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Methyl-beta-cyclodextrin does not preferentially target lipid raft cholesterol
Abstracts / Chemistry and Physics of Lipids 149S (2007) S74–S91
PO 143
PO 145
Methyl-beta-cyclodextrin does not preferentially target lipid raft cholesterol
Molecular dynamics simulations of peptide adsorption to a mineral surface
Saleemulla Mahammad, Ingela Parmryd
Jason O’Young, Bernd Grohe, Mikko Karttunen, Harvey Goldberg, Graeme Hunter
Department of Cell Biology, The Wenner-Gren Institute, Stockholm University, Sweden Methyl-beta-cyclodextrin (MBCD) is frequently used to acutely deplete cells of cholesterol and is a popular tool in lipid raft research, based on the idea that cholesterol is an absolute requirement for lipid raft integrity and that its depletion leads to lipid raft dispersion, i.e. if a cellular process is affected by cholesterol depletion it is assumed that lipid rafts are involved. Another widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts. To analyse this systematically, a protocol for progressive cholesterol depletion of Jurkat T cells was established using MBCD and [3H]-cholesterol. Up to 50% cholesterol depletion did not substantially affect cell viability of these cells. Depletion of 10, 20, 30, 40 and 50% total cholesterol was performed at 37 ◦ C and cells subjected to subcellular fractionation. MBCD extracts similar proportions of cholesterol from the Triton X-100 resistant (lipid raft enriched) as it does from other cellular fractions. Moreover, cholesterol is not exclusively extracted from the plasma membrane and the cells rapidly reestablish the relative differences in cholesterol concentration between different compartments. Moreover, they restore the cholesterol level in the plasma membrane by mobilising cholesterol from intracellular cholesterol stores. Interestingly, incubation of cells at 0 ◦ C causes loss of plasma membrane cholesterol with a concomitant increase in cholesteryl esters and adiposomes. By quantifying the changes, we estimate that about 25% of total free cholesterol found in the plasma membrane. The results clearly show that MBCD does not specifically extract cholesterol from lipid rafts and that intracellular cholesterol stores can be used to replenish plasma membrane cholesterol. doi:10.1016/j.chemphyslip.2007.06.194 PO 144 Withdrawn.
S85
University of Western Ontario, Canada Osteopontin (OPN) is an acidic phosphoglycoprotein that is believed to play a critical role in the prevention of calcium oxalate kidney stone formation. In vitro studies have shown that OPN can inhibit the formation of calcium oxalate monohydrate (COM) and that this inhibitory activity is dependent upon the phosphorylation of the protein. Our laboratory has previously synthesized peptides corresponding to a sequence 220–235 of rat OPN and containing 0, 1 or 3 phosphate groups (P0, P1, P3) Using these peptides, we investigated the effects of varying degrees of phosphorylation on COM crystals. Our results show that all three peptides bind to the {1 0 0} faces of the COM crystals. However, the degree of growth inhibition increases with peptide phosphorylation. Atomic scale molecular dynamic simulations were performed using the GROMACS suite. Simulations were performed in which P0, P1 or P3 was initially placed in an extended conformation parallel to the surface at a distance of approximately 3 nm. Each system was solvated with explicit water and counter-ions were added to maintain a charge–neutral state. All three peptides demonstrated an affinity for the {1 0 0} face in the simulations performed. Centre of mass trajectory analysis showed that P3 settles closer to the crystal surface than P1 or P0. Root mean squared distance analysis showed that peptide conformational stability is positively correlated with the degree of phosphorylation. Residue analysis showed that carboxylate-containing residues form closer and more stable contacts to the crystal surface than phosphorylated residues. The results indicate that phosphorylations increase the charge density of the peptide to facilitate transport to the surface whereas carboxylate-containing residues form close, stable interactions. This suggests separate roles for both phosphate and carboxylate groups, providing a possible explanation for the extensive phosphorylation found in many mineral-binding proteins. doi:10.1016/j.chemphyslip.2007.06.196
