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Rhamnolipids (RLs) are among the most important biosurfactants produced by microorganisms, and have been widely investigated because of their multiple biological activities. Their action appears to depend on their structural interference with lipid membranes, therefore several studies have been performed to investigate this aspect. We studied by X-ray scattering, neutron reflectometry and molecular dynamic simulations the insertion of dirhamnolipid (diRL), the most abundant RL, in model cellular membranes made of phospholipids and glycosphingolipids. In our model systems the affinity of diRL to the membrane is highly promoted by the presence of the glycosphingolipids and molecular dynamics simulations unveil that this evidence is related to sugar-sugar attractive interactions at the membrane surface. Our results improve the understanding of the plethora of activities associated with RLs, also opening new perspectives in their selective use for pharmaceutical and cosmetics formulations. Additionally, they shed light on the still debated role of carbohydrate-carbohydrate interactions as driving force for molecular contacts at membrane surface.

Immunotherapy using antibodies to target the aggregation of flexible proteins holds promise for therapeutic interventions in neurodegenerative diseases caused by protein misfolding. Prions or PrP

Hypothesis Adsorption of weak polyelectrolytes onto charged nanoparticles, and concurrent effects such as spatial partitioning of ions may be influenced by details of the polyelectrolyte structure (linear or star–like) and size, by the mobility of the nanoparticle surface charge, or the valence of the nanoparticle counterions. Experiments Ionization and complexation of weak polyelectrolytes on spherical macroions with monovalent and divalent countrions has been studied with constant–pH Monte Carlo titrations and primitive electrolyte models for linear and star–like polymers capable, also, of forming charged hydrogen bonds. Nanoparticles surface charge has been represented either as a single colloid–centered total charge (CCTC) or as surface–tethered mobile monovalent spherical charges (SMMSC). Findings Differences in the average number of adsorbed polyelectrolyte arms and their average charge, and in the relative amount of macroion counterions (m–CI’s) released upon polymer adsorption are found between CCTC and SMMSC nanoparticles. The amount of the counterions released also depends on the polymer structure. As CCTC adsorbs a lower number of star–like species arms, the degree of condensation of polymer counterions (p–CI’s) onto the polyelectrolyte is also substantially higher for the CCTC colloid, with a concurrent decrease of the osmotic coefficient values.

Hypothesis The critical concentration above which micelles form from zwitterionic surfactant solutions and their thermodynamic stability is affected by the interaction with weak Bronsted polyacid chains (An) via the formation of charged hydrogen bonds between the latter and anionic moieties. Experiments The interaction between zwitterionic micelles and polyacids capable of forming hydrogen bonds, and its dependence on the environmental pH and polymer structure, has been studied with constant–pH simulations and a restricted primitive model for all electrolytes. Findings At low pH, the formation of polyacid/micelle complexes is witnessed independently of the polymer size or structure, so that the concentration above which micelles form is substantially decreased compared to polyacid–free cases. Upon rising pH, polymer desorption takes place within a narrow range of pH values, its location markedly depending on the size and structure of polyacids, and on the relative disposition between headgroup charged moieties. Thus, the desorption onset for long linear polyacids (A60) interacting with sulphobetainic headgroups is roughly two pH units higher than for six decameric chains (6A10) adsorbed onto micelles bearing phosphorylcholinic headgroups. This effect, together with the preferential desorption of chain ends at intermediate pH, may be exploited for drug delivery purposes or building advanced metamaterials.

