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In this study, the deamidation is involved to modify the structure of barley proteins in terms of prolamin and glutelin in order to improve the functional properties of protein. A wide range of deamidation degrees (0.1% to 45%) were prepared using alkaline method. The results suggested that the optimal deamidation degree of barley prolamin is around 2.4-4.7%, where the solubility, emulsifying and foaming properties of prolamin were significantly improved at both acidic and neutral pHs. The optimal deamidation degree for glutelin is around 2.2 to 5.6%, where deamidated glutelin demonstrated markedly improved solubility at both acidic and neutral pHs. Glutelin performed strong tendency to form aggregates with spherical shape and very large molecular weight. These aggregates are important in stabilizing the emulsions at a broad range of deamidation degree (5.6-43%). These results suggest that barley protein would be an excellent candidate to develop as an emulsifying and foaming ingredient. / Food Science and Technology

This work was initiated to acquire a better understanding of the mechanisms, regulations, and significances of deamidation, as well as its role in the aging process.

Yes / The level of glutamine (Gln) deamidation in bone collagen provides information on the diagenetic history of bone but, in order to accurately assess the extent of Gln deamidation, it is important to minimise the conditions that may induce deamidation during the sample preparation. Here we report the results of a preliminary investigation of the variability in glutamine deamidation levels in an archaeological bone due to: a) sampling location within a bone; b) localised diagenesis; and c) sample preparation methods. We then investigate the effects of pre-treatment on three bone samples: one modern, one Medieval and one Pleistocene. The treatment of bone with acidic solutions was found to both induce deamidation and break down the collagen fibril structure. This is particularly evident in the Pleistocene material (~80,000 years BP) considered in this study. We show that ethylenediaminetetraacetic acid (EDTA), when used as an alternative to hydrochloric acid (HCl) demineralisation, induces minimal levels of deamidation and maintains the collagen fibril structure. Areas of bone exhibiting localised degradation are shown to be correlated with an increase in the levels of Gln deamidation. This indicates that the extent of Gln deamidation could provide a marker for diagenesis but that sampling is important, and that, whenever possible, subsamples should be taken from areas of the bone that are visually representative of the bone as a whole. Although validation of our observations will require analysis of a larger sample set, deamidation measurements could be a valuable screening tool to evaluate the suitability of bone for further destructive collagen analyses such as isotopic or DNA analysis, as well as assessing the overall preservation of bone material at a site. The measure of bone preservation may be useful to help conservators identify bones that may require special long-term storage conditions. / NERC (NE/J500197/1), Yorkshire Forward - Northern Way Initiative, Science City York, Gordon and Betty Moore Foundation, Leverhulme Trust

