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Quantitative Interactomics in Primary T Cells Unveils TCR Signal

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Quantitative Interactomics in Primary T Cells Unveils TCR Signal Quantitative interactomics in primary T cells unveils TCR signal diversification extent and dynamics Guillaume Voisinne, Kristof Kersse, Karima Chaoui, Liaoxun Lu, Julie Chaix, Lichen Zhang, Marisa Goncalves Menoita, Laura Girard, Youcef Ounoughene, Hui Wang, et al. To cite this version: Guillaume Voisinne, Kristof Kersse, Karima Chaoui, Liaoxun Lu, Julie Chaix, et al.. Quantitative interactomics in primary T cells unveils TCR signal diversification extent and dynamics. Nature Immunology, Nature Publishing Group, 2019, 20 (11), pp.1530 - 1541. 10.1038/s41590-019-0489-8. hal-03013469 HAL Id: hal-03013469 https://hal.archives-ouvertes.fr/hal-03013469 Submitted on 23 Nov 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. RESOURCE https://doi.org/10.1038/s41590-019-0489-8 Quantitative interactomics in primary T cells unveils TCR signal diversification extent and dynamics Guillaume Voisinne 1, Kristof Kersse1, Karima Chaoui2, Liaoxun Lu3,4, Julie Chaix1, Lichen Zhang3, Marisa Goncalves Menoita1, Laura Girard1,5, Youcef Ounoughene1, Hui Wang3, Odile Burlet-Schiltz2, Hervé Luche5,6, Frédéric Fiore5, Marie Malissen1,5,6, Anne Gonzalez de Peredo 2, Yinming Liang 3,6*, Romain Roncagalli 1* and Bernard Malissen 1,5,6* The activation of T cells by the T cell antigen receptor (TCR) results in the formation of signaling protein complexes (signalo­ somes), the composition of which has not been analyzed at a systems level. Here, we isolated primary CD4+ T cells from 15 gene­targeted mice, each expressing one tagged form of a canonical protein of the TCR­signaling pathway. Using affinity puri­ fication coupled with mass spectrometry, we analyzed the composition and dynamics of the signalosomes assembling around each of the tagged proteins over 600 s of TCR engagement. We showed that the TCR signal­transduction network comprises at least 277 unique proteins involved in 366 high­confidence interactions, and that TCR signals diversify extensively at the level of the plasma membrane. Integrating the cellular abundance of the interacting proteins and their interaction stoichiometry provided a quantitative and contextual view of each documented interaction, permitting anticipation of whether ablation of a single interacting protein can impinge on the whole TCR signal­transduction network. cells express TCRs on their surface, through which they points after anti-TCR plus anti-CD4 stimulation. A total of 277 detect antigens. The initiation of TCR signals relies on the unique proteins involved in 366 high-confidence protein–protein LCK and ZAP70 protein tyrosine kinases (PTKs) and gener- interactions (PPIs) were identified within the proximal TCR signal- T 1–3 ates protein assemblages of considerable complexity . Most previ- transduction network, a complexity that led us to revisit the mode ous approaches aiming at disentangling such complexity addressed of action of several signalosomes used by the TCR. one protein at a time, with limited quantitative insight. As a result, TCR signals are classically described as proceeding from the it remains difficult to understand how the TCR signal-transduction TCR to the inside of T cells via the LAT transmembrane adaptor, network processes signals and to predict the effects resulting from which is thought to serve as the earliest and often sole point of sig- a mutation or a drug. nal diversification downstream of the TCR2. In our original interac- Affinity purification of a protein of interest (the ‘bait’) with its tomics study, we showed that the transmembrane receptor CD6 was interacting partners (the ‘preys’), coupled with mass spectrometry also able to nucleate its own signalosome in response to TCR signal- (AP–MS), permits the definition of the composition of the corre- ing, and independently of LAT4. However, the lack of information sponding protein complex as a set of binary bait–prey interactions, on the numbers of complexes nucleating around LAT and CD6 pre- termed an ‘interactome’. We provided proof-of-concept for interac- cluded assessing their respective quantitative contribution to early tomics in primary CD4+ T cells by determining the composition TCR signal propagation and diversification. Here, by capitalizing on of the multiprotein complexes that formed around ZAP70 and the the recent possibilities to measure both the numbers of copies per adaptors LAT and SLP-76 (ref. 4). However, that pilot study was lim- cell (cellular