The stereochemistry of the amino acid side chain influences the inflammatory potential of muramyl dipeptide in experimental meningitis.

Intrathecal injections of 50 to 100 micro g of (N-acetylmuramyl-L-alanyl-D-isoglutamine) muramyl dipeptide (MDP)/rabbit dose-dependently triggered tumor necrosis factor alpha (TNF-alpha) secretion (12 to 40,000 pg/ml) preceding the influx of leukocytes in the subarachnoid space of rabbits. Intrathecal instillation of heat-killed unencapsulated R6 pneumococci produced a comparable leukocyte influx but only a minimal level of preceding TNF-alpha secretion. The stereochemistry of the first amino acid (L-alanine) of the MDP played a crucial role with regard to its inflammatory potential. Isomers harboring D-alanine in first position did not induce TNF-alpha secretion and influx of leukocytes. This stereospecificity of MDPs was also confirmed by measuring TNF-alpha release from human peripheral mononuclear blood cells stimulated in vitro. These data show that the inflammatory potential of MDPs depends on the stereochemistry of the first amino acid of the peptide side chain and suggest that intact pneumococci and MDPs induce inflammation by different pathways.

Differential roles of T cell receptor alpha and beta chains in ligand binding among H-2Kd-restricted cytolytic T lymphocyte clones specific for a photoreactive Plasmodium berghei circumsporozoite peptide derivative.

To study the interaction of T cell receptor with its ligand, a complex of a major histocompatibility complex molecule and a peptide, we derived H-2Kd-restricted cytolytic T lymphocyte clones from mice immunized with a Plasmodium berghei circumsporozoite peptide (PbCS) 252-260 (SYIPSAEKI) derivative containing photoreactive Nepsilon-[4-azidobenzoyl] lysine in place of Pro-255. This residue and Lys-259 were essential parts of the epitope recognized by these clones. Most of the clones expressed BV1S1A1 encoded beta chains along with specific complementary determining region (CDR) 3beta regions but diverse alpha chain sequences. Surprisingly, all T cell receptors were preferentially photoaffinity labeled on the alpha chain. For a representative T cell receptor, the photoaffinity labeled site was located in the Valpha C-strand. Computer modeling suggested the presence of a hydrophobic pocket, which is formed by parts of the Valpha/Jalpha C-, F-, and G-strands and adjacent CDR3alpha residues and structured to be able to avidly bind the photoreactive ligand side chain. We previously found that a T cell receptor specific for a PbCS peptide derivative containing this photoreactive side chain in position 259 similarly used a hydrophobic pocket located between the junctional CDR3 loops. We propose that this nonpolar domain in these locations allow T cell receptors to avidly and specifically bind epitopes containing non-peptidic side chains.

Comparison between computational alanine scanning and per-residue binding free energy decomposition for protein-protein association using MM-GBSA: application to the TCR-p-MHC complex.

Recognition by the T-cell receptor (TCR) of immunogenic peptides (p) presented by Class I major histocompatibility complexes (MHC) is the key event in the immune response against virus-infected cells or tumor cells. A study of the 2C TCR/SIYR/H-2K(b) system using a computational alanine scanning and a much faster binding free energy decomposition based on the Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) method is presented. The results show that the TCR-p-MHC binding free energy decomposition using this approach and including entropic terms provides a detailed and reliable description of the interactions between the molecules at an atomistic level. Comparison of the decomposition results with experimentally determined activity differences for alanine mutants yields a correlation of 0.67 when the entropy is neglected and 0.72 when the entropy is taken into account. Similarly, comparison of experimental activities with variations in binding free energies determined by computational alanine scanning yields correlations of 0.72 and 0.74 when the entropy is neglected or taken into account, respectively. Some key interactions for the TCR-p-MHC binding are analyzed and some possible side chains replacements are proposed in the context of TCR protein engineering. In addition, a comparison of the two theoretical approaches for estimating the role of each side chain in the complexation is given, and a new ad hoc approach to decompose the vibrational entropy term into atomic contributions, the linear decomposition of the vibrational entropy (LDVE), is introduced. The latter allows the rapid calculation of the entropic contribution of interesting side chains to the binding. This new method is based on the idea that the most important contributions to the vibrational entropy of a molecule originate from residues that contribute most to the vibrational amplitude of the normal modes. The LDVE approach is shown to provide results very similar to those of the exact but highly computationally demanding method.

