Structural Basis for the Association of MAP6 Protein with Microtubules and Its Regulation by Calmodulin: Microtubule and calmodulin binding on Mn modules of MAP6

International audienceMicrotubules are highly dynamic αβ-tubulin polymers. In vitro and in living cells, microtubules are most often cold- and nocodazole-sensitive. When present, the MAP6/STOP family of proteins protects microtubules from cold- and nocodazole-induced depolymerization but the molecular and structure determinants by which these proteins stabilize microtubules remain under debate. We show here that a short protein fragment from MAP6-N, which encompasses its Mn1 and Mn2 modules (MAP6(90-177)), recapitulates the function of the full-length MAP6-N protein toward microtubules, i.e. its ability to stabilize microtubules in vitro and in cultured cells in ice-cold conditions or in the presence of nocodazole. We further show for the first time, using biochemical assays and NMR spectroscopy, that these effects result from the binding of MAP6(90-177) to microtubules with a 1:1 MAP6(90-177):tubulin heterodimer stoichiometry. NMR data demonstrate that the binding of MAP6(90-177) to microtubules involve its two Mn modules but that a single one is also able to interact with microtubules in a closely similar manner. This suggests that the Mn modules represent each a full microtubule binding domain and that MAP6 proteins may stabilize microtubules by bridging tubulin heterodimers from adjacent protofilaments or within a protofilament. Finally, we demonstrate that Ca(2+)-calmodulin competes with microtubules for MAP6(90-177) binding and that the binding mode of MAP6(90-177) to microtubules and Ca(2+)-calmodulin involves a common stretch of amino acid residues on the MAP6(90-177) side. This result accounts for the regulation of microtubule stability in cold condition by Ca(2+)-calmodulin

ATTRIBUTION AUTOMATIQUE DES NOES LORS DE LA DETERMINATION DE STRUCTURES DE PROTEINES PAR RMN HOMONUCLEAIRE. APPLICATION A DES LIGANDS PEPTIDIQUES DE CANAUX IONIQUES

CHATENAY MALABRY-Ecole centrale (920192301) / SudocSudocFranceF

Rapid Assembly and Collective Behavior of Microtubule Bundles in the Presence of Polyamines

International audienceMicrotubules (MTs) are cylindrical cytoskeleton polymers composed of α-β tubulin heterodimers whose dynamic properties are essential to fulfill their numerous cellular functions. In response to spatial confinement, dynamic MTs, even in the absence of protein partners, were shown to self-organize into higher order structures (spindle or striped structures) which lead to interesting dynamical properties (MT oscillations). In this study, we considered the assembly and sensitivity of dynamic MTs when in bundles. To perform this study, spermine, a natural tetravalent polyamine present at high concentrations in all eukaryote cells, was used to trigger MT bundling while preserving MT dynamics. Interestingly, we first show that, near physiological ionic strengths, spermine promotes the bundling of MTs whereas it does not lead to aggregation of free tubulin, which would have been detrimental to MT polymerization. Experimental and theoretical results also indicate that, to obtain a high rate of bundle assembly, bundling should take place at the beginning of assembly when rapid rotational movements of short and newly nucleated MTs are still possible. On the other hand, the bundling process is significantly slowed down for long MTs. Finally, we found that short MT bundles exhibit a higher sensitivity to cold exposure than do isolated MTs. To account for this phenomenon, we suggest that a collective behavior takes place within MT bundles because an MT entering into a phase of shortening could increase the probability of the other MTs in the same bundle to enter into shortening phase due to their close proximity. We then elaborate on some putative applications of our findings to in vivo conditions including neurons

Caractérisation structurale de l'éliciteur viral du Potato Virus X (PVX) et recherche de gènes partenaires impliqués dans la résistance liée, chez les espèces cultivées, au gène dominant Rx

National audienc

An Endoscopic Training Model to Improve Accuracy of Colonic Polyp Size Measurement

Most studies of colonic polyps rely on visual estimation when regarding polyp size; however, the reliability of a visual estimate is questionable. Our study aims to develop a training model to improve the accuracy of size estimation of colonic polyps in vivo. Colon polyps were recorded on 160 video clips during colonoscopy. The size of each polyp was estimated by visual inspection and subsequently measured with a flexible linear measuring probe. The study included a pretest, an intervention, and a posttest. The pretest included 160 video clips, which comprised the visual - estimation portion of the study. The intervention was an educational model consisting of 30 video clips which included a visual-estimation section and a linear-measuring- probe section, designed to help the endoscopists to compare their visual estimate of size with the measured size of the polyps. The posttest included the 160 video clips used in the pretest, presented in random order. Intraobserver agreement and diagnostic accuracy were compared before and after the training session. Eight beginners and four experienced colonoscopists were enrolled. The overall kappa( kappa) values of intraobserver agreement for pretest and posttest were 0.74 and 0.85 for beginner group as well as 0. 83 and 0.88 for experienced group, respectively. The overall diagnostic accuracy improved from 0.52 to 0.78 for beginner group and 0.71 to 0.87 for experienced group (P < 0 .05) after education with the training model. This training model could help endoscopists improve the accuracy of measurement of polyps on colonoscopy in a short period. The durability of learning effect needs further investigation

