Eukaryotic translation initiation factor 5A (eIF5A) is the only known protein to contain hypusine, formed by post-translational modification of a highly conserved lysine residue. Hypusination is essential for eIF5A function, being required for binding of a specific subset of mRNAs necessary for progression of eukaryotic cells through the G1-S checkpoint. Little structural information is available for eIF5A other than that derived from archaeal homologues. The aim of this study was to conduct structure-function studies on Saccharomyces cerevisiae (yeast) eIF5A, encoded by TIF51A. Homology models of eIF5A were generated from the Methanococcus jannaschii archaeal homologue (aIF5A) and two Leishmania eIF5As. The models, along with secondary structure predictions identified an a-helix on the C-terminal domain, unique to eukaryote eIF5A. The Neurospora crassa structural analogue, HEX-1, which dimerises in three configurations, was used to generate similar dimeric model configurations of eIF5A. A biochemical and functional analysis was used to validate the homology models of eIF5A.Since the crystal structures of aIF5A and eIF5A were solved from unhypusinated protein produced in Escherichia coli, 6 x His-tagged eIF5A (His-eIF5A) was used for biochemical analysis. This analysis revealed that eIF5A existed as a dimer in solution, dependent on the presence of the highly conserved Cys 39 residue. A yeast TIF51A/TIF51B null yeast strain, with a chromosomal copy of TIF51A under control of PGAL1, was used to confirm that HiseIF5A and selected eIF5A mutants were functional in vivo. Biochemical analysis showed that hypusinated His-eIF5A also exists as a dimer, but neither the dimerisation, nor the function of eIF5A are dependent on the presence of Cys 39. Rather they depend on the presence of hypusine (Hpu) 51 and the presence of RNA leading to the conclusion that RNA and hypusine are required for dimerisation and hence function, of native eIF5A in vivo. In contrast, a Lys 51 to Arg 51 substitution or RNase treatment of His-eIF5A produced in E. coli did not destabilize the dimeric form, suggesting different folding/dimerisation mechanisms in E. coli and yeast cells. The information obtained from the initial homology models, together with the results of the biochemical analysis was used to propose a mechanism for dimerisation of yeast eIF5A involving both hypusine and RNA.