<p>Articular cartilage is a highly specialized avascular tissue and consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. The chondrocyte morphology and extracellular matrix properties vary with the depth of cartilage. Some past studies have defined the zonal distribution of a broad range of cartilage proteins in different layers. Based on the variations within each layer, the extracellular matrix can be further distinguished to pericellular, territorial and interterritorial regions. However, most of these studies used guanidine-HCl extraction that leaves an unextracted residual with a substantial amount of collagen. The high abundance of anionic polysaccharide molecules from cartilage adversely affects the chromatographic separation. Scatter oriented chondrocytes only account for the small proportion of the whole tissue protein extraction. However, the density of the cell varies with depth of cartilage as well. Moreover, the physiological status may also altered the extracellular matrix properties. Therefore, a comprehensive strategy to solve all these difficulties are necessary to elucidate the molecular structure of cartilage. </p><p>In this study, we used quantitative and qualitative proteomic analysis to investigate various cartilage tissue processing protocols. We established a method for removing chondrocytes from cartilage sections that minimized matrix protein loss. Quantitative and qualitative proteomic analyses were used to evaluate different cartilage extraction methodologies. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. The different extraction methods yielded different protein profiles. For instance, an overwhelming number of collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction. </p><p>Subsequently we applied these methods to extract cartilage sections from different cartilage layers (superficial, intermediate and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic). We also utilized lase capture microscopy (LCM) to harvest cartilage sample from individual subregions (territorial and interterritorial regions). The results suggested that there is more unique proteins existed in the superficial layer. By removing the chondrocytes, we were able to identify more extracellular matrix proteins. The phenotyping of cartilage subregions provided the chance to precisely localize the protein distribution, such as clusterin protein. We observed that the guanidine-HCl extractability (guanidine-HCl/ guanidine-HCl + in situ digestion extracts) of cartilage proteins. Proteoglycans showed high extractability while collagen and non-collagenous proteins had lower extractability. We also observed that the extractability might differ with depth of cartilage and also disease states might alter the characters as well. </p><p>Laser capture microscopy provides us the access to the cartilage subregions in which only few studies have investigated because of the difficulties to separate them. We established the proteomic analysis compatible-protocol to prepare the cartilage section for LCM application. The results showed that most of the proteoglycans and other proteins were enriched in the interterritorial regions. Type III and VI collagens, and fibrillin-1 were enriched in the territorial regions. We demonstrated that this distribution difference also varied with depth of cartilage. The difference of protein abundance between subregions might be altered because of disease states. </p><p>Last we were looking for the post-transliational modification existed in these subregions of cartilage. Deamidation is one of the modification without the enzyme involved. Previous studies have showed that deamidation may accumulated in the tissue with low turnover rate. Our proteomic analysis results suggests that abundance of deamidated peptides also varied in different layers and subregions of cartilage. </p><p>We have developed the monoclonal antibody based immunoassay to quantify the deamidated cartilage oligomeric matrix protein within cartilage tissue from different joints (hip and knee) and disease states (healthy, para-lesion, and remote lesion). The results suggests that the highest concentration of deamidated COMP was identified in arthritic hip cartilage. </p><p>The results of this study generated several reliable protocols to perform cartilage matrix proteomic analysis and provided data on the cartilage matrix proteome, without confounding by intracellular proteins and an overwhelming abundance of collagens. The discovery results elucidated the molecular architecture of cartilage tissue at different joint sites and disease states. The similarities among these cartilages suggested a constitutive role of some proteins such as collagen, prolargin, biglycan and decorin. Differences in abundance or distribution patterns, for other proteins such as for cartilage oligomaric matrix protein, aggrecan and hyaluronan and proteoglycan link protein, point to intriguing biological difference by joint site and disease state. Decellularization and a combination of extraction methodologies provides a holistic approach in characterizing the cartilage extracellular matrix. Guanidine-HCl extractability is an important marker to characterize the statue of cartilage; however it has not been fully understand. The protein distributions in matrix subregions may also serve as an index to characterize the metabolic status of cartilage in different disease states. A large sample cohort will be necessary to elucidate these characters.</p> / Dissertation

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19517号 / 理博第4177号 / 新制||理||1600(附属図書館) / 32553 / 京都大学大学院理学研究科化学専攻 / (主査)教授 藤井 紀子, 教授 三木 邦夫, 教授 杉山 弘 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