The exposure to pathogens triggers the activation of adaptive immune responses through antigens bound to surface receptors of antigen presenting cells (APCs). T cell receptors (TCR) are responsible for initiating the immune response through their physical direct interaction with antigen-bound receptors on the APCs surface. The study of T cell interactions with antigens is considered of crucial importance for the comprehension of the role of immune responses in cancer growth and for the subsequent design of immunomodulating anticancer drugs. RNA sequencing experiments performed on T cells represented a major breakthrough for this branch of experimental molecular biology. Apart from the gene expression levels, the hypervariable CDR3α/β sequences of the TCR loops can now be easily determined and modelled in the three dimensions, being the portions of TCR mainly responsible for the interaction with APC receptors. The most direct experimental method for the investigation of antigens would be based on peptide libraries, but their huge combinatorial nature, size, cost, and the difficulty of experimental fine tuning makes this approach complicated time consuming, and costly. We have implemented in silico methodology with the aim of moving from CDR3α/β sequences to a library of potentially antigenic peptides that can be used in immunologically oriented experiments to study T cells’ reactivity. To reduce the size of the library, we have verified the reproducibility of experimental benchmarks using the permutation of only six residues that can be considered representative of all ensembles of 20 natural amino acids. Such a simplified alphabet is able to correctly find the poses and chemical nature of original antigens within a small subset of ligands of potential interest. The newly generated library would have the advantage of leading to potentially antigenic ligands that would contribute to a better understanding of the chemical nature of TCR-antigen interactions. This step is crucial in the design of immunomodulators targeted towards T-cells response as well as in understanding the first principles of an immune response in several diseases, from cancer to autoimmune disorders.

Traditionally, a drug potency is expressed in terms of thermodynamic quantities, mostly Kd, and empirical IC50 values. Although binding affinity as an estimate of drug activity remains relevant, it is increasingly clear that it is also important to include (un)binding kinetic parameters in the characterization of potential drug-like molecules. Herein, we used standard in silico screening to identify a series of structurally related inhibitors of hDAAO, a flavoprotein involved in schizophrenia and neuropathic pain. We applied a novel methodology, based on scaled molecular dynamics, to rank them according to their residence times. Notably, we challenged the application in a prospective fashion for the first time. The good agreement between experimental residence times and the predicted residence times highlighted the procedure's reliability in both predictive and refinement scenarios. Additionally, through further inspection of the performed simulations, we substantiated a previous hypothesis on the involvement of a protein loop during ligand unbinding.

Intrinsically disordered proteins (IDPs) are emerging as an important class of the proteome. Being able to interact with different molecular targets, they participate in many physiological and pathological activities. However, due to their intrinsically heterogeneous nature, determining the equilibrium properties of IDPs is still a challenge for biophysics. Herein, we applied state-of-the-art enhanced sampling methods to Sev NTAIL, a test case of IDPs, and constructed a bin-based kinetic model to unveil the underlying kinetics. To validate our simulation strategy, we compared the predicted NMR properties against available experimental data. Our simulations reveal a rough free-energy surface comprising multiple local minima, which are separated by low energy barriers. Moreover, we identified interconversion rates between the main kinetic states, which lie in the sub-μs time scales, as suggested in previous works for Sev NTAIL. Therefore, the emerging picture is in agreement with the atomic-level properties possessed by the free IDP in solution. By providing both a thermodynamic and kinetic characterization of this IDP test case, our study demonstrates how computational methods can be effective tools for studying this challenging class of proteins.

In the last three decades, protein and nucleic acid structure determination and comprehension of the mechanisms, leading to their physiological and pathological functions, have become a cornerstone of biomedical sciences. A deep understanding of the principles governing the fates of cells and tissue at the molecular level has been gained over the years, offering a solid basis for the rational design of drugs aimed at the pharmacological treatment of numerous diseases. Historically, affinity indicators (i.e. Kd and IC50/EC50) have been assumed to be valid indicators of the in vivo efficacy of a drug. However, recent studies pointed out that the kinetics of the drug-receptor binding process could be as important or even more important than affinity in determining the drug efficacy. This eventually led to a growing interest in the characterisation and prediction of the rate constants of protein-ligand association and dissociation. For instance, a drug with a longer residence time can kinetically select a given receptor over another, even if the affinity for both receptors is comparable, thus increasing its therapeutic index. Therefore, understanding the molecular features underlying binding and unbinding processes is of central interest towards the rational control of drug binding kinetics. In this review, we report the theoretical framework behind protein-ligand association and highlight the latest advances in the experimental and computational approaches exploited to investigate the binding kinetics.

Ligand-target residence time is emerging as a key drug discovery parameter because it can reliably predict drug efficacy in vivo. Experimental approaches to binding and unbinding kinetics are nowadays available, but we still lack reliable computational tools for predicting kinetics and residence time. Most attempts have been based on brute-force molecular dynamics (MD) simulations, which are CPU-demanding and not yet particularly accurate. We recently reported a new scaled-MD-based protocol, which showed potential for residence time prediction in drug discovery. Here, we further challenged our procedure's predictive ability by applying our methodology to a series of glucokinase activators that could be useful for treating type 2 diabetes mellitus. We combined scaled MD with experimental kinetics measurements and X-ray crystallography, promptly checking the protocol's reliability by directly comparing computational predictions and experimental measures. The good agreement highlights the potential of our scaled-MD-based approach as an innovative method for computationally estimating and predicting drug residence times.