Radiosensibilité individuelle : les tests vont changer nos pratiques [Intrinsic radiosensitivity: predictive assays that will change the daily practice].

The impact of curative radiotherapy depends mainly on the total dose delivered homogenously in the targeted volume. Nevertheless, the dose delivered to the surrounding healthy tissues may reduce the therapeutic ratio of many radiation treatments. In a same population treated in one center with the same technique, it appears that individual radiosensitivity clearly exists, namely in terms of late side effects that are in principle non-reversible. This review details the different radiobiological approaches that have been developed to better understand the mechanisms of radiation-induced late effects. We also present the possibilities of clinical use of predictive assays in the close future.

Structural analysis of TCR-ligand interactions studied on H-2Kd-restricted cloned CTL specific for a photoreactive peptide derivative.

To study the interaction of the TCR with its ligand, the complex of a MHC molecule and an antigenic peptide, we modified a TCR contact residue of a H-2Kd-restricted antigenic peptide with photoreactive 4-azidobenzoic acid. The photoreactive group was a critical component of the epitope recognized by CTL clones derived from mice immunized with such a peptide derivative. The majority of these clones expressed V beta 1-encoded beta chains that were paired with J alpha TA28-encoded alpha chains. For one of these TCR, the photoaffinity labeled sites were mapped on the alpha chain as a J alpha TA28-encoded tryptophan and on the beta chain as a residue of the C' strand of V beta 1. Molecular modeling of this TCR suggested the presence of a hydrophobic pocket that harbors this tryptophan as well as a tyrosine on the C' strand of V beta 1 between which the photoreactive side chain inserts. It is concluded that this avid binding principle may account for the preferential selection of V beta 1 and J alpha TA28-encoded TCR.

Enhanced production of indole-3-acetic acid by a genetically modified strain of Pseudomonas fluorescens CHA0 affects root growth of cucumber but does not improve protection of the plant against Pythium root rot

The biocontrol strain CHA0 of Pseudomonas fluorescens produces small amounts of indole-3-acetic acid via the tryptophan side chain oxidase and the tryptophan transaminase pathways. A recombinant plasmid (pME3468) expressing the tryptophan monooxygenase pathway was introduced into strain CHA0; this resulted in elevated synthesis of indole-3-acetic acid in vitro, especially after addition of -tryptophan. In natural soil, strain CHA0/pME3468 increased fresh root weight of cucumber by 17-36%, compared to the effect of strain CHA0; root colonization was about 106 cells per g of root. However, both strains gave similar protection of cucumber against Pythium ultimum. In autoclaved soil, at 6×107 cells per g of root, strain CHA0 stimulated growth of roots and shoots, whereas strain CHA0/pME3468 caused root stunting and strong reduction of plant weight. These results are in agreement with the known effects of exogenous indole-3-acetic acid on plant roots and suggest that in the system examined, indole-3-acetic acid does not contribute to the biocontrol properties of strain CHA0.

H-2Kd-restricted antigenic peptides share a simple binding motif.

We have defined structural features that are apparently important for the binding of four different, unrelated antigenic epitopes to the same major histocompatibility complex (MHC) class I molecule, H-2Kd. The four epitopes are recognized in the form of synthetic peptides by cytotoxic T lymphocytes of the appropriate specificity. By analysis of the relative potency of truncated peptides, we demonstrated that for each of the four epitopes, optimal antigenic activity was present in a peptide of 9 or 10 amino acid residues. A comparison of the relative competitor activity of the different-length peptides in a functional competition assay, as well as in a direct binding assay based on photoaffinity labeling of the Kd molecule, indicated that the enhanced potency of the peptides upon reduction in length was most likely due to a higher affinity of the shorter peptides for the Kd molecule. A remarkably simple motif that appears to be important for the specific binding of Kd-restricted peptides was identified by the analysis of peptides containing amino acid substitutions or deletions. The motif consists of two elements, a Tyr in the second position relative to the NH2 terminus and a hydrophobic residue with a large aliphatic side chain (Leu, Ile, or Val) at the COOH-terminal end of the optimal 9- or 10-mer peptides. We demonstrated that a simple peptide analogue (AYP6L) that incorporates the motif can effectively and specifically interact with the Kd molecule. Moreover, all of the additional Kd-restricted epitopes defined thus far in the literature contain the motif, and it may thus be useful for the prediction of new epitopes recognized by T cells in the context of this MHC class I molecule.

