Ca2+, a signal for death and life, is closely involved in the regulation of numerous important cellular events. Ca2+ carries out its function through its binding to Ca2+-receptors or Ca2+-binding proteins. The EF-hand protein, with a helix-loop-helix Ca2+-binding motif, constitutes one of the largest protein families. To facilitate our understanding of the role of Ca2+ in biological systems (denoted as calciomics) using genomic information, an improved pattern search method (http://www.chemistry.gsu.edu/faculty/Yang/Calciomics.htm) for the identification of EF-hand and EF-like Ca2+-binding proteins was developed. This fast and robust method allows us to analyze putative EF-hand proteins at the genome-wide level and further visualize the evolutionary scenario of the EF-hand protein family. This prediction method further enables us to locate a putative viral EF-hand Ca2+-binding motif within the rubella virus nonstructural protease that cleaves the nonstructural protein precursor into two active replicase components. A novel grafting approach has been used to probe the metal-binding properties of this motif by engineering the predicted 12-residue Ca2+-coordinating loop into a non-Ca2+-binding scaffold protein, CD2 domain 1. Structural and conformational studies were further performed on a purified, bacterially-expressed NS protease minimal metal-binding domain spanning the Zn2+- and EF-hand Ca2+-binding motif. It was revealed that Ca2+ binding induced local conformational changes and increased thermal stability. Furthermore, functional studies were carried out using RUB infectious cDNA clone and replicon constructs. Our studies have shown that the Ca2+ binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions. In addition, we have predicted and characterized a calmodulin-binding domain in the gap junction proteins connexin43 and connexin44. Peptides encompassing the CaM binding motifs were synthesized and their ability to bind CaM was determined using various biophysical approaches. Transient expression in HeLa cells of two mutant Cx43-EYFP constructs without the putative CaM-binding site eliminated the Ca2+-dependent inhibition of gap junction permeability. These results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 and Cx44 in a Ca2+-dependent manner, providing a molecular basis for the well-characterized Ca2+-dependent inhibition of Cx43-containing gap junctions.

Ca2+, both as a first and a second messenger, is closely involved in the modulation and regulation of numerous important cellular events, such as cell proliferation, differentiation and cell death. Fine-tuned Ca2+ signaling is achieved by its reversible or irreversible binding to a repertoire of Ca2+ signaling molecules. Among them, the extracellular calcium sensing receptor (CaSR) senses Ca2+ concentration ([Ca2+]o) in the milieu outside of cells where Ca2+ serves as a first messenger. An array of naturally-occurring mutations in CaSR has been found in patients with inherited disorders of Ca2+ homeostasis, leading to abnormal intracellular responses toward [Ca2+]o. In the present study, we have computationally predicted and experimentally characterized the metal-binding properties of five Ca2+-binding sites within CaSR and the accompanying metal--induced conformational changes by using two complementary methods-the grafting approach and the subdomain approach. Based on our results, a model has been proposed to explain the distinct CaSR-mediated responses toward abnormally ¡°high¡± or ¡°low¡± extracellular Ca2+ levels. In addition, we predicted and verified the interaction between CaSR with the most ubiquitously expressed four EF-hand-containing intracellular Ca2+ sensor protein, calmodulin (CaM). Our results demonstrate that the C-terminal CaM-binding domain of the CaSR is essential for proper intracellular Ca2+ response to external signals. Furthermore, we have applied the grafting approach to study the metal-binding properties and oligomeric state of the single EF-hand containing protein, STIM1. Our studies confirmed that the single EF-hand motif in STIM1, which resides in an equilibratium between its monomeric and dimeric forms, was capable of binding Ca2+ with a dissociation constant comparable to the ER Ca2+ concentration, suggesting it could function as a ER Ca2+ sensor responsible for sensing the Ca2+ filling state of ER.

No description available.

Un modèle d'endommagement dédié aux composites bobinés est développé à partir des outils de lamécanique de l'endommagement continu, de la thermodynamique des processus irréversibles et dela théorie de représentation des fonctions tensorielles. La particularité de ce modèle est l'utilisationd'une approche à directions fixes de l'endommagement qui associe à chaque mode de dégradationdes variables internes scalaires et des tenseurs directionnels. La rupture des fibres (considéréecomme probabiliste), les fissurations tant matricielles, que hors - plan ou provoquées par lecisaillement sont ainsi prises en compte. Le modèle est capable de reproduire, dans un contextetridimensionnel imposé par les fortes épaisseurs de composite, la perte de rigidité, l'interaction entreanisotropies initiale et induite, la non linéarité du comportement, les déformations résiduelles et laviscosité en cisaillement. Afin de valider cette approche, le comportement thermomécaniqued'éprouvettes issues de structures bobinées a été caractérisé grâce à des essais de traction multi -instrumentés (vidéo - traction, émission acoustique,...). On montre que le modèle est capable nonseulement de simuler la réponse mécanique macroscopique de ces échantillons, mais également dereproduire l'émission acoustique enregistrée, de distinguer les différentes formesd'endommagement et de prédire précisément l'éclatement des réservoirs hyperbares.