Selected members of the large rolipram-related GEBR family of type 4 phosphodiesterase (PDE4) inhibitors have been shown to facilitate long-term potentiation and to improve memory functions without causing emetic-like behavior in rodents. Despite their micromolar-range binding affinities and their promising pharmacological and toxicological profiles, few if any structure-activity relationship studies have been performed to elucidate the molecular bases of their action. Here, we report the crystal structure of a number of GEBR library compounds in complex with the catalytic domain of PDE4D as well as their inhibitory profiles for both the long PDE4D3 isoform and the catalytic domain alone. Furthermore, we assessed the stability of the observed ligand conformations in the context of the intact enzyme using molecular dynamics simulations. The longer and more flexible ligands appear to be capable of forming contacts with the regulatory portion of the enzyme, thus possibly allowing some degree of selectivity between the different PDE4 isoforms.

For weak polyelectrolytes, the interplay between pH, solvent properties, and polymer structure affects the amount of charges, their distribution, and hence their confor- mations via Coloumb repulsion. Attractive interactions can also develop between charged and neutral sites counteracting the expected Coulomb-induced expansion. To gauge how such competition affects polyelectrolyte structure and ionization, the titration of a single polyelectrolyte chain, isolated or close to a charged sphere, mimicked with a novel many-body potential model is simulated with Monte Carlo. Apart from showing a 10-fold higher ionization than isolated monomers at low pH, interacting species contracted forming short-range clusters of charged and neutral ionizable groups. The presence of a charged sphere synergically boosted both effects due to mono- mer interactions, forcing the chains to condense onto its sur- face at much lower pH. Structural properties, however, seem to be controlled only by the ionization degree despite the pres- ence of the topological restraint represented by the spherical surface. Using Monte Carlo titration results, the equilibrium ionization of isolated chains is also estimated; the results evi- dence that even weak interactions can easily lead to a doubling of the total charge. V C 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2015, 00, 000-000

Intrinsically disordered proteins (IDPs) are emerging as an important class of the proteome. Being able to interact with different molecular targets, they participate in many physiological and pathological activities. However, due to their intrinsically heterogeneous nature, determining the equilibrium properties of IDPs is still a challenge for biophysics. Herein, we applied state-of-the-art enhanced sampling methods to Sev N

The genome of measles virus is encapsidated by multiple copies of the nucleoprotein (N), forming helical nucleocapsids of molecular mass approaching 150 Megadalton. The intrinsically disordered C-terminal domain of N (N TAIL ) is essential for transcription and replication of the virus via interaction with the phosphoprotein P of the viral polymerase complex. The molecular recognition element (MoRE) of N TAIL that binds P is situated 90 amino acids from the folded RNA-binding domain (N CORE ) of N, raising questions about the functional role of this disordered chain. Here we report the first in situ structural characterization of N TAIL in the context of the entire N-RNA capsid. Using nuclear magnetic resonance spectroscopy, small angle scattering, and electron microscopy, we demonstrate that N TAIL is highly flexible in intact nucleocapsids and that the MoRE is in transient interaction with N CORE . We present a model in which the first 50 disordered amino acids of N TAIL are conformationally restricted as the chain escapes to the outside of the nucleocapsid via the interstitial space between successive N CORE helical turns. The model provides a structural framework for understanding the role of N TAIL in the initiation of viral transcription and replication, placing the flexible MoRE close to the viral RNA and, thus, positioning the polymerase complex in its functional environment.

