No description available.

No description available.

La déamidation est la modification post-traductionnelle de l'asparagine (Asn) et de la glutamine (Glu). Elle est communèment observée dans les peptides et les protéines. Il a été démontré que la déamidation limite la durée de vie de ces macromolécules. Dans ce travail, la déamidation de l'asparagine dans des petits peptides et dans l'enzyme triosephosphate isomérase a été modélisée. La déamidation dans la triosephosphate isomérase de mammifères a été observée sur deux sites distincts: Asn15 et Asn71. Asn71 a une vitesse de déamidation plus élevée que Asn15 et moins grande que pour un petit peptide. Il a été suggéré que la déamidation de Asn15 se produit sous l'influence de la déamidation de Asn71. Pour expliquer ces résultats expérimentaux, des simulations de dynamiques moléculaires classiques à l'échelle de la microseconde et des calculs d'énergie libre, de type umbrella sampling, à l'aide de méthodes combinées mécanique quantique/mécanique moléculaire ont été réalisés. Nous montrons que la déamidation séquentielle dans la triosephosphate isomérase est due à la fois à des effets locaux et globaux. Ces résultats apporte une nouvelle perspective sur l'impact de l'ordre structurel sur la vitesse de déamidation Nous avons également déterminé la voie la plus plausible de cette reaction ainsi que l'influence de la variation du pKa, dans la chaîne principale, de la partie amide du résidu adjacent de l'asparagine sur la vitesse de déamidation. En regard de l'importance des variations de pKa dans l'environnement protéique, nous avons élaboré un protocole informatique permettant d'évaluer de manière rapide et précise des pKa . Ce protocole a été appliqué à des petites molécules organiques et nous avons montré qu'il était également applicable à des études relatives à la prédiction de pKa dans les protéines / Deamidation is the posttranslational modification of asparagine (Asn) and glutamine (Glu) residues, which is observed in several proteins and peptides. It has been shown that deamidation limits the lifetime of these macromolecules. In this work, deamidation of asparagine in small peptides and in the enzyme triosephosphate isomerase has been modeled. Deamidation in mammalian triosephosphate isomerase has been observed at two distinct deamidation sites: Asn15 and Asn71. Asn71 deamidates faster than Asn15 and slower than a small peptide. It has been suggested that, deamidation at Asn15 occurs with the influence of deamidated Asn71. In order to explain these experimental findings, microsecond long classical molecular dynamics simulations and free energy calculations using quantum mechanics/molecular mechanics tools combined with umbrella sampling technique have been performed. The sequential deamidation in triosephosphate isomerase has been shown to be related with both global and local effects. These results bring a new perspective to the impact of the high-order structure on deamidation rate. The most plausible route of this reaction was also determined. The pKa shift of backbone amide of the residue adjacent to asparagine has been found to be one of the most crucial factor determining the rate of deamidation. Considering the importance of pKa shifts in protein environment, a computational protocol was suggested in order to obtain accurate and fast pKa predictions. This protocol was applied to small organic molecules, and it has been shown to be applicable to studies concerning aminoacid pKa predictions

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20008号 / 農博第2192号 / 新制||農||1045(附属図書館) / 学位論文||H28||N5017(農学部図書室) / 33104 / 京都大学大学院農学研究科農学専攻 / (主査)教授 裏出 令子, 教授 松村 康生, 教授 三上 文三 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20431号 / 農博第2216号 / 新制||農||1048(附属図書館) / 学位論文||H29||N5052(農学部図書室) / 京都大学大学院農学研究科農学専攻 / (主査)教授 裏出 令子, 教授 松村 康生, 教授 三上 文三 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

A method has been developed for the rapid, quantitative separation of normal and abnormal phosphoglucose isoemrase allozymes from individuals heterozygous for genetic variant forms of the enzyme. The method utilizes a substrate gradient elution of the enzyme from carboxymethyl Biogel and is far superior in terms of resolution and recovery to methods based on electrophoresis and isoelectric focusing. Four different genetic variant forms of the enzyme were isolated and subjected to a systematic comparison of their physical, catalytic and stability properties. The physical and catalytic properties of the variants were similar; however, clear differences in the stability of the allozymes were apparent.

No description available.

No description available.