The plant homeodomain 6 gene (PHF6) is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL); however, its specific functional role in leukemia development remains to be established. Here, we show that loss of PHF6 is an early mutational event in leukemia transformation. Mechanistically, genetic inactivation of Phf6 in the hematopoietic system enhances hematopoietic stem cell (HSC) long-term self-renewal and hematopoietic recovery after chemotherapy by rendering Phf6 knockout HSCs more quiescent and less prone to stress-induced activation. Consistent with a leukemia-initiating tumor suppressor role, inactivation of Phf6 in hematopoietic progenitors lowers the threshold for the development of NOTCH1-induced T-ALL. Moreover, loss of Phf6 in leukemia lymphoblasts activates a leukemia stem cell transcriptional program and drives enhanced T-ALL leukemia-initiating cell activity. These results implicate Phf6 in the control of HSC homeostasis and long-term self-renewal and support a role for PHF6 loss as a driver of leukemia-initiating cell activity in T-ALL. Phf6 controls HSC homeostasis, leukemia initiation, and T-ALL leukemia-initiating cell self-renewal. These results substantiate a role for PHF6 mutations as early events and drivers of leukemia stem cell activity in the pathogenesis of T-ALL. Further, in the hematopoietic system stem cell aging is characterized by accumulation HSCs with poor self-renewal capacity and myeloid biased differentiation. Despite improved appreciation of the cell intrinsic and cell extrinsic mechanisms driving age-associated HSC functional exhaustion, no interventions have proven effective in delaying HSC aging to date. Here, we show that genetic inactivation of the Phf6 prevents age- associated HSC functional decline. Immunophenotypic and single cell transcriptomics profiling demonstrated markedly decreased accumulation of immunophenotypically-defined HSCs, reduced myeloid bias and decreased upregulation of transcriptional programs associated with stem cell aging in old hematopoietic-specific Phf6 knockout mice. Functionally, Phf6 knockout HSCs from aged mice demonstrated increased hematopoietic reconstitution capacity and preservation of lymphoid differentiation potential. Mechanistically, analysis of long-term HSCs from old Phf6 knockout mice revealed reduced levels of ongoing DNA damage and downregulation of genotoxic stress-induced transcriptional signaturesconducive of HSC aging. These results identify Phf6 as an important epigenetic regulator of HSC aging, whose inactivation counters the functional deterioration of HSC activity induced with age. Microprotein encoding genes are a class of genes which encode poly-peptide gene products comprised by 100 or fewer amino acids. Until recently, many such genes had been considered of low- or no-coding potential given the technical limitations associated with identification of such small proteins. However, recently prominent examples of microprotein encoding genes have been reported with a wide variety of regulatory functions. Therefore, we hypothesized that novel microprotein genes exist within the human genome with oncogenic and tumor suppressive roles. To test this hypothesis, we developed a pipeline for identification of microproteins based on conservation of the open reading frame. Leveraging PLATE-seq to generate a high-dimensional readout in a loss-of-function screen, we then screened for microproteins with potential tumor suppressive or oncogenic function. From this, we identified a brain- specific, 65 amino-acid microprotein encoded in within LINC00617 (TUNAR) which is conserved at the protein level across vertebrates. We experimentally validated the protein-level expression of the TUNAR microprotein. In vitro and in vivo knockout and overexpression experiments demonstrate a role for TUNAR as a tumor suppressor in glioma. Specifically, we show that loss of Tunar in the mouse brain results in lower expression of Fermt1 and genes in the integrin signaling pathway. Consistently, overexpression of TUNAR in human glioblastoma multiforme cell lines significantly impeded cellular migration suggesting a role of Tunar in glioma cell dissemination. Finally, human glioma sequencing and copy number data were mined to determine the prognostic significance of the loss of TUNAR in human gliomas. These analyses demonstrated that copy number loss of TUNAR is associated with poor outcomes in lower grade gliomas and that TUNAR expression and glioma grade are strongly, negatively correlated suggesting that TUNAR likely has tumor suppressive effects in human glioma.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21778号 / 工博第4595号 / 新制||工||1716(附属図書館) / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 木村 俊作, 教授 瀧川 敏算, 教授 大内 誠 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Elastin-like polypeptides (ELP) are artificially designed protein biopolymers that can be produced by living organisms. These proteins have the unique ability to undergo reversible inverse phase transition, in response to changes in temperature and/or addition of chaotropic salts. Below the transition temperature (T1) , ELP is soluble in water. Increasing the temperature above Ti, ELP coacervates into an aqeous ELP-rich phase. In this thesis, this unique feature of ELP was used in for recombinant protein purification and for the formation of aqueous multiple-phase systems. For protein purification, ELP was fused with an intein and a model protein (thioredoxin), to demonstrate a simple and inexpensive approach for recombinant protein purification. The ELP tags replace the chromatographic media and the intein replaces the use of the protease in conventional methods. Using ELP tags was found to be consistent with large -scale recombinant protein production/purification by purifying an ELP tagged protein using a stirred cell equipped with a microfiltration membrane. When the temperature and/or salt concer.tration is increased for mixtures containing free ELP and ELP tagged proteins, simultaneous phase transition takes place. This served as the basis for the development of a method suitable for selectively recovering molecules from complex mixtures with high specificity, full reversibility, and virtually unlimited affinity. The second parts of this thesis focus on the ability of ELP to form aqueous twophase systems (A TPS) in vitro and most importantly, in vivo- with the formation of aqueous microcompartments in living cells. These compartments exclude the protein making machinery of the cell, acting as depots for newly expressed protein. It is also shown (in vitro) that ELP bastd droplets exclude proteases, protecting proteins from degradation. These observations are important for high-level production of recombinant proteins. Also described, is the formation of protein based aqueous multiphasic systems, with tunable morphologies. / Thesis / Doctor of Philosophy (PhD)

No description available.

