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, Immunoprecipitations were performed using 4 ?g of mouse monoclonal anti-Flag or rabbit polyclonal anti-NEU1 pre-adsorbed on protein G sepharose beads (GE Healthcare) for 2 h at 4 °C. Immunoprecipitated proteins were eluted with Laemmli buffer and subjected to SDS-PAGE and immunoblotting. Immunoblottings were performed using the indicated antibodies and immunoreactivity was revealed using a HRP-conjugated secondary antibodies (1/10,000) followed by enhanced chemiluminescence detection reagents and visualized with the Odyssey Fc LI-COR scanner

?. Csm-?leu, ?. Trp, and ?. Ade, After sonication, crude membranes were pelleted by centrifugation at 20,000 g during 45 min at 4 °C and resuspended in 20 mM MES, pH4.5. Sialidase activity was assessed using the 2?-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (BioSynth) as substrate. Assays were performed in triplicate (each run in duplicate) with 50 ?g of crude membranes proteins and 200 ?M of substrate for 2 h at 37 °C. Reactions were stopped by adding 1 M Na 2 CO 3 and the fluorescence of each well was measured in triplicate using an Infinite F200 PRO microplate reader (TECAN), Split-ubiquitin yeast two hybrid. The NEU1 cDNA was subcloned into pDONR221 before integration in the Split-Ubiquitin destination vectors. The Split-Ubiquitin vectors, pMetYC-DEST and pNX35-DEST, were used to produce the Met-repressible bait construct NEU1-Cub-PLV and prey construct NEU1-NubG, respectively. NubWT fragment, pMetYC-DEST and pNX35-DEST vectors were kindly provided by Dr F. Chaumont (Institut des Sciences de la Vie

, NEU1 homology model generation. Prediction of the human NEU1 3D structure was done by homology modeling using the SWISS-MODEL software 59-61 (accessible via the ExPASy web server) and the human NEU2 crystal structure

E. Monti, Sialidases in vertebrates: a family of enzymes tailored for several cell functions, Adv Carbohydr Chem Biochem, vol.64, pp.403-479, 2010.

E. Giacopuzzi, R. Bresciani, R. Schauer, E. Monti, and G. Borsani, New insights on the sialidase protein family revealed by a phylogenetic analysis in metazoa, PLoS One, vol.7, p.44193, 2012.

V. Lombard, H. Golaconda-ramulu, E. Drula, P. M. Coutinho, and B. Henrissat, The carbohydrate-active enzymes database (CAZy) in 2013, Nucleic Acids Res, vol.42, pp.490-495, 2014.

T. Miyagi and K. Yamaguchi, Mammalian sialidases: physiological and pathological roles in cellular functions, Glycobiology, vol.22, pp.880-896, 2012.

S. Magesh, T. Suzuki, T. Miyagi, H. Ishida, and M. Kiso, Homology modeling of human sialidase enzymes NEU1, NEU3 and NEU4 based on the crystal structure of NEU2: hints for the design of selective NEU3 inhibitors, J Mol Graph Model, vol.25, pp.196-207, 2006.

E. Bonten, A. Van-der-spoel, M. Fornerod, G. Grosveld, and A. Azzo, Characterization of human lysosomal neuraminidase defines the molecular basis of the metabolic storage disorder sialidosis, Genes Dev, vol.10, pp.3156-3169, 1996.

E. J. Bonten, I. Annunziata, and A. Azzo, Lysosomal multienzyme complex: pros and cons of working together, Cell Mol Life Sci, vol.71, pp.2017-2032, 2014.

K. E. Lukong, Intracellular distribution of lysosomal sialidase is controlled by the internalization signal in its cytoplasmic tail, J Biol Chem, vol.276, pp.46172-46181, 2001.

L. Duca, The elastin receptor complex transduces signals through the catalytic activity of its Neu-1 subunit, J Biol Chem, vol.282, pp.12484-12491, 2007.
URL : https://hal.archives-ouvertes.fr/hal-00144208

A. Rusciani, Elastin peptides signaling relies on neuraminidase-1-dependent lactosylceramide generation, PLoS One, vol.5, p.14010, 2010.
URL : https://hal.archives-ouvertes.fr/hal-00554845

A. Hinek, A. V. Pshezhetsky, M. Von-itzstein, and B. Starcher, Lysosomal sialidase (neuraminidase-1) is targeted to the cell surface in a multiprotein complex that facilitates elastic fiber assembly, J Biol Chem, vol.281, pp.3698-3710, 2006.

S. Blaise, Elastin-derived peptides are new regulators of insulin resistance development in mice, Diabetes, vol.62, pp.3807-3816, 2013.

L. Dridi, Positive regulation of insulin signaling by neuraminidase 1, Diabetes, vol.62, pp.2338-2346, 2013.

T. Uemura, Contribution of sialidase NEU1 to suppression of metastasis of human colon cancer cells through desialylation of integrin beta4, Oncogene, vol.28, pp.1218-1229, 2009.

