+ 5 0.025 mM versus GluM+ 5 4 mM: 0.0215; GlnM+ 5 0.6 mM versus GlnM+ 5 4 mM: 0.9297; GlnM+ 5 0.025 mM + MSO versus GlnM+ 5 4 mM: 0.9961; GlnM+ 5 0.025 mM versus GlnM+ 5 4 mM: 0.0268 (e）;α-keto 0.6 mM versus Gln 0.6 mM: 0.0001; Glu 0.6 mM versus Gln 0.6 mM: 0.0001; Gln 0 mM versus Gln 0.6 mM: 0.0285 (g）;Gln 0.5 min: control versus MSO: 0.4846; control versusGLULKD: 0.5904; Gln 10 min: control versus MSO: 0.6709; control versusGLULKD: 0.6910; Gln 20 min: control versus MSO: 0.5896; control versusGLULKD: 0.6784; Glu 0.5 min: control versus MSO: 0.9774; control versusGLULKD: 0.8810; Glu 10 min: control versus MSO: 0.0502; control versusGLULKD: 0.9598; Glu 20 min: control versus MSO: 0.9782; control versusGLULKD: 0.7783 (i）;0.6623 (j）;0.6704 (k）;control versusGLULKD0.1 mM: 0.0054; control versusGLULKD0.6 mM: 0.0247 control versusGLULKD2 mM: 0.0017 (l）;control versusGLULKD0.6 mM and 10 mM: < 0.0001 (m）;control versusGLULKD0.6 mM and 10 mM: < 0.0001 (n）;0.0313 (o）;0.0075 (p）;0.0019 (q）;0.0116 (r）;0.0091 (s）;0.5110 (t). α-keto, α-ketoglutarate; GDH, glutamate dehydrogenase.
Extended Data Fig. 6 Rescuing the phenotype associated withGLULKDin vitro.
a, Schematic of the DORA–RHOA–FRET biosensor, depicting from N- to C-terminal the circular permutated RHOA effector protein kinase N (cpPKN), the dimeric circular permutated Venus (dcpVen), the ribosomal protein-based linkers (L9), the dimeric Cerulean3 (dCer3) and RHOA.b–m, Representative images of control (b–d), MSO-treated (e–g),GLULKD(h–j) andRHOJKD(k–m) ECs after staining for F-actin (phalloidin) (b,d,e,g,h,j,k,m) and pMLC (c,d,f,g,i,j,l,m).n, Quantification of the pMLC immunoreactivity (mean ± s.e.m.;n = 5 independent experiments;*P< 0.05 versus control, one-samplet-test).o–t, Representative images of control (o,q,s) andGLULKD(p,r,t) EC spheroids treated with vehicle (o,p) or the ROCK inhibitors Y27632 (q,r) or fasudil hydrochloride (Fasu.;s,t).u,v, Quantification of the number of sprouts per spheroid (u) and sprout length (v) (mean ± s.e.m.;n = 3 independent experiments;*P< 0.05 and NS,P> 0.05与未经处理的控制,单向的ANOVA with Dunnett’s multiple comparisons versus untreated control).w, Quantification of the lamellipodial area in vehicle- or fasudil-hydrochloride-treated control andGLULKDECs (mean ± s.e.m.;n = 6 independent experiments;*P< 0.05 and NS,P> 0.05与未经处理的控制,单向的ANOVA with Dunnett’s multiple comparisons versus untreated control).x, Quantification of the lamellipodial area in vehicle-, ML7- or peptide-18 (pep.18)-treatedGLULKDand control ECs (mean ± s.e.m.;n = 4 independent experiments of which 3 experiments included the ML7-treatment;*P< 0.05 versus untreated control, one-way ANOVA with Dunnett’s multiple comparisons versus untreated control).y, Scratch wound closure in vehicle-, ML7- or peptide-18-treatedGLULKDand control ECs (mean ± s.e.m.;n = 3 independent experiments;*P< 0.05 versus untreated control, one-way ANOVA with Dunnett’s multiple comparisons versus untreated control).z, Fold changes (versus untreated control ECs) in F-actin levels from phalloidin-stained vehicle-, ML7- or peptide-18-treatedGLULKDECs (mean ± s.e.m.;n = 4 independent experiments of which 3 included the peptide 18-treatment;*P< 0.05 versus untreated control, one-samplet-test).aa, Fold changes (versus untreated control ECs) in pMLC levels from pMLC-immunostained vehicle-, ML7- or peptide-18-treatedGLULKDECs (mean ± s.e.m.;n = 4 independent experiments of which 3 included the peptide 18-treatment;*P< 0.05 versus untreated