PO 143
PO 145
Methyl-beta-cyclodextrin does not preferentially target lipid raft cholesterol
Molecular dynamics simulations of peptide adsorption to a mineral surface
Saleemulla Mahammad, Ingela Parmryd
Jason O’Young, Bernd Grohe, Mikko Karttunen, Harvey Goldberg, Graeme Hunter
Department of Cell Biology, The Wenner-Gren Institute, Stockholm University, Sweden Methyl-beta-cyclodextrin (MBCD) is frequently used to acutely deplete cells of cholesterol and is a popular tool in lipid raft research, based on the idea that cholesterol is an absolute requirement for lipid raft integrity and that its depletion leads to lipid raft dispersion, i.e. if a cellular process is affected by cholesterol depletion it is assumed that lipid rafts are involved. Another widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts. To analyse this systematically, a protocol for progressive cholesterol depletion of Jurkat T cells was established using MBCD and [3H]-cholesterol. Up to 50% cholesterol depletion did not substantially affect cell viability of these cells. Depletion of 10, 20, 30, 40 and 50% total cholesterol was performed at 37 ◦ C and cells subjected to subcellular fractionation. MBCD extracts similar proportions of cholesterol from the Triton X-100 resistant (lipid raft enriched) as it does from other cellular fractions. Moreover, cholesterol is not exclusively extracted from the plasma membrane and the cells rapidly reestablish the relative differences in cholesterol concentration between different compartments. Moreover, they restore the cholesterol level in the plasma membrane by mobilising cholesterol from intracellular cholesterol stores. Interestingly, incubation of cells at 0 ◦ C causes loss of plasma membrane cholesterol with a concomitant increase in cholesteryl esters and adiposomes. By quantifying the changes, we estimate that about 25% of total free cholesterol found in the plasma membrane. The results clearly show that MBCD does not specifically extract cholesterol from lipid rafts and that intracellular cholesterol stores can be used to replenish plasma membrane cholesterol. doi:10.1016/j.chemphyslip.2007.06.194 PO 144 Withdrawn.
S85
University of Western Ontario, Canada Osteopontin (OPN) is an acidic phosphoglycoprotein that is believed to play a critical role in the prevention of calcium oxalate kidney stone formation. In vitro studies have shown that OPN can inhibit the formation of calcium oxalate monohydrate (COM) and that this inhibitory activity is dependent upon the phosphorylation of the protein. Our laboratory has previously synthesized peptides corresponding to a sequence 220–235 of rat OPN and containing 0, 1 or 3 phosphate groups (P0, P1, P3) Using these peptides, we investigated the effects of varying degrees of phosphorylation on COM crystals. Our results show that all three peptides bind to the {1 0 0} faces of the COM crystals. However, the degree of growth inhibition increases with peptide phosphorylation. Atomic scale molecular dynamic simulations were performed using the GROMACS suite. Simulations were performed in which P0, P1 or P3 was initially placed in an extended conformation parallel to the surface at a distance of approximately 3 nm. Each system was solvated with explicit water and counter-ions were added to maintain a charge–neutral state. All three peptides demonstrated an affinity for the {1 0 0} face in the simulations performed. Centre of mass trajectory analysis showed that P3 settles closer to the crystal surface than P1 or P0. Root mean squared distance analysis showed that peptide conformational stability is positively correlated with the degree of phosphorylation. Residue analysis showed that carboxylate-containing residues form closer and more stable contacts to the crystal surface than phosphorylated residues. The results indicate that phosphorylations increase the charge density of the peptide to facilitate transport to the surface whereas carboxylate-containing residues form close, stable interactions. This suggests separate roles for both phosphate and carboxylate groups, providing a possible explanation for the extensive phosphorylation found in many mineral-binding proteins. doi:10.1016/j.chemphyslip.2007.06.196