S. R. Amith, Neu1 desialylation of sialyl alpha-2,3-linked beta-galactosyl residues of TOLL-like receptor 4 is essential for receptor activation and cellular signaling, Cell Signal, vol.22, pp.314-324, 2010.

P. Jayanth, S. R. Amith, K. Gee, and M. R. Szewczuk, Neu1 sialidase and matrix metalloproteinase-9 cross-talk is essential for neurotrophin activation of Trk receptors and cellular signaling, Cell Signal, vol.22, pp.1193-1205, 2010.

A. Hinek, T. D. Bodnaruk, S. Bunda, Y. Wang, and K. Liu, Neuraminidase-1, a subunit of the cell surface elastin receptor, desialylates and functionally inactivates adjacent receptors interacting with the mitogenic growth factors PDGF-BB and IGF-2, Am J Pathol, vol.173, pp.1042-1056, 2008.

, Scientific RepoRts |, vol.6, p.38363

E. P. Lillehoj, NEU1 sialidase expressed in human airway epithelia regulates epidermal growth factor receptor (EGFR) and MUC1 protein signaling, J Biol Chem, vol.287, pp.8214-8231, 2012.

C. Lee, NEU1 sialidase regulates the sialylation state of CD31 and disrupts CD31-driven capillary-like tube formation in human lung microvascular endothelia, J Biol Chem, vol.289, pp.9121-9135, 2014.

A. V. Pshezhetsky and A. Hinek, Where catabolism meets signalling: neuraminidase 1 as a modulator of cell receptors, Glycoconj J, vol.28, pp.441-452, 2011.

E. J. Bonten, Heterodimerization of the sialidase NEU1 with the chaperone protective protein/cathepsin A prevents its premature oligomerization, J Biol Chem, vol.284, pp.28430-28441, 2009.

A. Van-der-spoel, E. Bonten, and A. Azzo, Transport of human lysosomal neuraminidase to mature lysosomes requires protective protein/cathepsin A, EMBO J, vol.17, pp.1588-1597, 1998.

A. V. Pshezhetsky, Cloning, expression and chromosomal mapping of human lysosomal sialidase and characterization of mutations in sialidosis, Nat Genet, vol.15, pp.316-320, 1997.

E. J. Bonten and A. Azzo, Lysosomal neuraminidase. Catalytic activation in insect cells is controlled by the protective protein/ cathepsin A, J Biol Chem, vol.275, pp.37657-37663, 2000.
URL : https://hal.archives-ouvertes.fr/hal-00562944

C. L. Ried, C. Scharnagl, and D. Langosch, Entrapment of Water at the Transmembrane Helix-Helix Interface of Quiescin Sulfhydryl Oxidase 2, Biochemistry, vol.55, pp.1287-1290, 2016.

E. V. Bocharov, K. S. Mineev, M. V. Goncharuk, and A. S. Arseniev, Structural and thermodynamic insight into the process of "weak" dimerization of the ErbB4 transmembrane domain by solution NMR, Biochim Biophys Acta, vol.1818, pp.2158-2170, 2012.

K. D. Nadezhdin, O. V. Bocharova, E. V. Bocharov, and A. S. Arseniev, Dimeric structure of transmembrane domain of amyloid precursor protein in micellar environment, FEBS Lett, vol.586, pp.1687-1692, 2012.

E. V. Bocharov, Structure of FGFR3 transmembrane domain dimer: implications for signaling and human pathologies, Structure, vol.21, pp.2087-2093, 2013.

H. Yamamoto, Novel germline mutation in the transmembrane domain of HER2 in familial lung adenocarcinomas, J Natl Cancer Inst, vol.106, p.338, 2014.

F. N. Barrera, Roles of carboxyl groups in the transmembrane insertion of peptides, J Mol Biol, vol.413, pp.359-371, 2011.

A. A. Polyansky, PREDDIMER: a web server for prediction of transmembrane helical dimers, Bioinformatics, vol.30, pp.889-890, 2014.

E. M. Lynes, Palmitoylation is the switch that assigns calnexin to quality control or ER Ca 2+ signaling, J Cell Sci, vol.126, pp.3893-3903, 2013.

F. D'-avila, Identification of lysosomal sialidase NEU1 and plasma membrane sialidase NEU3 in human erythrocytes, J Cell Biochem, vol.114, pp.204-211, 2013.

P. L. Clark, How to Build a Complex, Functional Propeller Protein From Parts, Trends Biochem Sci, 2016.

R. G. Smock, I. Yadid, O. Dym, J. Clarke, D. S. Tawfik et al., Novo Evolutionary Emergence of a Symmetrical Protein Is Shaped by Folding Constraints, Cell, vol.164, pp.476-486, 2016.

F. W. Studier, Protein production by auto-induction in high density shaking cultures, Protein Expr Purif, vol.41, pp.207-234, 2005.

D. Schwarz, Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems, Nat Protoc, vol.2, pp.2945-2957, 2007.

S. M. Kelly, T. J. Jess, and N. C. Price, How to study proteins by circular dichroism, Biochim Biophys Acta, vol.1751, pp.119-139, 2005.