control, one-samplet-test. ExactPvalues: MSO: 0.0372;GLULKD: 0.0060;RHOJKD: 0.0051 (n）;GLULKDversus control: 0.0045; Fasu. versus control: 0.9596;GLULKD+ Fasu. versus control: 0.8857 (u）;GLULKDversus control: 0.0199; Fasu. versus control: 0.8309;GLULKD+ Fasu. versus control: 0.9327 (v)GLULKDversus control: 0.0074; Fasu. versus control: 0.5906;GLULKD+ Fasu. versus control: 0.9900; (w）;GLULKDversus control: 0.0011;GLULKD+ ML7 versus control: 0.0079;GLULKD+ pep.18 versus control: 0.0017 (x）;GLULKDversus control: 0.0034;GLULKD+ ML7 versus control: 0.0022;GLULKD+ pep.18 versus control: 0.0040 (y）;GLULKD: 0.0058; ML7: 0.0072; pep.18: 0.0888 (z）;GLULKD: 0.0369; ML7: 0.0021; pep.18: 0.1672 (aa). Scale bars: 20 μm (b–m), 100 μm (o–t). For gel source images, see Supplementary Fig.1.
Extended Data Fig. 7 Rho GTPase localization and interaction with GS.
a, Co-IP assays showing no detectable interaction between GS and RHOA or RHOC (red asterisk indicates a non-specific band (also present in the IgG controls and unaffected by silencing ofRHOAorRHOC)). Image shown is representative of 3 independent experiments.b, Co-IP of overexpressedGLULandRHOJ-eGFPorΔN20-RHOJ-eGFPin ECs. Quantifications are mean ± s.e.m.;n = 4 independent experiments;*P< 0.05, one-samplet-test. In some of the experiments, the expression of ΔN20-RHOJ–eGFP was lower than the expression of RHOJ–eGFP. To correct for this, densitometric quantification was performed and signals in immunoprecipitation lanes were normalized to input signals.c, Immunoblotting for RHOA and RHOC on cytosolic (c) and membrane (m) fractions of ECs with NaK as membrane marker and GAPDH as cytosolic marker. Image shown is representative of 3 independent experiments.d, BiFC化验与氨基哈尔GS耦合f of eGFP, and RHOJ coupled to the C-terminal half of eGFP. Only when GS and RHOJ are in close proximity do the two eGFP half-sites complement each other and form a functional eGFP.e, Percentage of ECs displaying BiFC upon overexpression ofGLUL-eGFP1/2andRHOJ-eGFP2/2orGLUL-eGFP1/2andΔN20-RHOJ-eGFP2/2. Data are mean ± s.e.m.;n = 3 independent experiments; *P< 0.05; Student’st-test.f, Schematic of SPT-PALM imaging under TIRF illumination with the plasma membrane depicted at the top. The TIRF region is bright (whereas the part outside the TIRF region is greyed out) and contains the plasma membrane and its immediately adjacent space (not shown at exact relative dimensions). Weight and number of arrowheads represent the velocity of single particles (the photoswitchable fluorescent protein (PSFP) or the PSFP coupled to the protein of interest (here GS)). The PSFP is activated upon entry into the TIRF region and is colour-coded differently inside and outside of the TIRF region. PSFP–GS displays reduced velocity in the TIRF region, presumably because of palmitoylation and membrane association of GS.g, Scheme for the in-cell labelling of proteins with clickable alkyne-containing palmitoylation probes and subsequent biotin-azide clicking. X represents a palmitoylated protein, N3is the biotin-coupled azide.h,i, Rate of CoA release from palmitoyl-CoA as a readout for recombinant human GS autopalmitoylation while varying either the doses of palmitoyl-CoA (h) or the amounts of recombinant GS (i) (mean ± s.e.m.;n = 4 independent experiments forhandn = 5 fori;*P< 0.05, one-way ANOVA with Dunnett’s multiple comparisons versus 0 µM palmitoyl-CoA or versus 0.5 µg recombinant GS).j, Representative GS immunoblot (of 3 independent experiments) for the binding of