Lydie Lethier

The protease activity of transthyretin reverses the effect of pH on the amyloid-β protein/heparan sulfate proteoglycan interaction: A biochromatographic study

Patients suffering of Alzheimers disease (AD) are characterized by a low transthyretin (TTR) level in the brain. The effect of pH and TTR concentration in the medium on the β-amyloid protein (Aβ)/heparan sulfate proteoglycan (HSPG) association mechanism were studied using a biochromatographic approach. For this purpose, HSPG was immobilized via amino groups onto the amino propyl silica pre-packed column, activated with glutaraldehyde, by using the Schiff base method. Using an equilibrium perturbation method, it was clearly shown that Aβ can be bound with HSPG. This approach allowed the determination of the thermodynamic data of this binding mechanism. The role of the pH was also analyzed. Results from enthalpy-entropy compensation and the plot of the number of protons exchanged versus pH showed that the binding mechanism was dependent on pH with a critical value at pH=6.5. This value agreed with a histidine protonation as an imidazolium cation. Moreover, the corresponding thermodynamical data showed that at pH>6.5, van der Waals and hydrogen bonds due to aromatic amino acids as tyrosine or phenylalanine present in the N-terminal (NT) part governed the Aβ/HSPG association. Aβ remained in its physiological structure in a random coil form (i.e. the non-amyloidogenic structure) because van der Waals interactions and hydrogen bonds were preponderant. At acidic pH (pH<6.5), ionic and hydrophobic interactions, created by histidine protonation and hydrophobic amino acids, appeared in the Aβ/HSPG binding. These hydrophobic and ionic interactions led to the conversion of the random coil form of Aβ into a β-sheet structure which was the amyloidogenic folding. When TTR was incubated with Aβ, the Aβ/HSPG association mechanism was enthalpy driven at all pH values. The affinity of Aβ for HSPG decreased when TTR concentration increased due to the complexation of Aβ with TTR. Also, the decrease of the peak area with the increase of TTR concentration demonstrated that this Aβ/TTR association led to the cleavage of Aβ full length to a smaller fragment. For acidic pH (pH<6.5), it was shown that the importance of the hydrophobic and ionic interactions decreased when TTR concentration increased. This result confirmed that Aβ was cleaved by TTR in a part containing only the NT part. Our results demonstrated clearly that TTR reversed the effect of acidic pH and thus played a protective role in AD.

Development and Evaluation of a New Fluorinated Double-Wall Carbon Nanotube HPLC Stationary Phase

A novel column based on silica-containing immobilized fluorinated double-wall carbon nanotubes (F-DWCNTs) was developed. This F-DWCNT stationary phase was synthesized to combine the analytical performance of carbon nanotubes and the fluorine-based unique selectivity for polar compounds. First, the chromatographic support was coated with DWCNTs in a noncovalent way to preserve the sp2 internal nanotube structure. Second, the DWCNT silica particles were functionalized with fluorine atoms via a solution of Br2 and BrF3 at room temperature. This F-DWCNT stationary phase was applied for a variety of separations. The solute retention behaviour was particularly studied under isocratic conditions with a high fraction of ACN in the ACN/water (v/v) mobile phase. The retention factors of the solute molecule do not depend linearly on the ACN fraction, but follow a quadratic relationship. This fluorinated stationary phase separated compounds based upon a combination of hydrophobic and polar selective stationary phase interactions. This F-DWCNT appeared to work best when fluorinated or halogenated compounds were encountered. They have longer retention time, better selectivity and work well with high fraction of organic modifiers. This novel stationary phase could thus be a good choice for LC–MS experiments.

Carbon nanotube poroshell silica as a novel stationary phase for fast HPLC analysis of monoclonal antibodies

A new carbon nanotube porous silica poroshell stationary phase was developed. The chromatographic support was coated with ultrashort single-wall carbon nanotubes (SWCNTs) in a noncovalent way. It was demonstrated that the porous amino silica surface of the 300 NH2 poroshell column stabilized with 1-methyl-2-pyrrolidinone efficiently and stably adsorbed SWCNTs onto the chromatographic support. It was shown that this novel poroshell carbon nanotube (CNT) stationary phase was very useful for the HPLC separation of a series of monoclonal antibodies (mAbs) in a short analysis time (<3xa0min). The high-performance liquid chromatography (HPLC) method was validated and was successfully tested for the fast quantitative and qualitative control of chemotherapeutic bags fabricated in a hospital pharmacy.

A Novel Fluorinated Boron Nitride Nanotube Organic Monolithic Column for Capillary Liquid Chromatography

A novel HPLC stationary phase based on fluorinated boron nitride nanotubes (F-BNNTs) incorporated into a monolithic polymeric material was developed. This F-BNNT stationary phase was synthesized to combine the analytical performance of boron nitride nanotubes and the fluorine-based unique selectivity for polar compounds. This F-BNNT column appeared to work well when fluorinated or halogenated compounds were encountered.