protein abundance) of each interacting protein, and ited to three baits and relied on pervanadate-based T cell activation, the quantitative relationship existing between a bait and a prey in a a stimulation condition that is less physiological than that result- given complex (interaction stoichiometry)6, we succeeded in iden- ing from engagement of the TCR in combination with CD4 or CD8 tifying and quantifying the TCR-inducible signalosomes that form coreceptors. Here, we extended our interactomics approach to sig- at the inner face of the plasma membrane. Unexpectedly, the CD5 naling complexes (‘signalosomes’) that assemble around 15 canoni- and CD6 transmembrane receptors assembled signalosomes with cal proteins used by the proximal TCR signal-transduction network. kinetics and in numbers comparable to those nucleated by the LAT We avoided the pitfalls associated with transformed T cells5 by using adaptor, demonstrating that the breadth of early TCR signal diversi- primary CD4+ T cells, and we captured signaling dynamics by ana- fication is larger than expected. Finally, to decipher the function of lyzing each of the 15 signalosomes before and at four different time the poorly characterized interacting proteins identified within the 1Centre d’Immunologie de Marseille-Luminy, Aix Marseille Université, INSERM, CNRS, Marseille, France. 2Institut de Pharmacologie et de Biologie Structurale, Département Biologie Structurale Biophysique, Protéomique Génopole Toulouse Midi Pyrénées CNRS UMR 5089, Toulouse, France. 3School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China. 4Laboratory of Mouse Genetics, Institute of Psychiatry and Neuroscience, Xinxiang Medical University, Xinxiang, China. 5Centre d’Immunophénomique, Aix Marseille Université, INSERM, CNRS UMR, Marseille, France. 6Laboratory of Immunophenomics, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China. *e-mail: [email protected]; [email protected]; [email protected] 1530 NatURE IMMUnology | VOL 20 | NOVEMBER 2019 | 1530–1541 | www.nature.com/natureimmunology NATURE IMMUNOLOGY RESOURCE a Stimulation Interaction TCR stoichiometry Primary Prey CD4+ T cells A Prey Bait B Relative OST tag Ost Interaction Time protein sequence specificity - abundance Gene-targeted mice expressing Affinity purification and quantitative Composition, stoichiometry and tagged form of 15 canonical proteomics analysis of the complexes dynamics of 15 signalosomes involved components of the TCR assembling around the tagged in the propagation of proximal TCR signaling pathway proteins over stimulation time signals at unprecedented resolution b Rnmt Arhgap27 Nxf1 Prpf31 Sla Ccar2 Chd5 Smg1 Ccm2l Dnah2 Nxt1 Kdelc1 Traf7 Cd28 Ppp1cb Sord Smarca5 Zscan29 Nt5dc3 Smarca4 Hck Cct7 Ddx3x Vav3 Dnm1 Cct5 Abl1 Ppp1r12a Akap9 Prrc2a Ptpra Sos2 Mcm5 Gon4l Vav1 Hist1h2bc Khdrbs1 Rasa1 Dctn1 Sec11a Ccdc146 Nfatc2 Evl Vasp Vapb Skap2 Sos1 Trim28 Erich5 Ppp3cb Ppil1 Creb1 Snw1 Grb2 Wipf1 Xrcc6 Kbtbd11 Slp-76 Mcm3 Map4k1 Gm9476 Fyb Pdzd8 Lat Dbnl Skap1 Fyn Themis Inppl1 Arap1 H2-K1 Grap Ship1 Dok2 B4galnt1 Vapa Grap2 Btla Itk Plcγ1 Eif3c Rasal3 Cd3g Pstpip1 Nck1 Ywhae Shp1 Shc1 Ywhaq Ywhaz Ptpn22 Ubash3a Ywhab Ywhah Sh3kbp1 Trbc1 Hsp90ab1 Hspa8 Ywhag Cdc37 Tapbp Hsp90aa1 Csk Cd6 Trac Ipo5 Fkbp5 Dock2 Smc4 Ptrh2 Cd5 Esyt1 Rpn1 Cd247 Elmo1 Rab5c Psmd3 Ppp2r1a Capzb Mccc1 St13 Cd3e Rab11b Sun2 Lrrc59 Ighv5-6 Pcbp1 Zap70 Copg1 Pgrmc2 Pik3ca Gm10250 Atp1a1 Stoml2 Gnai2 Tubb5 Hadhb Tuba1b Psmc2 Mogs Cand1 Faah Psmd13 Acaca Tardbp Pik3r1 Far1 Sugt1 Slc25a5 Vdac2 Canx Hsd17b12 Ncapd2 Phb Rplp0 Ptprcap Smc2 Phb2 Pdcd4 Ruvbl1 Ifggd1 Hspa9 Tmx1 Umps Cnp Dnaja2 Pds5a Ptprc Cd2ap Tfrc Ecm29 Stat1 Cbl Pik3cd Capza2 Fkbp8 Cd2 M6pr Psmc3 Atp5c1 Atp5b Rpn2 Hsdl1 Psmd14 Ddost Samhd1 Lck Lrpprc Psmd12 Trim21 Pik3r2 Rpl6 Xpo1 Cd4 Vdac1 Gnb2 Irgm1 Ncaph Hnrnpm Tubb4b Slc25a12 Lime1 Ankrd13a Capza1 Ipo7 Psmc5 Tuba4a Dgka Psmd11 H2-D1 Hist1h1a Ly9 Ifi47 Sh2d2a Tmpo Rpl10 Nfkb1 Nucb1 Crkl Rpl27a Crk Mccc2 Tmed10 Tnpo1 COX2 Psmd7 Ostc Rac2 Pccb Atp5a1 Acadl Faf2 Cblb Eps15l1 Psmc6 Itgb2 Pik3cb Mlec Pfkp Raver1 Nop9 Atp5f1 Tap1 Psmd6 Sdha Rpl7a Bait Prey Fmnl1 Cyb5b Rps14 Ubash3b Itsn2 Uqcrc1 Phgdh Mybbp1a Tap2 Slc5a4b Mms19 Gimap5 Rps16 Rps19 Rps20 Interaction Lax1 Atp5d Psmc1Rps10 Rps17 Rpl23 Reported Rpl30 Not reported Actr1a Snrpd3 Gsr c d e Annotation term AAA ATPase family Interaction Bait AccD/PCCB family CF(1) Reported CRK family Lck F-actin-capping protein alpha subunit family Not reported Mitochondrion inner membrane Cbl PI3/PI4-kinase family PI3K p85 subunit family Fyb Prohibitin family Proteasome Cblb norm_Nint 3D-structure
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