The interplay of protein dynamics and molecular recognition is of fundamental importance in biological processes. Atomic-resolution insights into these phenomena may provide new opportunities for drug discovery. Herein, we have combined NMR relaxation experiments and residual dipolar coupling (RDC) measurements with molecular dynamics (MD) simulations to study the effects of the anti-inflammatory drug carbenoxolone (CBNX) on the conformational properties and on the internal dynamics of a subdomain (box A) of high-mobility group B protein (HMGB1). (15) N relaxation data show that CBNX binding enhances the fast pico- to nanosecond motions of a loop and partially removes the internal motional anisotropy of the first two helices of box A. Dipolar wave analysis of amide RDC data shows that ligand binding induces helical distortions. In parallel, increased mobility of the loop upon ligand binding is highlighted by the essential dynamics analysis (EDA) of MD simulations. Moreover, simulations detect two possible orientations for CBNX, which induces two possible conformations of helix H3, one being similar to the free form and the second one causing a partial helical distortion. Finally, we introduce a new approach for the analysis of the internal coordination of protein residues that is consistent with experimental data and allows us to pinpoint which substructures of box A are dynamically affected by CBNX. The observations reported here may be useful for understanding the role of protein dynamics in binding at atomic resolution.

In this work we present the investigation of the structural disorder induced by Al doping in SmBa 2 Cu 2.67 Al 0.33 O 6+δ superconductors in direct correlation with their transport properties. The interest in such superconducting materials lies in the possibility of extending the δ range of the underdoped region of REBa 2 Cu 3 O 6+δ superconductors, where phenomena such as pseudogap and superconducting diamagnetic fluctuations above T c appear. The pair distribution function analysis of high-resolution powder diffraction data has shown that local distortions are present in the proximity of the Al dopant ions. Disorder spreads to the superconducting Cu2-03 planes producing corrugations in these planes, associated with a decrease of superconducting charge carriers. Conductivity and magnetization measurements have demonstrated that Al-doping affects the charge transport mechanism: Anderson localization and superconductivity coexist, and the T and δ superconducting domains are strongly reduced. Al doping allows not only controlling chemically the charge carrier concentration but also the potential fluctuations within the Cu2-02/3 planes and appears to be a powerful tool for the investigation of the underdoped zone of REBa 2 Cu 3 O 6+δ superconductors.

Correct folding is critical for the biological activities of proteins. As a contribution to a better understanding of the protein (un)folding problem, we studied the effect of temperature and of urea on peptostreptococcal Protein L destructuration. We performed standard molecular dynamics simulations at 300 K, 350 K, 400 K, and 480 K, both in 10 M urea and in water. Protein L followed at least two alternative unfolding pathways. Urea caused the loss of secondary structure acting preferentially on the beta-sheets, while leaving the alpha-helices almost intact; on the contrary, high temperature preserved the beta-sheets and led to a complete loss of the alpha-helices. These data suggest that urea and high temperature act through different unfolding mechanisms, and protein secondary motives reveal a differential sensitivity to various denaturant treatments. As further validation of our results, replica-exchange molecular dynamics simulations of the temperature-induced unfolding process in the presence of urea were performed. This set of simulations allowed us to compute the thermodynamical parameters of the process and confirmed that, in the configurational space of Protein L unfolding, both of the above pathways are accessible, although to a different relative extent.

High-mobility group box 1 protein (HMGB1) is a nuclear component, but extracellularly it serves as a signaling molecule involved in acute and chronic inflammation, for example in sepsis and arthritis. The identification of HMGB1 inhibitors is therefore of significant experimental and clinical interest. We show that glycyrrhizin, a natural anti-inflammatory and antiviral triterpene in clinical use, inhibits HMGB1 chemoattractant and mitogenic activities, and has a weak inhibitory effect on its intranuclear DNA-binding function. NMR and fluorescence studies indicate that glycyrrhizin binds directly to HMGB1 (K(d) approximately 150 microM), interacting with two shallow concave surfaces formed by the two arms of both HMG boxes. Our results explain in part the anti-inflammatory properties of glycyrrhizin, and might direct the design of new derivatives with improved HMGB1-binding properties.

Drug discovery is expensive and high-risk. Its main reasons of failure are lack of efficacy and toxicity of a drug candidate. Binding affinity for the biological target has been usually considered one of the most relevant figures of merit to judge a drug candidate along with bioavailability, selectivity and metabolic properties, which could depend on off-target interactions. Nevertheless, affinity does not always satisfactorily correlate with in vivo drug efficacy. It is indeed becoming increasingly evident that the time a drug spends in contact with its target (aka residence time) can be a more reliable figure of merit. Experimental kinetic measurements are operatively limited by the cost and the time needed to synthesize compounds to be tested, to express and purify the target, and to setup the assays. We present here a simple and efficient molecular-dynamics-based computational approach to prioritize compounds according to their residence time. We devised a multiple-replica scaled molecular dynamics protocol with suitably defined harmonic restraints to accelerate the unbinding events while preserving the native fold. Ligands are ranked according to the mean observed scaled unbinding time. The approach, trivially parallel and easily implementable, was validated against experimental information available on biological systems of pharmacological relevance.