recombinant human GS to palmitoyl-CoA agarose. IF, input fraction; FT, flow through; W8, wash fraction 8; SDS is the eluate.k–m, Representative images of RHOJ–eGFP localization in ECs under vehicle-treatment (k) or treatment with 2BP (a pan-palmitoylation inhibitor) (l). Red arrowheads indicate eGFP signal at membrane ruffles, which was quantified as the percentage of the total cellular area (m) (mean ± s.e.m.;n = 4 independent experiments;*P< 0.05 versus vehicle-treated, paired Student’st-test).n–p, Representative images of ECs overexpressing wild-typeRHOJ-eGFP(n),RHOJ-eGFPencoding the point mutation C3A (o) orRHOJ-eGFPencoding the point mutation C11A (p). Red arrowheads indicate RHOJ at the plasma membrane. ECs that are not completely in the field of view have been masked out in blue.q, Quantification of the RHOJ–eGFP positive area at the plasma membrane as a percentage of the total cell area. Data are mean ± s.e.m.;n = 5 independent experiments; *P< 0.05; one-way ANOVA with Dunnett’s comparison versus wild-type RHOJ.r, RHOJ immunoblotting on membrane versus cytosolic fractions from ECs overexpressing wild-typeRHOJ-eGFP(RHOJWT),RHOJ-eGFPencoding the point mutation C3A (RHOJC3A) orRHOJ-eGFPencoding the point mutation C11A (RHOJC11A), with NaK as membrane marker and GAPDH and α-tubulin as cytosolic markers.s, Densitometric quantification of RHOJ/NaK as determined inr. Data are mean ± s.e.m.;n = 6 independent experiments; *P< 0.05; one-samplet-test.t, RHOJ activity in ECs under treatment with vehicle or 2BP (blots are representative of 3 independent experiments; densitometric quantification in arbitrary units is mean ± s.e.m.;*P< 0.05, paired Student’st-test versus vehicle-treated).u, RHOJ immunoblotting of control andGLULKDECs overexpressing RHOJ (RHOJOE) subjected to acyl-resin-assisted capture. The cleaved bound fraction (cBF) represents palmitoylated RHOJ. IF is the input fraction, whereas the cleaved unbound fraction (cUF) and the preserved bound fraction (pBF) are controls showing the depletion of RHOJ from the thioester-cleaving reagent and the near absence of non-specific binding of RHOJ to the resin (seeMethods). Densitometric quantification of cBF/IF is shown (mean ± s.e.m.;n = 3 independent experiments;*P< 0.05, one-samplet-test versus control).v, Left, autopalmitoylation enables endothelial GS to interact directly (or indirectly) with the Rho GTPase RHOJ and to sustain the palmitoylation, membrane localization and activity of RHOJ (reflected by GTP binding). RHOJ activity then sustains normal EC migration and lamellipodia formation, and keeps actin stress-fibre formation at levels that promote normal EC migration and vessel branching in vivo. Through mechanisms that are not completely understood, active RHOJ inhibits signalling of the RHOA/B/C–ROCK–(p)MLC pathway (itself known to promote stress-fibre formation). The relative contribution of a direct effect of RHOJ on migration versus the indirect effect through RHOA/B/C–ROCK–(p)MLC is yet to be determined. Reduced opacity of RHOA/B/C, ROCK and (p)MLC indicates reduced signalling of this pathway. Right, loss of endothelial GS renders RHOJ less active (visually reflected by fewer palmitoylated, membrane-bound RHOJ proteins), and reduces the inhibition of the RHOA/B/C–ROCK–(p)MLC pathway. The resulting excessive stress-fibre formation causes ECs to lose migratory capacity and reduces vessel branching in vivo. Dashed lines indicate reduced activity; the red cross indicates GS blockade; the