Permeability properties of ENaC selectivity filter mutants.

The epithelial Na(+) channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na(+) absorption. The amiloride-sensitive ENaC is highly selective for Na(+) and Li(+) ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the alphaS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K(+) ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted alphaS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position alpha589, indicating that alphaS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na(+) and Li(+). These findings demonstrate the critical role of the pore size at the alphaS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the alphaS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the alphaS589 side chain is oriented toward the subunit-subunit interface and that substitution of alphaS589 by larger residues increases the pore diameter by adding extra volume at the subunit-subunit interface.

Role of intracellular cysteines in ENaC function

The epithelial sodium channel (ENaC) regulates the sodium reabsorption in the collecting duct principal cells of the nephron. ENaC is mainly regulated by hormones such as aldosterone and vasopressin, but also by serine proteases, Na+ and divalent cations. The crystallization of an ENaC/Deg member, the Acid Sensing Ion Channel, has been recently published but the pore-lining residues constitution of ENaC internal pore remains unclear. It has been reported that mutation aS589C of the selectivity filter on the aENaC subunit, a three residues G/SxS sequence, renders the channel permeant to divalent cations and sensitive to extracellular Cd2+. We have shown in the first part of my work that the side chain of aSer589 residue is not pointing toward the pore lumen, permitting the Cd2+ to permeate through the ion pore and to coordinate with a native cysteine, gCys546, located in the second transmembrane domain of the gENaC subunit. In a second part, we were interested in the sulfhydryl-reagent intracellular inhibition of ENaC-mediated Na+ current. Kellenberger et al. have shown that ENaC is rapidly and reversibly inhibited by internal sulfhydryl reagents underlying the involvement of intracellular cysteines in the internal regulation of ENaC. We set up a new approach comprising a Substituted Cysteine Analysis Method (SCAM) using intracellular MTSEA-biotin perfusion coupled to functional and biochemical assays. We were thus able to correlate the cysteine-modification of ENaC by methanethiosulfonate (MTS) and its effect on sodium current. This allowed us to determine the amino acids that are accessible to intracellular MTS and the one important for the inhibition of the channel. RESUME : Le canal épithélial sodique ENaC est responsable de la réabsorption du sodium dans les cellules principales du tubule collecteur rénal. Ce canal est essentiellement régulé par voie hormonale via l'aldostérone et la vasopressine mais également par des sérines protéases, le Na+ lui-même et certains cations divalents. La cristallisation du canal sodique sensible au pH acide, ASIC, un autre membre de la famille ENaC/Deg, a été publiée mais les acides aminés constituant le pore interne d'ENaC restent indéterminés. Il a été montré que la mutation aS589C du filtre de sélectivité de la sous-unité aENaC permet le passage de cations divalents et l'inhibition du canal par le Cd2+ extracellulaire. Dans un premier temps, nous avons montré que la chaîne latérale de la aSer589 n'est pas orientée vers l'intérieur du pore, permettant au Cd2+ de traverser le canal et d'interagir avec une cysteine native du second domaine transrnembranaire de la sous-unité γENaC, γCys546. Dans un second temps, nous nous sommes intéressés au mécanisme d'inhibition d'ENaC par les réactifs sulfhydryl internes. Kellenberger et al. ont montré l'implication de cystéines intracellulaires dans la régulation interne d'ENaC par les réactifs sulfhydryl. Nous avons mis en place une nouvelle approche couplant la méthode d'analyse par substitution de cystéines (SCAM) avec des perfusions intracellulaires de MTSEAbiotine. Ainsi, nous pouvons meure en corrélation les modifications des cystéines d'ENaC par les réactifs methanethiosulfonates (MTS) avec leur effet sur le courant sodique, et donc mettre en évidence les acides aminés accessibles aux MTS intracellulaires et ceux qui sont importants dans la fonction du canal.