The oxygen non-stoichiometry and diffusion in SmBa2Cu3−xAlxO6+δ superconductor were investigated by means of thermogravimetric analysis (TGA) in a wide P ( O 2 ) ( 1 ⩽ P ( O 2 ) ⩽ 10 − 4 atm ) and T ( 400 ⩽ T ⩽ 800 °C ) range. Thermogravimetric data at fixed oxygen partial pressures were collected using temperature steps and long isotherms. The samples used for TG analysis were previously sieved in order to obtain powders of known particle dimensions. This procedure allowed to determine both the oxygen non-stoichiometry and its diffusion coefficient in SmBa2Cu3−xAlxO6+δ. With respect to undoped SmBa2Cu3O6+δ, the corresponding δ values are quite similar in oxidising conditions, while the doped material loses less oxygen than the pure one in reducing conditions. For what concern the oxygen diffusion, aluminium doping does not affect the activation energy for that process; on the opposite the absolute D value is at least one order of magnitude lower than in the pure phase and the D varies as a function of P ( O 2 ) . All the above features are interpreted under the hypothesis that, in the thermodynamic conditions of the experiments, only O4 oxygen ions and sites surrounded by two Cu1 ions are involved into gas–solid equilibria and into diffusion process.

Prion protein (PrP) amyloid formation is a central feature of genetic and acquired forms of prion disease such as Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob disease. The major component of GSS amyloid is a PrP fragment spanning residues approximately 82-146. To investigate the determinants of the physicochemical properties of this fragment, we synthesized PrP-(82-146) and variants thereof, including entirely and partially scrambled peptides. PrP-(82-146) readily formed aggregates that were partially resistant to protease digestion. Peptide assemblies consisted of 9.8-nm-diameter fibrils having a parallel cross-beta-structure. Second derivative of infrared spectra indicated that PrP-(82-146) aggregates are primarily composed of beta-sheet (54%) and turn (24%) which is consistent with their amyloid-like properties. The peptide induced a remarkable increase in plasma membrane microviscosity of primary neurons. Modification of the amino acid sequence 106-126 caused a striking increase in aggregation rate, with formation of large amount of protease-resistant amorphous material and relatively few amyloid fibrils. Alteration of the 127-146 region had even more profound effects, with the inability to generate amyloid fibrils. These data indicate that the intrinsic properties of PrP-(82-146) are dependent upon the integrity of the C-terminal region and account for the massive deposition of PrP amyloid in GSS.

We present here a study on the effect of Al doping on the stucture of SmBa 2 Cu 3-X Al X O 6+δ (Sm-123) superconductor. Electron MicroProbe Analysis (EMPA) and X-Ray Powder Diffraction (XRPD) have revealed that the limit of a aluminium solubility x is between 0.5 and 0.6. For further doping BaAl 2 O 4 appears besides the superconducting phase. XRPD analysis on samples annealed in both oxidising and reducing conditions have revealed that the Al doping inhibits the tetragonal to orthorhombic phase transition. Nuclear Magnetic Resonance (NMR) analysis has shown that almost all the Al ions are coordinated tetrahedrally. The comparison between oxygen non-stoichiometry in pure and Al doped SmBa 2 Cu 3-X Al X O 6+δ suggests that the Al ions are ordered in clusters. A model is proposed for short-range order around Al doping ions which allows us to interpret the phase transition inhibition.

International audience; Nuclear magnetic resonance (NMR) spectroscopy provides detailed understanding of the nature and extent of protein dynamics on physiologically important timescales. We present recent advances in the combination of NMR with state-of-the-art molecular simulation that are providing unique new insight into the motions on timescales from nanoseconds to milliseconds. In particular, we focus on methods based on residual dipolar couplings (RDCs) that allow for detailed mapping of the protein conformational energy landscape. A novel combination of RDCs with accelerated molecular dynamics allows for the development of ensemble representations of the underlying Boltzmann ensemble.