question mark indicates unknown mechanisms. ExactPvalues are as follows: 0.0153 (b）;0.0334 (e）;2 versus 0 μM: 0.6327; 5 versus 0 μM: 0.2841; 10 versus 0 μM: 0.1090; 20 versus 0 μM: 0.0339; 40 versus 0 μM: 0.0034 (h）;1 versus 0.5 μg: 0.5806; 2 versus 0.5 μg: 0.0319; 4 versus 0.5 μg: 0.0037; 8 versus 0.5 μg: 0.0001; 16 versus 0.5 μg: 0.0001 (i）;0.0313 (m）;RHOJ C3A versus RHOJ WT: 0.0001; RHOJ C11A versus RHOJ WT: 0.0001 (q）;RHOJ C3A versus RHOJ WT: 0.0015; RHOJ C11A versus RHOJ WT: 0.0007 (s）;0.0051 (t）;0.0461 (u). Scale bar, 200 μm (k,l,n–p). For gel source images, see Supplementary Fig.1.
Extended Data Fig. 8 Possible molecular model of GS autopalmitoylation.
a, Structure of human GS, showing its bifunnel-shaped catalytic site. A schematic of the GS decamer is shown from the top and front views with individual subunits A and B labelled and coloured grey and green, respectively. On the right is a close-up of the bifunnel catalytic site that is formed between subunits A and B. The GS decamer has ten active sites, each located at the interface of two adjacent subunits. ATP enters from the top, whereas glutamate enters from below; manganese ions (Mn2+) are shown as grey spheres.b, Molecular dynamics simulation of palmitoyl-CoA in the catalytic cleft of GS predicts that, whereas the head of palmitoyl-CoA is tightly bound to the adenine-binding site, the tail can point in opposing directions with respect to the principal axis of the protein. The most representative structures of the two alternative conformations (A and B) observed during the long molecular dynamics simulations for palmitoyl-CoA binding to GS (in blue, seen from two different perspectives) are shown in red (A, tail bending upwards) and green (B, tail bending downwards).c, Detailed view of conformation A, which is the main conformation. The sulfur atom of palmitoyl-CoA (which is immediately adjacent to the carbon on which the nucleophilic attack occurs) (coloured yellow) approaches the highly conserved cysteine 209 (also coloured yellow), with an interatomic distance (S–S) that, during the simulations, reversibly fluctuates between 3 and 8 Å. The hydrophobic tail positions itself along grooves characterized by the presence of hydrophobic residues. Colour coding is as follows: carbon, grey; nitrogen, blue; phosphorous, gold; oxygen, red. Cysteines and serines within 5 Å of the palmitoyl tail are highlighted in yellow and orange, respectively. The hydrophobic residues around the tail are shown in green.d, Detailed view of conformation B, in which the tail is found in a buried hydrophobic cleft, with the sulfur at a distance of 5 Å or less from the conserved serines 65 and 75 and the tail occupying the site of the GS inhibitor MSO. Details of the extensive steric clash between MSO and the secondary binding pose (B) observed in palmitoyl-CoA MD simulations are shown. Palmitoyl-CoA is represented as sticks, with standard atomic colours as stated inc. MSO is shown in cyan and its position is taken from entry 2QC8 of the Protein Data Bank. Cysteines and serines within 5 Å of the palmitoyl tail are highlighted in yellow and orange, respectively. The hydrophobic residues around the tail are shown in green.e链霉亲和素拉o后,GS免疫印迹f biotin-azide-clicked lysates from 16C-YA (palmitoylation probe) labelled HEK-293T cells overexpressing wild-typeGLULorGLULwith a point mutation in C209. The input shows the level of GS overexpression. A representative blot from 4 independent experiments is