La protéine Bcl xL, membre de la famille de Bcl 2, est essentiellement décrite pour son rôle dans l'inhibition de la mort des cellules. Récemment, un nouveau rôle lui a été attribué dans la régulation de la macro-autophagie, processus principalement décrit pour promouvoir la survie des cellules. Bcl xL exerce donc ses fonctions de survie à travers la régulation d'au moins deux processus différents.Si les fonctions anti-apoptotiques de Bcl xL ne sont plus à démontrer, ses fonctions dans la régulation de l'autophagie sont davantage débattues. Ainsi, nous avons centré ce travail sur la caractérisation des fonctions pro-autophagiques de Bcl xL afin de mieux comprendre ses fonctions de survie. Nos résultats suggèrent que Bcl xL subit in vivo et dans des cellules en culture une modification de type déamidation. Nous montrons que cette modification renforce les fonctions pro-autophagiques de Bcl xL sans affecter ses fonctions anti-apoptotiques. Par ailleurs, nous nous sommes intéressés à l'interaction entre Bcl xL et la petite GTPase Rab7, une protéine essentielle au processus autophagique et endocytique. Nous avons généré, et analysé d'un point de vue fonctionnel, des mutants de Bcl xL de type perte ou gain d'interaction avec Rab7. Notre principale conclusion est que Bcl xL stimule le trafic des vésicules médié par Rab7, et nous proposons que les fonctions pro-autophagiques de Bcl xL sont majoritairement dépendantes de son interaction avec Rab7. Cette étude contribue ainsi à mieux définir les fonctions pro-autophagiques de Bcl xL ainsi que les processus qui les régulent. Par ailleurs, elle approfondit nos connaissances des fonctions oncogéniques de Bcl xL en intégrant la composante supplémentaire de ses fonctions pro-autophagiques, et ouvre ainsi des perspectives pour l'élaboration de nouvelles stratégies thérapeutiques anti-cancéreuses notamment. / Bcl xL, a member of the Bcl-2 family, is mainly described for its role in the inhibition of cell death. Recently, Bcl xL was attributed a new role in the regulation of macro-autophagy, a process described mainly for its contribution to cell survival. Hence, Bcl xL wields its survival functions through the regulation of at least two different processes. If the anti-apoptotic functions of Bcl xL are now well established, its role in the regulation of autophagy is more debated. Therefore we focused this work on the characterization of Bcl xL pro-autophagic functions in order to get a better understanding of its survival functions. Our results suggest that Bcl xL undergoes in vivo and in cultured cells a modification called deamidation. We show that this modification enhances its pro-autophagic functions without affecting its anti-apoptotic functions. In addition, we characterized an interaction between Bcl xL and the small GTPase Rab7 which is essential for autophagy and endocytosis. We generated mutants of Bcl xL either gaining or loosing interaction with Rab7. The functional analysis of these mutants suggested that Bcl xL stimulates the vesicle trafficking mediated by Rab7, and prompts us to hypothesize that Bcl xL pro-autophagic functions are mainly dependent on its interaction with Rab7. This study helps to better define the pro-autophagic functions of Bcl xL and the processes regulating them. It provides further insights in the oncogenic functions of Bcl xL by implementing additional component of its pro-autophagic functions, and opens perspectives for the development of new therapeutic strategies against cancer progression.

In the last two decades, protein drugs have enjoyed a rapid growth and achieved a tremendous success in treating human diseases. However, the presence of physiological barriers greatly impedes the efficient delivery of such unconventional large molecule drugs, and therefore limits their clinical utility. An elegant way to address this challenge takes advantage of certain endogenous transporter proteins, such as human transferrin (Tf), whose ability to traverse physiological barriers has been extensively exploited. However, methods to investigate Tf-based drug delivery remained insufficient and unsatisfactory until recent development of quantitative mass spectrometry (MS). Hereby, MS-based methods have been developed and validated for quantitation of exogenous Tf in biological fluids. Particularly, different O18-labeling based approaches have been evaluated, modified and developed in this work, in order to achieve the most reliable quantitation. Alternatively, a novel approach based on indium labeling and inductively coupled plasma mass spectrometry (ICP-MS) detection has been developed for sensitive quantitation of Tf in biological fluids. The second aspect of this dissertation work focuses on the application of MS-based methods for characterization of protein drugs at different levels, ranging from protein identification, covalent structure, conformation, and interaction with physiological partners. Particularly, an O18-labeling assisted approach has been developed to identification of protein deamidation products. This new approach can readily distinguish between the two deamidated isomers. Also, an LC-MS based method has been developed for ranking the susceptibility of protein disulfide bonds to reduction, which could be applied to several disulfide bond-related analyses. Finally, a recently designed growth hormone transferrin fusion protein was studied using MS-based methods, and the molecular basis for its successful oral delivery was revealed.

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