Polyclonal antibodies against the first two enzymes of the Arabidopsis thaliana flavonoid biosynthetic pathway were developed using conventional and phage antibody technology. cDNAs from Arabidopsis coding regions of chalcone synthase (CHS) and chalcone isomerase (CHI) were sub-cloned in frame into a bacterial expression vector as fusions with glutathione Stransferase (GST) using standard directional cloning techniques. Analysis of crude extracts of Escherichia coli containing GST .. CHS or GST .. CHI fusion protein indicated that the cells expressed equivalent amounts per volume of culture. CHS and CHI were purified to near homogeneity, yielding approximately 100 micrograms of GST .. CHS and 1 milligram of GST-CHI per liter of culture. The purified fusion proteins were injected into chickens and polyclonal lgY·s were purified from egg yolk Accumulation of CHS and CHI, as well as products of the pathway, were compared during the first eight days of Arabidopsis development. CHS and CHI are sequentially induced and reach maximal accumulation levels by day 5. Anthocyanidin levels are offset by one reaching maximal levels at day 6. The fusion proteins were also used to screen a phage-display library for Fabl fragments that recognize CHS and CHI epitopes. Preliminary data indicated that enrichment of phage displaying antibodies against CHS and CHI was successful. Phage-derived antibodies against CHS and CHI provide valuable tools for future experiments addressing Western blot analysis, immunolocalization experiments, and disruption of the flavonoid biosynthetic pathway by introduction of the corresponding genes into transgenic Arabidopsis plants. / Master of Science

Neurotoxic aggregates of amyloid beta (Aβ) peptide contribute to the etiology of Alzheimer's disease (AD). Aβ1-42 forms oligomeric structures that undergo further aggregation into protofibrils and fibrils. Oligomeric Aβ1-42 is more toxic than monomers or mature fibrils. In this work, we used two distinct approaches to inhibit Aβ1-42 oligomerization and toxicity. First, seven distinct but overlapping Aβ fragments were used to identify their individual aggregation propensities and their effects on Aβ1-42 oligomerization and cytotoxicity. Studies on suppression of Aβ1-42 cytotoxicity by peptides, including those derived from Aβ1-42, have been conducted before, but peptides encompassing the whole Aβ1-42 sequence have not been systematically analyzed. Aβ1-42 was allowed to aggregate and form oligomeric assemblies in aqueous buffer for 4 h in the absence or presence of 2-fold molar excess of an Aβ fragment. Cytotoxicity analysis then recorded the impact of each fragment on Aβ1-42 cytotoxicity as well as the toxicity of the fragments themselves. An enzyme-linked immunosorbent assay that detects oligomeric Aβwas used to determine the effect of each fragment on Aβ1-42 oligomerization after 4 h of aggregation. Four fragments of Aβ1-42 inhibited the toxicity of oligomeric Aβ1-42 to various degrees, while two others conferred no cellular protection against Aβ1-42 toxicity. Interestingly, one fragment enhanced Aβ1-42 toxicity after 4 h of aggregation. Three of the four fragments that blocked Aβ1-42 toxicity partially disrupted oligomer formation, showing correlation between the inhibition of Aβ1-42 aggregation and the inhibition of cellular toxicity. Second, we examined whether protein disulfide isomerase (PDI), a chaperone mainly found in the endoplasmic reticulum, could reverse the oligomeric state of aggregated Aβ1-42 and thus its toxicity. Previous work has demonstrated that PDI inhibits Aβ1-42 aggregation at sub-stoichiometric concentrations. To assess PDI's effect on Aβ1-42 toxicity, Aβ1-42 was allowed to aggregate for 2 h before the addition of PDI at a 1:10 molar ratio of PDI to Aβ1-42 and then allowed to aggregate for another 2 h. MTS cytotoxicity assays using PC-12 cells showed that adding PDI 2 h after the start of aggregation improves cell survival. Through a differential centrifugation assay followed by Western blot, we qualitatively illustrated that PDI can reverse a 2 h aggregate of Aβ1-42 to the monomeric state. Overall, in this project we have learned that inhibiting the oligomeric assembly of Aβ1-42 directly decreases the effect of Aβ1-42 toxicity. Inhibition of Aβ1-42 toxicity was seen with both fragments derived from Aβ1-42 and PDI, shedding light into two novel approaches as possible therapeutics for AD.