J. Cavanagh, W. J. Fairbrother, A. G. Palmer, and N. J. Skelton, Protein NMR spectroscopy: principles and practice, 2006.

T. L. Hwang, P. C. Van-zijl, and S. Mori, Accurate quantitation of water-amide proton exchange rates using the phase-modulated CLEAN chemical EXchange (CLEANEX-PM) approach with a Fast-HSQC (FHSQC) detection scheme, J Biomol NMR, vol.11, pp.221-226, 1998.

D. H. De-jong, Improved parameters for the Martini Coarse-Grained protein force field, J Chem Theory Comput, vol.9, pp.687-697, 2013.

T. A. Wassenaar, H. I. Ingólfsson, R. A. Böckmann, D. P. Tieleman, and S. J. Marrink, Computational lipidomics with insane: a versatile tool for generating custom membranes for molecular simulations, J Chem Theory Comput, vol.11, pp.2144-2155, 2015.

T. A. Wassenaar, High-throughput simulations of dimer and trimer assembly of membrane proteins. The DAFT approach, J Chem Theory Comput, vol.11, pp.2278-2291, 2015.

C. Kandt, W. L. Ash, and D. P. Tieleman, Setting up and running molecular dynamics simulations of membrane proteins, Methods, vol.41, pp.475-488, 2007.

D. Van-der-spoel, GROMACS: fast, flexible, and free, J Comput Chem, vol.26, pp.1701-1718, 2005.

C. Oostenbrink, A. Villa, A. E. Mark, and W. F. Van-gunsteren, A biomolecular force field based on the free enthalpy of hydration and solvation: the GROMOS force-field parameter sets 53A5 and 53A6, J Comput Chem, vol.25, pp.1656-1676, 2004.

H. J. Berendsen, J. R. Grigera, and T. P. Straatsma, The missing term in effective pair potentials, J. Phys. Chem, vol.91, pp.6269-6271, 1987.

B. Hess, H. Bekker, H. J. Berendsen, and J. G. Fraaije, LINCS: A Linear Constraint Solver for Molecular Simulations, J. Computational. Chem, vol.18, pp.1463-1472, 1997.

T. Darden, Y. Darrin, and L. G. Pedersen, Particle mesh ewald-an n.log(n) method for ewald sums in large systems, J. Chem. Phys, vol.98, pp.10089-10092, 1993.

U. Essmann, L. Perera, M. L. Berkowitz, T. Darden, L. Hsing et al., A smooth particle mesh Ewald method, J. Chem. Phys, vol.103, pp.8577-8593, 1995.

T. V. Pyrkov, A. O. Chugunov, N. A. Krylov, D. E. Nolde, and R. G. Efremov, PLATINUM: a web tool for analysis of hydrophobic/ hydrophilic organization of biomolecular complexes, Bioinformatics, vol.25, pp.1201-1202, 2009.

W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W. Impey, and M. L. Klein, Comparison of Simple Potential Functions for Simulating Liquid Water, J. Chem. Phys, vol.79, pp.926-935, 1983.

J. P. Jambeck and A. P. Lyubartsev, Derivation and systematic validation of a refined all-atom force field for phosphatidylcholine lipids, J Phys Chem B, vol.116, pp.3164-3179, 2012.

K. Lindorff-larsen, Improved side-chain torsion potentials for the Amber ff99SB protein force field, Proteins, vol.78, pp.1950-1958, 2010.

M. Parrinello and A. Rahman, Polymorphic transitions in single crystals: A new molecular dynamics method, J Appl Phys, vol.52, pp.7182-7190, 1981.

J. Henin, A. Pohorille, and C. Chipot, Insights into the recognition and association of transmembrane alpha-helices. The free energy of alpha-helix dimerization in glycophorin A, J Am Chem Soc, vol.127, pp.8478-8484, 2005.

J. Lee and W. Im, Role of hydrogen bonding and helix-lipid interactions in transmembrane helix association, J Am Chem Soc, vol.130, pp.6456-6462, 2008.

C. Hachez, Arabidopsis SNAREs SYP61 and SYP121 coordinate the trafficking of plasma membrane aquaporin PIP2;7 to modulate the cell membrane water permeability, Plant Cell, vol.26, pp.3132-3147, 2014.

K. Arnold, L. Bordoli, J. Kopp, and T. Schwede, The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling, Bioinformatics, vol.22, pp.195-201, 2006.

, Scientific RepoRts |, vol.6, p.38363

P. Benkert, M. Biasini, and T. Schwede, Toward the estimation of the absolute quality of individual protein structure models, Bioinformatics, vol.27, pp.343-350, 2011.

M. Biasini, SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information, Nucleic Acids Res, vol.42, pp.252-258, 2014.

E. V. Bocharov, P. E. Volynsky, K. V. Pavlov, R. G. Efremov, and A. S. Arseniev, Structure elucidation of dimeric transmembrane domains of bitopic proteins, Cell Adh Migr, vol.4, pp.284-298, 2010.