shown.f,g, Quantification of total sprout length (f) and number of sprouts per spheroid (g) for control andGLULKDECs with or without overexpression of shRNA-resistantGLULencoding the point mutation C209A (rGLULC209A-OE) (mean ± s.e.m.;n = 4 independent experiments; *P< 0.05 versus control, one-way ANOVA with Dunnett’s multiple comparison versus control).h, Schematic of protein autopalmitoylation. Upon binding of palmitoyl-CoA to the protein, free CoA (grey oval) is released and can be detected.i,重组野生型mutat GS和g点ions R324C and R341C were incubated with different concentrations of palmitoyl-CoA in a cell-free system at physiological pH. The amount of CoA released per minute was determined as a direct readout for protein autopalmitoylation. Data are mean ± s.e.m. of 3 (R324C and R341C) and 4 (WT) independent experiments. NS,P > 0.05; *P< 0.05 according to two-way ANOVA comparing the entire dose–response to the dose–response of wild-type GS.j, Different amounts of recombinant wild-type, R324C and R341C GS were incubated with a fixed concentration of palmitoyl-CoA (40 μM), and the amount of CoA released per minute was determined as readout for autopalmitoylation. Data are mean ± s.e.m. of 4 (R324C and R341C) and 5 (wild-type) independent experiments. NS,P > 0.05;*P< 0.05 according to two-way ANOVA comparing the entire dose–response to the dose–response of wild-type GS. The data for wild-type GS fromiandjare also included in Extended Data Fig.7as stand-alone data, but are included here for comparison purposes.k, Boyden chamber migration for control,GLULKD,GLULKD+ rGLULOE,GLULKD+ rGLULR341C-OEandGLULKD+ rGLULR324C-OEECs, all under mitomycin C-treatment (mean ± s.e.m.;n = 3 independent experiments; NS,P > 0.05;*P< 0.05, one-way ANOVA with Dunnett’s multiple comparison versus control). ExactPvalues:GLULKDversus control: 0.0004;GLULKD+ rGLULC209A-OEversus control: 0.0004 (f）;GLULKDversus control: 0.0001;GLULKD+ rGLULC209A-OEversus control: 0.0001 (g）;R324C versus WT: 0.8228; R341C versus WT: 0.7530 (i）;R324C versus WT: 0.1331; R341C versus WT: 0.0003 (j）;GLULKDversus control: 0.0054;GLULKD+ rGLULOEversus control: 0.8152;GLULKD+ rGLULR341C-OEversus control: 0.3645;GLULKD+ rGLULR324C-OEversus control: 0.2118 (k). For gel source images, see Supplementary Fig.1.
Supplementary information
Supplementary Discussion
This file contains Supplementary discussions 1-5, all of which contain additional rationale and discussion of experimental data and were not included in the main manuscript due to space restrictions.
Supplementary Figures
This file contains uncropped gel pictures and blots with size markers
Video 1
Time lapse imaging of randomly moving control ECs Time lapse video of sparsely seeded control ECs. Video shown is representative of nine independent experiments
Video 2
Time lapse imaging of randomly movingGLULKDECs Time lapse video of sparsely seededGLULKDECs. Video shown is representative of nine independent experiments
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Eelen, G., Dubois, C., Cantelmo, A.R.et al.Role of glutamine synthetase in angiogenesis beyond glutamine synthesis.Nature561, 63–69 (2018). https://doi.org/10.1038/s41586-018-0466-7
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DOI:https://doi.org/10.1038/s41586-018-0466-7
Keywords
- Glutamate-ammonia Ligase (GLUL)
- Glutamine Synthetase (GS)
- Rhosis
- Human Umbilical Vein ECs (HUVECs)
- Bimolecular Fluorescence Complementation (BiFC)
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