The SLC2 family of facilitated hexose and polyol transporters.

The SLC2 family of glucose and polyol transporters comprises 13 members, the glucose transporters (GLUT) 1-12 and the H(+)- myo-inositol cotransporter (HMIT). These proteins all contain 12 transmembrane domains with both the amino and carboxy-terminal ends located on the cytoplasmic side of the plasma membrane and a N-linked oligosaccharide side-chain located either on the first or fifth extracellular loop. Based on sequence comparison, the GLUT isoforms can be grouped into three classes: class I comprises GLUT1-4; class II, GLUT6, 8, 10, and 12 and class III, GLUT5, 7, 9, 11 and HMIT. Despite their sequence similarity and the presence of class-specific signature sequences, these transporters carry various hexoses and HMIT is a H(+)/ myo-inositol co-transporter. Furthermore, the substrate transported by some isoforms has not yet been identified. Tissue- and cell-specific expression of the well-characterized GLUT isoforms underlies their specific role in the control of whole-body glucose homeostasis. Numerous studies with transgenic or knockout mice indeed support an important role for these transporters in the control of glucose utilization, glucose storage and glucose sensing. Much remains to be learned about the transport functions of the recently discovered isoforms (GLUT6-13 and HMIT) and their physiological role in the metabolism of glucose, myo-inositol and perhaps other substrates.

Assignment strategies for large proteins by magic-angle spinning NMR: the 21-kDa disulfide-bond-forming enzyme DsbA.

We present strategies for chemical shift assignments of large proteins by magic-angle spinning solid-state NMR, using the 21-kDa disulfide-bond-forming enzyme DsbA as prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to approximately 17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy, and three-dimensional (3D) and four-dimensional (4D) backbone correlation experiments yield highly confident assignments for more than 90% of backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths below 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, transferred echo double resonance experiments enhanced sensitivity for Pro and Gly residues; [2-(13)C]glycerol labeling clarified Val, Ile, and Leu assignments; in-phase anti-phase correlation spectra enabled interpretation of otherwise crowded Glx/Asx side-chain regions; and 3D NCACX experiments on [2-(13)C]glycerol samples provided unique sets of aromatic (Phe, Tyr, and Trp) correlations. Together with high-sensitivity CANCOCA 4D experiments and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and will enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution two-dimensional (13)C-(13)C spectrum.

Origin of the spectral shifts among the early intermediates of the rhodopsin photocycle.

A combined strategy based on the computation of absorption energies, using the ZINDO/S semiempirical method, for a statistically relevant number of thermally sampled configurations extracted from QM/MM trajectories is used to establish a one-to-one correspondence between the structures of the different early intermediates (dark, batho, BSI, lumi) involved in the initial steps of the rhodopsin photoactivation mechanism and their optical spectra. A systematic analysis of the results based on a correlation-based feature selection algorithm shows that the origin of the color shifts among these intermediates can be mainly ascribed to alterations in intrinsic properties of the chromophore structure, which are tuned by several residues located in the protein binding pocket. In addition to the expected electrostatic and dipolar effects caused by the charged residues (Glu113, Glu181) and to strong hydrogen bonding with Glu113, other interactions such as π-stacking with Ala117 and Thr118 backbone atoms, van der Waals contacts with Gly114 and Ala292, and CH/π weak interactions with Tyr268, Ala117, Thr118, and Ser186 side chains are found to make non-negligible contributions to the modulation of the color tuning among the different rhodopsin photointermediates.

Oxidation of proteins: Basic principles and perspectives for blood proteomics.

Protein oxidation mechanisms result in a wide array of modifications, from backbone cleavage or protein crosslinking to more subtle modifications such as side chain oxidations. Protein oxidation occurs as part of normal regulatory processes, as a defence mechanism against oxidative stress, or as a deleterious processes when antioxidant defences are overcome. Because blood is continually exposed to reactive oxygen and nitrogen species, blood proteomics should inherently adopt redox proteomic strategies. In this review, we recall the biochemical basis of protein oxidation, review the proteomic methodologies applied to analyse redox modifications, and highlight some physiological and in vitro responses to oxidative stress of various blood components.