In order to understand the conformational behaviour of Intrinsically Disordered Proteins (IDPs), it is essential to develop a molecular representation of the partially folded state. Due to the very large number of degrees of conformational freedom available to such a disordered system, this problem is highly underdetermined. Characterisation therefore requires extensive experimental data, and novel analytical tools are required to exploit the specific conformational sensitivity of different experimental parameters. In this review we concentrate on the use of nuclear magnetic resonance (NMR) spectroscopy for the study of conformational behaviour of IDPs at atomic resolution. Each experimental NMR parameter is sensitive to different aspects of the structural and dynamic behaviour of the disordered state and requires specific consideration of the relevant averaging properties of the physical interaction. In this review we present recent advances in the description of disordered proteins and the selection of representative ensembles on the basis of experimental data using statistical coil sampling from flexible-meccano and ensemble selection using ASTEROIDS. Using these tools we aim to develop a unified molecular representation of the disordered state, combining complementary data sets to extract a meaningful description of the conformational behaviour of the protein.

Background: The Wiskott-Aldrich syndrome (WAS) is a rare genetic disease characterized by thrombocytopenia, immunodeficiency, autoimmunity, and hematologic malignancies. Secondary mutations leading to re-expression of WAS protein (WASP) are relatively frequent in patients with WAS. Objective: The tissue distribution and function of revertant cells were investigated in a novel case of WAS gene secondary mutation. Methods: A vast combination of approaches was used to characterize the second-site mutation, to investigate revertant cell function, and to track their distribution over a 18-year clinical follow-up. Results: The WAS gene secondary mutation was a 4-nucleotide insertion, 4 nucleotides downstream of the original deletion. This somatic mutation allowed the T-cell-restricted expression of a stable, full-length WASP with a 3-amino acid change compared with the wild-type protein. WASP(+) T cells appeared early in the spleen (age 10 years) and were highly enriched in a mesenteric lymph node at a later time (age 23 years). Revertant T cells had a diversified T-cell-receptor repertoire and displayed in vitro and in vivo selective advantage. They proliferated and produced cytokines normally on T-cell-receptor stimulation. Consistently, the revertant WASP correctly localized to the immunologic synapse and to the leading edge of migrating T cells. Conclusion: Despite the high proportion of functional revertant T cells, the patient still has severe infections and autoimmune disorders, suggesting that re-expression of WASP in T cells is not sufficient to normalize immune functions fully in patients with WAS. (J Allergy Clin Immunol 2010;125:439-48.) Background: The Wiskott-Aldrich syndrome (WAS) is a rare genetic disease characterized by thrombocytopenia, immunodeficiency, autoimmunity, and hematologic malignancies. Secondary mutations leading to re-expression of WAS protein (WASP) are relatively frequent in patients with WAS. Objective: The tissue distribution and function of revertant cells were investigated in a novel case of WAS gene secondary mutation. Methods: A vast combination of approaches was used to characterize the second-site mutation, to investigate revertant cell function, and to track their distribution over a 18-year clinical follow-up. Results: The WAS gene secondary mutation was a 4-nucleotide insertion, 4 nucleotides downstream of the original deletion. This somatic mutation allowed the T-cell-restricted expression of a stable, full-length WASP with a 3-amino acid change compared with the wild-type protein. WASP(+) T cells appeared early in the spleen (age 10 years) and were highly enriched in a mesenteric lymph node at a later time (age 23 years). Revertant T cells had a diversified T-cell-receptor repertoire and displayed in vitro and in vivo selective advantage. They proliferated and produced cytokines normally on T-cell-receptor stimulation. Consistently, the revertant WASP correctly localized to the immunologic synapse and to the leading edge of migrating T cells. Conclusion: Despite the high proportion of functional revertant T cells, the patient still has severe infections and autoimmune disorders, suggesting that re-expression of WASP in T cells is not sufficient to normalize immune functions fully in patients with WAS. (J Allergy Clin Immunol 2010;125:439-48.) BACKGROUND: The Wiskott-Aldrich syndrome (WAS) is a rare genetic disease characterized by thrombocytopenia, immunodeficiency, autoimmunity, and hematologic malignancies. Secondary mutations leading to re-expression of WAS protein (WASP) are relatively frequent in patients with WAS. OBJECTIVE: The tissue distribution and function of revertant cells were investigated in a novel case of WAS gene secondary mutation. METHODS: A vast combination of approaches was used to characterize the second-site mutation, to investigate revertant cell function, and to track their distribution over a 18-year clinical follow-up. RESULTS: The WAS gene secondary mutation was a 4-nucleotide insertion, 4 nucleotides downstream of the original deletion. This somatic mutation allowed the T-cell-restricted expression of a stable, full-length WASP with a 3-amino acid change compared with the wild-type protein. WASP(+) T cells appeared early in the spleen (age 10 years) and were highly enriched in a mesenteric lymph node at a later time (age 23 years). Revertant T cells had a diversified T-cell-receptor repertoire and displayed in vitro and in vivo selective advantage. They proliferated and produced cytokines normally on T-cell-receptor stimulation. Consistently, the revertant WASP correctly localized to the immunologic synapse and to the leading edge of migrating T cells. CONCLUSION: Despite the high proportion of functional revertant T cells, the patient still has severe infections and autoimmune disorders, suggesting that re-expression of WASP in T cells is not sufficient to normalize immune functions fully in patients with WAS. Copyright 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Mutations in the gene autoimmune regulator (AIRE) cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy. AIRE is expressed in thymic medullary epithelial cells, where it promotes the expression of tissue-restricted antigens. By the combined use of biochemical and biophysical methods, we show that AIRE selectively interacts with histone H3 through its first plant homeodomain (PHD) finger (AIRE–PHD1) and preferentially binds to non-methylated H3K4 (H3K4me0). Accordingly, in vivo AIRE binds to and activates promoters containing low levels of H3K4me3 in human embryonic kidney 293 cells. We conclude that AIRE–PHD1 is an important member of a newly identified class of PHD fingers that specifically recognize H3K4me0, thus providing a new link between the status of histone modifications and the regulation of tissue-restricted antigen expression in thymus.