Solution structure of the anticancer p28 peptide in biomimetic medium

The p28 peptide derived from Pseudomonas aeruginosa azurin shows an anticancer activity after binding to p53 protein and is currently in Phase I of clinical trials. We have studied its structure in water and in a biomimetic media and show that the peptide is unstructured in water but when studied in a biomimetic medium assumes a structure very similar to the one observed in azurin, suggesting a high propensity of this peptide to maintain this secondary structure. Analysis of p28 sequences from different bacterial species indicates conservation of the secondary structure despite amino acid replacement in different positions, suggesting that others, similar peptides could be tested for binding to p53

Application of LC-MS-based metabolomics method in differentiating septic survivors from non-survivors

Septic shock is the most severe form of sepsis, which is still one of the leading causes of death in the intensive care unit (ICU). Even though early prognosis and diagnosis are known to be indispensable for reaching an optimistic outcome, pathogenic complexities and the lack of specific treatment make it difficult to predict the outcome individually. In the present study, serum samples from surviving and non-surviving septic shock patients were drawn before clinical intervention at admission. Metabolic profiles of all the samples were analyzed by liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. One thousand four hundred nineteen peaks in positive mode and 1878 peaks in negative mode were retained with their relative standard deviation (RSD) below 30 %, in which 187 metabolites were initially identified by retention time and database in the light of the exact molecular mass. Differences between samples from the survivors and the non-survivors were investigated using multivariate and univariate analysis. Finally, 43 significantly varied metabolites were found in the comparison between survivors and non-survivors. Concretely, metabolites in the tricarboxylic acid (TCA) cycle, amino acids, and several energy metabolism-related metabolites were up-regulated in the non-survivors, whereas those in the urea cycle and fatty acids were generally down-regulated. Metabolites such as lysine, alanine, and methionine did not present significant changes in the comparison. Six metabolites were further defined as primary discriminators differentiating the survivors from the non-survivors at the early stage of septic shock. Our findings reveal that LC-MS-based metabolomics is a useful tool for studying septic shock

NMR metabolomic analysis of breast cancer risk in the SU.VI.MAX prospective cohort study

International audienceBackground: Application of metabolomics to the field of nutritional epidemiology opens new perspectives for ground-breaking discoveries. To our knowledge, no prospective study had been conducted to investigate the relationship between baseline non-targeted metabolomics profiles and subsequent long-term breast cancer risk. This project investigates whether metabolomic signatures, established from a simple blood draw, could contribute to better understand and predict the risk of developing female breast cancer in the following decade.Methods: A nested case-control study was set up in the SU.VI.MAX cohort (1994-2007), involving 206 breast cancer cases and 396 matched controls. Non-targeted NMR metabolomic profiles were assessed on baseline plasma samples. Multivariable conditional logistic regression models were used. Predictive performance was assessed using the NRI indicator (Net Reclassification Improvement). Results: For the NOESY sequence, 237 buckets were obtained after NMR spectrum division with “intelligent bucketing”, among which 25 were significantly associated with breast cancer risk in logistic models (228/27 buckets for the CPMG sequence, respectively). The corresponding P-values ranged from 0.00007 (for the bucket 5.1869 ppm, corresponding to methine moieties of glyceryl, ORT3vsT1=0.37 [0.23-0.61]) to 0.04 (for the bucket 2.429 ppm, corresponding to glutamine, ORT3vsT1=1.62 [1.02-2.57]). Lipoproteins, lipids (including unsaturated fatty acids, glycerides and glycerophospholipids and derived compounds) and glycoproteins were associated with decreased breast cancer risk, whereas several amino acids and derived compounds (valine, leucine, glutamine, creatine, creatinine and threonine) and beta-glucose were associated with increased risk. Most metabolites significantly improved the predictive performance of the models.Conclusion: This pioneering study suggests that several metabolites (some of which pertaining to the food metabolome) would be involved in breast cancer etiology. Similar analyses based on mass spectrometry metabolomics are underway in the study, as well as the assessment of the correlations between metabolomic profiles and detailed food and nutrient intakes