Voltage-gated Cav2.1 (P/Q-type) and Cav2.2 (N-type) channels are two closely related calcium channels that play indispensable roles in signal transduction pathways by regulating neurotransmitter release. Despite having highly conserved amino acid sequences, they are differentially modulated by calmodulin, which mediate two important feedback mechanisms known as Ca2+-dependent inactivation (CDI) and Ca2+-dependent facilitation (CDF). These dual regulatory mechanisms contribute to synaptic plasticity, but only CDI is observed in Cav2.2 channel, while both CDI and CDF are present in Cav2.1 channel. Previously, it was hypothesized that the lack of CDF in Cav2.2 channel is due to the pre-IQ-IQ domain of the channel’s lower binding affinity for calmodulin compared to that of Cav2.1 channel. Now that the EF-hand domain of calcium channels is identified as one of the two minimally required molecular determinants that are responsible for supporting CDF in Cav2.1 channel and preventing CDF in Cav2.2 channel, it was necessary to determine the role of EF-hand domain in calmodulin binding of Cav2.1 and Cav2.2 channels. Using pull-down binding assays, this study finds that the EF-hand domain enhances calmodulin binding to the proximal C-terminal domain of Cav2.2 channel, which suggests that the lack of CDF in Cav2.2 does not result from the channel’s weak interaction with CaM, but from the EF-pre-IQ-IQ domain of the channel’s inability to allow calmodulin from fully exerting its effects.

Cette étude aborde le problème de l’aggravation du risque de fractures de côtes chez les automobilistesâgés en choc frontal. L’analyse de la bibliographie fait ressortir que les moyens actuels d’évaluationdu risque de fractures ne permettent pas de prendre en compte les différences anatomiques et depropriétés mécaniques du thorax observées chez les personnes âgées. Les modèles éléments finis (EF)personnalisés du corps humain offrent un grand potentiel en tant qu’outil avancé d’évaluation durisque de blessures. Toutefois, des données expérimentales sont nécessaires pour valider ces modèlesdans des conditions réalistes. De plus, le choix du niveau de personnalisation et la sensibilité de laréponse du modèle à celle-ci doivent être évaluées.Des expérimentations in vivo menés sur des volontaires ceinturés en choc léger, de différents âges etanthropométries, ont été réalisées. Ces tests ont permis d’étudier l’influence de l’âge et de lacorpulence sur la réponse mécanique du thorax et ont permis l’obtention de corridors nécessaires à lavalidation de modèles EF personnalisés. La géométrie du modèle numérique THUMS a été adaptée àcelle des volontaires et les propriétés mécaniques du thorax ont été modifiées au vu du vieillissementpour effectuer une analyse similaire dans le domaine lésionnel. Les simulations numériques ont mis enévidence un risque accru de fracture de côtes pour certains modèles personnalisés.Cette étude devrait permettre de mieux estimer le risque de blessure pour les automobilistesvulnérables. Elle devrait contribuer ainsi à promouvoir les modèles personnalisés du corps humaincomme outil avancé d’évaluation du risque de blessures. / This study deals with the topic of increased risk of rib fractures among elderly drivers infrontal impact. The analysis of the literature reveals that actual thorax injury assessment tools do nottake into account for the differences in anatomical features and biological material properties observedbetween adults and elderly. Personalized human body finite element (FE) models have great potentialas improved thorax injury assessment tools. However, experimental data are needed to validate thesemodels under real-world conditions. In addition, the choice of the level of personalization of the modeland the sensibility of the model response to this personalization must be assessed to predict thoracicinjury risk.In vivo sled tests were performed on belted volunteers of various anthropometries and age. These testswere used to assess the influence of age and corpulence on thorax mechanical response and allowed toobtain corridor responses needed to validate personalized FE models. The geometry of the FE modelTHUMS was adapted to the volunteers and the thorax material properties were modified consideringaging to carry out a similar analysis in the injurious domain. Numerical simulations highlighted anincreased risk of rib fractures for specific personalized models.This study should help to better estimate the injury risk for car occupants. It should contribute topromote personalized human body models as attractive thorax injury assessment tool ofvulnerable individuals.

No description available.

The position of the cell division plane in plants is determined by the position of the preprophase band. The pre prophase band (PPB) is a ring of microtubules centered around the nucleus on the inner side of plasma membrane that establishes the cortical division site. The PPB forms at the end of G2 and breaks down at the end of prophase leaving behind protein markers of its position that are collectively called the cortical division site. During cytokinesis the phragmoplast expands towards the cortical division site and mediates the fusion of the new cell plate with the mother cell at that position. Several proteins necessary for PPB formation in plants have been identified, including maize DCD1 and ADD1 and Arabidopsis TON2, which are all type 2A protein phosphatase (PP2A)B" regulatory subunits. DCD1, ADD1, and TON2 localize to the PPB and the cortical division site through metaphase. The PP2A subunits each have two EF-hand domains, which are predicted to bind calcium ions. Since calcium ions are important for some aspects of cell division, we designed a series of constructs to test if TON2 binds calcium. TON2 protein was cloned into expression vectors, pET42a, and expression of TON2 protein was confirmed via Western blotting and immunodetection using a GST antibody. Site directed mutagenesis was used to mutate the TON2 EF-hand domains and mutated cDNAs were also cloned into expression vectors. These were then expressed in bacterial systems. Finally, the GST tagged proteins were purified. In the future, wild-type and mutated proteins TON2 proteins will used in calcium binding assays to determine if TON2 binds calcium.

No description available.

Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami. / Russian Foundation for Basic Research / Revisión por pares