Thermodynamic study of transthyretin association (wild‐type and senile forms) with heparan sulfate proteoglycan: pH effect and implication of the reactive histidine residue

The tetramer destabilization of transthyretin into monomers and its fibrillation are phenomena leading to amyloid deposition. Heparan sulfate proteoglycan (HSPG) has been found in all amyloid deposits. A chromatographic approach was developed to compare binding parameters between wild-type transthyretin (wtTTR) and an amyloidogenic transthyretin (sTTR). Results showed a greater affinity of sTTR for HSPG at pHu20097.4 compared with wtTTR owing to the monomeric form of sTTR. Analysis of the thermodynamic parameters showed that van der Waals interactions were involved at the complex interface for both transthyretin forms. For sTTR, results from the plot representing the number of protons exchanged vs pH showed that the binding mechanism was pH-dependent with a critical value at a pHu20096.5. This observation was due to the protonation of a histidine residue as an imidazolium cation, which was not accessible when TTR was in its tetrameric structure. At pH >6.5, dehydration at the binding interface and several contacts between nonpolar groups of sTTR and HSPG were also coupled to binding for an optimal hydrogen-bond network. At pH <6.5, the protonation of the His residue from sTTR monomer when pH decreased broke the hydrogen-bond network, leading to its destabilization and thus producing slight conformational changes in the sTTR monomer structure.

An HPLC chromatographic framework to analyze the β-cyclodextrin/solute complexation mechanism using a carbon nanotube stationary phase

A carbon nanotube (CNT) stationary phase was used for the first time to study the β-cyclodextrin (β-CD) solute complexation mechanism using high performance liquid chromatography (HPLC). For this, the β-CD was added at various concentrations in the mobile phase and the effect of column temperature was studied on both the retention of a series of aniline and benzoic acid derivatives with the CNT stationary phase and their complexation mechanism with β-CD. A decrease in the solute retention factor was observed for all the studied molecules without change in the retention order. The apparent formation constant KF of the inclusion complex β-CD/solute was determined at various temperatures. Our results showed that the interaction of β-CD with both the mobile phase and the stationary phase interfered in the complex formation. The enthalpy and entropy of the complex formation (ΔHF and ΔSF) between the solute molecule and CD were determined using a thermodynamic approach. Negative enthalpies and entropies indicated that the inclusion process of the studied molecule in the CD cavity was enthalpically driven and that the hydrogen bonds between carboxylic or aniline groups and the functional groups on the β-CD rim play an important role in the complex formation.

Heparan Sulfate Proteoglycans Promote Telomerase Internalization and MHC Class II Presentation on Dendritic Cells

Telomerase is a prototype-shared tumor Ag and represents an attractive target for anticancer immunotherapy. We have previously described promiscuous and immunogenic HLA-DR–restricted peptides derived from human telomerase reverse transcriptase (hTERT) and referred as universal cancer peptide (UCP). In nonsmall cell lung cancer, the presence of spontaneous UCP-specific CD4 T cell responses increases the survival of chemotherapy-responding patients. However, the precise mechanisms of hTERT’s uptake, processing, and presentation on MHC-II molecules to stimulate CD4 T cells are poorly understood. In this work, by using well-characterized UCP-specific CD4 T cell clones, we showed that hTERT processing and presentation on MHC-II involve both classical endolysosomal and nonclassical cytosolic pathways. Furthermore, to our knowledge, we demonstrated for the first time that hTERT’s internalization by dendritic cells requires its interaction with surface heparan sulfate proteoglycans. Altogether, our findings provide a novel mechanism of tumor-specific CD4 T cell activation and will be useful for the development of novel cancer immunotherapies that harness CD4 T cells.

HPLC Characterization of the Association Mechanism of Organic Compounds with β Cyclodextrin Using a Novel Boron Nitride Nanotube Particulate Stationary Phase

In this paper, a simple homogeneous coating of silica spherical particles with pristine boron nitride nanotubes (BNNTs) was described. BNNTs dissolved in dimethylacetamide (DMAc) were mixed with amino-functionalized silica particles having a 5 μm diameter. Favorable interaction between the amino group and the BNNT surfaces induces the absorption of the BNNTs on the silica. The BNNT-coated silica particles were used as stationary phase for HPLC. For the first time, it was demonstrated that this new particulate BNNT stationary phase can be used for the study of the complexation of solute molecules (terpene molecules used as test drugs in this work) with β cyclodextrin (βCD). The apparent formation constants Kf of terpene derivative/βCD were in the same magnitude as those reported in the literature. The plot of Kf versus the water fraction in the methanol/water mobile phase showed that the BNNT surface played an active role in the complex formation due to terpene/BNNT-specific polar interactions. This work demonstrated that our novel particulate BNNT HPLC stationary phase was an efficient tool to study molecular recognition mechanism and more specifically the association between a drug substance and a target molecule with the aim of reaching biopharmaceutic and clinical applications.

