Sialic acids and diabetes : impact of desialylation on insulin receptor s functionality
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1 Journée scientifique du Centre de Calcul et de la Maison de la Simulation Sialic acids and diabetes : impact of desialylation on insulin receptor s functionality GUILLOT Alexandre Supervisors: DURLACH Vincent BLAISE Sébastien CNRS UMR 7369 MEDyC Team matrix aging and vascular remodelling 11/06/2015
2 2 Supervisors In vivo / In vitro In silico Dr Sébastien BLAISE Pr Vincent DURLACH Dr Stéphanie BAUD Pr Manuel DAUCHEZ
3 3 Diabetes 360 million diabetics France : 3 million each day Abnormal and chronic increase of blood sugar level Type 1 diabetes Low insulin production Genetic factors Type 2 diabetes Ineficient insulin Aging, obesity 80 % of all diabetes
4 Extracellular Intracellular 4 Insulin receptor Insulin fixation α-chains (red and green) β-chains (orange and blue) Plasma membrane Insulin receptor IR activation Reduce blood glucose level
5 5 Aim of the study Sialic acids and diabetes : impact of desialylation on insulin receptor s functionality Desialylation (Removes sialic acids) Plasma membrane Insulin receptor Normal functionality Insulin receptor Altered functionality
6 N-glycan chains Protein synthesis Small and simple protein e.g. insulin (51 AA) Ready to use or few maturation steps Many maturations steps needed! N-glycan chains addition Large and complex protein e.g. insulin receptor (1355 AA) 6
7 N-glycan chains Sugar or modified sugar linked to proteins specific residues (asparagine) Glycan chain Asparagine (protein) + H2O Various purposes Protein folding Reduce protein degradation Protein-protein interactions... depending on : Glycan chain localisation Protein s partners Glycan type... 7
8 8 N-glycan chains 3 groups : High mannose Hybrid Complex Many mannose, 2 N- acetylglucosamines Mix between high mannose and complex types Various types of saccharides : - N-acetylglucosamine - Mannose - Galactose - Fucose - Sialic acids + Various lengths and antenna number in each group
9 9 Insulin receptor and sialic acids Sialic acids Glycan chain Plasma membrane Insulin receptor What are the consequences of removing sialic acids over the insulin receptor mechanisms? First step : will the removal of sialic acids change the glycan chain dynamics
10 10 Choice of the structure According to literature and the most common structures found : 4 chains have been selected Tri-antennary Bi-antennary With Sialic acids Fuc Fuc Without Sialic acids Fuc Fuc
11 11 Force field GLYCAM, AMBER Already used for glycan chains Rarely used for both glycan + protein OPLS (all atoms) Often used for proteins Miss glycan atoms description Missing atoms added by homology with already described atoms
12 Construction Each chain link is separately built using Avogadro software Residues are then linked 1 by 1 with energy minimisation at each step until the final glycan chain is obtained N-acetylglucosamine Sialic acid Galactose Asparagine Mannose 12
13 13 Molecular dynamic simulations (GROMACS) Energy minimisation Reduce energy in vacuum Creation of the box Delimiting the simulation Addition of water molecules For physiological-like environment Addition of ions If necessary, the total system charge must be neutralised Energy minimisation Reduce energy in solvent System equilibration For correct water repartition Molecular dynamic simulation 500 ns, physiological temperature 310 K
14 14 Molecular dynamic simulations About 13,000 atoms during simulation Computation time (500ns) : 10 nodes 160 cores 60 h Output files : 12 GB 500,000 positions saved for each atom (1/picosecond) Light version : no more water and 50,000 positions
15 Without SA With SA Results analysis : clustering Cluster #1 Cluster #2 Objective: identify mains conformations that the chain will adopt. Cluster #1 Cluster #2 Other With SA 72% 14% 14% (18 clusters) Without SA 28% 24% 48% (41 clusters) Cluster size : Less and bigger clusters for chain with SA More and smaller clusters for chain without SA 15
16 Results analysis : clustering Cluster #1 With SA 72 % Cluster #2 Without SA 24 % Cluster #1 with SA = cluster #2 without SA Cluster #2 With SA 14 % Cluster #1 Without SA 28 % Cluster #2 with SA = cluster #1 without SA 16
17 Results analysis : dihedral angle Objective: identify flexibility points by measuring phi, psi and omega dihedral angles angle types: single thin pic: less flexibility 1 single large pic: more flexibility More than 1 pic: many conformations Antenna fixation sites are more flexible Removing sialic acid can change dihedral angle
18 RMSF (Å) 18 Results analysis : antenna movements (RMSF) 3 2,5 2 Objective: measure the motion of each antenna RMSF : root mean square fluctuation Removing sialic acids increase antenna motions 1,5 1 0,5 0 Antenna 1 Antenna 2 With SA Without SA
19 19 Results analysis : umbrella visualisation Objective: visualise the covered zone by each antenna Protein
20 20 Results analysis : umbrella visualisation Antenna 1 With SA Without SA Cluster #1 72 % Cluster #2 24 % ASN ASN Cluster #2 14 % Cluster #1 28 % 1 nm Antenna 1 displays the 2 previously identified clusters
21 21 Results analysis : umbrella visualisation Antenna 2 With SA Without SA ASN ASN 1 nm Antenna 2 is more flexible when sialic acids are removed
22 22 Conclusion Impact of desialylation on glycan chains Changes average conformations Increases antenna flexibility Covered zone displays different profiles Desialylation
23 23 Further work Impact of desialylated N-glycan chains on insulin receptor Objective: show conformational modifications of IR when SA are removed Stucture Insulin binding site Dimerization Insulin receptor activity N-glycan chain linked to the receptor New molecular dynamic simulations Plasma membrane Insulin receptor
24 Further work A whole new challenge : Insulin receptor: 18 potential glycan sites Addition of plasma membrane HUGE system 620,000 atoms 20 nodes 320 cores 130 h for a 500 ns simulation Terabytes of data 13,000 atoms 620,000 atoms 24
25 SA level Activation level 25 IR SA Further work IR P-IR ke : induce the removal of sialic acids from insulin receptor P-IR : phosphorylation activation of the IR Correlation between insulin receptor sialylation and activity Insulin ke Insulin ke We need in silico studies to understand this mechanism at a molecular level
26 26 Acknowledgements UMR CNRS/URCA 7369 MEDyC Sébastien BLAISE Vincent DURLACH Manuel DAUCHEZ Laurent DUCA Laurent DEBELLE Laurent MARTINY Maison de la simulation de Champagne-Ardenne Stéphanie BAUD François-Xavier MAQUART Team 2 Matrix aging and Vascular remodelling SiRMa Laboratory Funding
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