Biomedical subjects

Y Aubin

Publications and source records attributed to Y Aubin.

5 recordsLinked to original sources

PubMed1999

Hsp60 accelerates the maturation of pro-caspase-3 by upstream activator proteases during apoptosis.

The activation of caspases represents a critical step in the pathways leading to the biochemical and morphological changes that underlie apoptosis. Multiple pathways leading to caspase activation appear to exist and vary depending on the death-inducing stimulus. We demonstrate that the activation of caspase-3, in Jurkat cells stimulated to undergo apoptosis by a Fas-independent pathway, is catalyzed by caspase-6. Caspase-6 was found to co-purify with caspase-3 as part of a multiprotein activation complex from extracts of camptothecin-treated Jurkat cells. A biochemical analysis of the protein constituents of the activation complex showed that Hsp60 was also present. Furthermore, an interaction between Hsp60 and caspase-3 could be demonstrated by co-immunoprecipitation experiments using HeLa as well as Jurkat cell extracts. Using a reconstituted in vitro system, Hsp60 was able to substantially accelerate the maturation of procaspase-3 by different upstream activator caspases and this effect was dependent on ATP hydrolysis. We propose that the ATP-dependent 'foldase' activity of Hsp60 improves the vulnerability of pro-caspase-3 to proteolytic maturation by upstream caspases and that this represents an important regulatory event in apoptotic cell death.

Amino Acid Sequence↗

PubMed1996

Correlation between dynamics and high affinity binding in an SH2 domain interaction.

Protein-protein interfaces can consist of interactions between large numbers of residues of each molecule; some of these interactions are critical in determining binding affinity and conferring specificity, while others appear to play only a marginal role. Src-homology-2 (SH2) domains bind to proteins containing phosphorylated tyrosines, with additional specificity provided by interactions with residues C-terminal to the phosphotyrosine (pTyr) residue. While the C-terminal SH2 domain of phospholipase C-gamma 1 (PLCC SH2) interacts with eight residues of a pTyr-containing peptide from its high affinity binding site on the beta-platelet-derived growth factor receptor, it can still bind tightly to a phosphopeptide containing only three residues. Novel deuterium (2H) based nuclear magnetic resonance (NMR) spin relaxation experiments which probe the nanosecond-picosecond time scale dynamics of methyl containing side chain residues have established that certain regions of the PLCC SH2 domain contacting the residues C-terminal to the pTyr have a high degree of mobility in both the free and peptide complexed states. In contrast, there is significant restriction of motion in the pTyr binding site. These results suggest a correlation between the dynamic behavior of certain groups in the PLCC SH2 complex and their contribution to high affinity binding and binding specificity.

Amino Acid Sequence↗

PubMed1994

13C NMR studies of wheat germ agglutinin interactions with N-acetylglucosamine at a magnetically oriented bilayer surface.

The orientation of synthetic 13C-labeled glycolipid receptors and their interaction with the plant lectin wheat germ agglutinin have been studied in an oriented membrane system using NMR spectroscopy. A series of 2-[1,2-13C2]acetamido-2-deoxy-beta-D-glucopyranosides were synthesized with between zero and four hydrophilic ethoxy units between the headgroup and an alkyl chain which anchors the receptors in the bilayers. The chemical shift anisotropy of the 13C carbonyl and a 13C-13C dipolar coupling between the labeled carbons provide information about the orientation and dynamics of the receptor headgroup in oriented membrane systems. It was found that the headgroups of the receptors with two, three, or four ethoxy units appeared isotropic when incorporated in the oriented bilayers, but those of the receptors with zero or one ethoxy units were significantly ordered by the bilayers. The average orientations consistent with measured spectral parameters were determined for the receptors with zero and one ethoxy units and were found to coincide with low-energy conformations from molecular modeling. When the plant lectin wheat germ agglutinin was added to the sample, only the receptors with two, three, or four ethoxy units separating the headgroup from the alkyl chain showed evidence of binding by the lectin. Although the 13C-labeled resonances broadened when the protein bound, no changes in dipolar couplings or chemical shift anisotropies could be detected, suggesting that the motion of the headgroup was slowed by protein binding, but average orientation and overall order changed little. Competition studies demonstrated that none of the lectin/receptor complexes are more stable than the complex of the lectin and N-acetylglucosamine in solution. These results suggest that the membrane does not stabilize the interactions of wheat germ agglutinin with these cell-surface receptors. Furthermore, molecular modeling demonstrates that the zero- and one-spacer receptors may not bind wheat germ agglutinin because the orientations of the N-acetyl groups in these receptors would result in significant steric contacts between the lectin/receptor complex and the membrane.

Acetylglucosamine↗

PubMed1993

Structure and dynamics of the sialic acid moiety of GM3-ganglioside at the surface of a magnetically oriented membrane.

13C-NMR techniques in oriented bilayer systems composed of DMPC and CHAPSO have been used to measure dipolar interactions between 13C-13C and 13C-1H pairs located in the sialic acid moiety of ganglioside GM3. These interactions are reduced to a structural and motional model for the headgroup of this glycolipid using an order matrix approach. The analysis shows an average structure possessing a high degree of order and is most consistent with structures that are well extended from the membrane surface. Saturation of the ganglioside environment with high concentrations of Ca2+ (0.28 M) produces a small perturbation of the headgroup. GM3 dissolved in a DMPC/CHAPSO system can also be demonstrated to bind wheat germ agglutinin (WGA). The minimal perturbation of structure-dependent parameters suggests that the dominant structure of the sialic acid moiety in the isolated membrane system is favored for binding by the protein.

Chemical Phenomena↗

PubMed1993

Structure and dynamics of sialic acid at the surface of a magnetically oriented membrane system.

A structural analysis of a sialic acid containing glycolipid alpha-dodecyl-N-acetylneuraminic acid anchored to the surface of a phospholipid-based membrane-like fragment is presented. The analysis is based on measurement of dipolar interactions between 13C-13C and 13C-1H spin pairs in a magnetically oriented membrane phase and the interpretation of data using an order matrix formalism. Structural analysis in this medium allows an assessment of properties in an environment approximating the environment where similar molecules function as cell surface receptors. The results indicate an extended headgroup conformation with the carboxyl group of the sialic acid near the membrane interface. Anisotropic motion of the sialic acid headgroup occurs with the most restriction along an axis which is significantly out of the plane of the sialic acid ring suggesting an important role for the carboxylate group in membrane surface interaction. The effect of calcium on the headgroup orientation was also investigated, but the results showed no significant change in the preferred conformation, possibly due to a weak binding under the conditions of the experiment.

Glycolipids↗