An HPLC method associated with a thermodynamic analysis to compare the binding of TRAIL and its nanovectorized form to death receptors DR4 and DR5 and their relationship to cytotoxicity.

TRAIL is a member of the TNF family of cytokines which induces apoptosis of cancer cells via its binding to its cognate receptors, DR5 a high affinity site and DR4 a site of low affinity. Our working group has recently demonstrated that nanovectorization of TRAIL with single wall carbon nanotubes (abbreviated NPT) enhanced TRAIL affinity to the high affinity site DR5 and increased pro apoptotic potential in different human tumor cell lines. In this paper, the DR4 low affinity site was immobilized on a chromatographic support and the effect of temperature on a wide temperature range 1°C-50°C was studied to calculate the thermodynamic parameters of the binding of TRAIL and NPT to DR4 and DR5 receptors. For the first time the heat capacity changes for the different binding processes were determined. At a physiological pH (7.4) the heat capacity changes for the binding of NPT to DR4 and DR5 were respectively equal to -0.91kJ/molK and -0.28kJ/molK and those obtained for the binding of TRAIL to DR4 and DR5 were respectively equal to -1.54kJ/molK and -1.05kJ/molK. By the use of differential scanning calorimetry (DSC), a phase transition (∼12°C for DR5, ∼4°C for DR4) between a disordered (low temperature) and an ordered (high temperature) solid like state visualized in the receptor structure confirmed the temperature dependence of binding affinity enthalpy ΔH for soluble TRAIL and its nanovectorized form to its cognate receptors. In the low temperature domain, the positive ΔH values contribute non-favourably to the free energy of binding, TRAIL and NPT described similar affinities for DR4 and DR5. For the high temperature domain, negative ΔH values indicated that van der Waals interactions and hydrogen bonding are engaged favourably at the ligand - receptor interface. Above 30°C, their rank-ordered affinities were thus strongly different in the sequence: TRAILDR4<NPTDR4<TRAILDR5< NPTDR5. The nanovectorization of TRAIL enhanced its binding to both DR4 and DR5 receptors at 37°C and could potentially sensitized cancer cells to TRAIL induced apoptosis through simultaneous activation of DR4 and DR5 as described in this paper for the non-small lung carcinoma cell line (H1703), the two hepatocarcinoma cell lines (SK-Hep1, HUH) and the colon carcinoma cell line (HCT116WT). The excellent linear coefficient (r2=1) for the plot comparing NPT cytotoxicity to TRAIL cytotoxicity confirmed a high degree of similarity for the mechanism of cytotoxicity of TRAIL and NPT between these four cell lines expressing DR4 and DR5 receptors. The slope (0.10) and the negative intercept (-1.23) of this plot indicated that NPT was much more efficient in these four cell lines as compared to TRAIL alone. As well, it was shown that the extra cellular acidosis led to the protonation of the TRAIL residue histidine by flipping the His switch to the on position with a concomitant decrease in affinity for DR4 and DR5 receptors. The highest affinity for NPT to DR4 and DR5 receptors observed at low pHs was due to the less accessibility of the His molecular switch to be protonated when TRAIL was immobilized on CNTs.

H2O2: a Ca2+ or Mg2+‐sensing function in statin passive diffusion

In a previous paper Guillaumes group demonstrated that magnesium (Mg(2+) concentration range 0.00-2.60 mm) increased the passive diffusion of statins and thus played a role in their potential toxicity. In order to confirm an increase in this passive diffusion by divalent salt cations, the role of calcium chloride (CaCl2) on the statin-immobilized artificial membrane (IAM) association was studied. It was demonstrated that calcium supplementation (Ca(2+) concentration range 0.00-3.25 mm) increases the statin passive diffusion. In addition, it was shown that the Ca(2+) effect on the statin-IAM association is higher than that of Mg(2+). These results show that Ca(2+) enhances the passive diffusion of drugs into biological membranes and thus their potential toxicity. Also, addition of H2O2 to the medium showed a hyperbolic response for the statin passive diffusion and this effect was enhanced for the highest Ca(2+) or Mg(2+) concentrations in the medium. H2O2 is likely to interact with the polar head groups of the IAM through dipole-dipole interactions. The conformational changes in H2O2-IAM result in a higher degree of exposure of hydrophobic areas, thus explaining why the binding of pravastatin, which showed the lowest logP value, was less affected by H2O2. This result shows the significant contribution of H2O2 and thus the oxidative stress on the statin passive diffusion. Much of the sensitivity derives from the action of Ca(2+) or Mg(2+), in turn supported the idea that H2O2 may serve a Ca(2+) or Mg(2+) sensing function in statin passive diffusion.