Activated coagulation Factor V is an important cofactor of the coagulation cascade that catalyzes the formation of the prothrombinase complex on the surface of membranes rich in phosphatidyl-L-serine (PS). Here we report molecular dynamics simulations of the two crystallographic structures (the open and closed conformations) of domain C2 of coagulation Factor V (FaVC2). The calculations were performed in water (1.5 ns for each conformation) and in the presence of a neutral phospholipid bilayer model (POPE; 10 ns for each conformation) in order to describe the dynamics of the free (plasma circulating) and membrane bound forms of FaVC2. Water simulations confirmed the hypothesis that the plasma circulating form is in the closed conformation. In contrast, the membrane simulations showed that both conformations are energetically compatible with membrane binding. We have investigated the mechanism, the dynamics, and the energetics of the binding process. Our data are consistent with published estimates of the immersion depth of the aromatic residues (W26 and W27), and with mutagenesis studies involving specific residues located on the spikes at the bottom of the FaVC2 structure. Electrostatic interactions between the phospholipid head groups and hydrophilic residues at the bottom of the structure play a key role in the binding process by creating a large number of hydrogen bonds that anchor the protein to the membrane. The simulations identified a stable phospholipid binding pocket reminiscent of a previously suggested PS interaction site. Our structural data could contribute to the design of potential inhibitors able to disrupt membrane association.

Using redox proteomics techniques to characterize the thiol status of proteins in human T lymphocytes, we identified cyclophilin A (CypA) as a specifically oxidized protein early after mitogen activation. CypA is an abundantly expressed cytosolic protein, target of the immunosuppressive drug cyclosporin A (CsA), for which a variety of functions has been described. In this study, we could identify CypA as a protein undergoing glutathionylation in vivo. Using MALDI-MS we identified Cys52 and Cys62 as targets of glutathionylation in T lymphocytes, and, using bioinformatic tools, we defined the reasons for the susceptibility of these residues to the modification. In addition, we found by circular dichroism spectroscopy that glutathionylation has an important impact on the secondary structure of CypA. Finally, we suggest that glutathionylation of CypA may have biological implications and that CypA may play a key role in redox regulation of immunity.