No description available.

Collagen is one of the most important and abundant proteins in mammals. It consists of three left-handed PPII helixes coiled along a common axis to form a very compact right-handed super helix. The primary structure is shown to be (Gly-Xaa-Yaa)n repeats with high content of prolyl residues at both Xaa and Yaa positions. <i>Cis-trans</i> isomerization of the prolyl amide bonds is one of the rate-limiting steps during collagen triple helix folding. The conformationally locked alkene isosteres Fmoc-Gly-Ψ[(E)CH=C]-Pro-Hyp(tBu)-OH and Fmoc-Pro-Ψ[(E)CH=C]-Pro-OH were designed and synthesized. The synthesis of the Gly-Pro isostere had no stereo-control, and the two diastereomers of the tripeptide isostere Fmoc-Gly-Ψ[(E)CH=C]-Pro-Hyp(tBu)-OBn were separated by normal phase HPLC. Although the stereoselectivity of the asymmetric reduction was not good for the Pro-Pro isostere, the resulting diastereomers was separable by flash chromatography, and the absolute stereochemistry of the two diastereomers was determined by Mosher's method. The Gly-Pro alkenyl peptides, and their control peptide Ac-(Gly-Pro-Hyp)8-Gly-Gly-Tyr-NH2 were synthesized and purified. All three peptides showed a maximum around 225 nm and a minimum close to 200 nm in the CD spectra, which indicated the formation of PPII helixes. The Tm value of the control peptide was determined to be 50.0 °C. The peptide with Gly-Ψ[(E)CH=C]-L-Pro-Hyp as the guest triplet formed a stable triple helix with a Tm value of 28.3 °C. The peptide with Gly-Ψ[(E)CH=C]-D-Pro-Hyp as the guest triplet showed a linear decrease in the ellipticity with increasing temperature, which indicated that no triple helix was formed. The Pro-Pro alkenyl peptide and its control peptide H-(Pro-Pro-Gly)₁₀-OH were synthesized and purified. The T_m value of control peptide was determined to be 31.6 °C by extrapolation to 0 M TMAO in PBS buffer, which was very close to the measured value of 31.5 °C. The Pro-Pro alkenyl peptide began to show a maximum around 225 nm in the CD spectra when the concentration of TMAO was higher than 2.5 M. After extrapolation to 0 M TMAO, the T_m value was determined to be –22.0 °C. These results indicate that the backbone inter-chain hydrogen bond is one of the major forces in stabilizing the collagen triple helix, while <i>cis-trans</i> isomerization has limited contribution. The intrinsic properties of the amide bond may have huge influence on the stability of the collagen triple helix. The helix-turn-helix motif is an important tertiary structure in DNA-binding proteins. Stepwise modifications of the Antennapedia HTH peptide (27-55) were performed to improve the helicity and stability. The peptide with more side-chain ion-pairs was over 4 times more helical than the native Antp peptide, while the Ala-based peptide was over 9 times more helical than the native peptide. A 12-membered ring, Fmoc-protected HTH-turn mimic was designed and synthesized, and was ready for solid phase peptide synthesis. The solubility of the cyclic peptide was very poor, and the purification of the final product was very difficult. The solubility problem might also affect solid phase peptide synthesis in the future. / Ph. D.

Les vésicules géantes unilamellaires (GUV) permettent d’étudier efficacement les interactions entre les lipides et les peptides. Dans ce manuscrit, il a été montré que les interactions attractives lipides-peptides sont supprimées par l’attachement de polypeptides de type élastine (ELP) sur des peptides riches en arginine et peuvent être modulées par l’auto-assemblage en micelles ainsi que par le nombre de groupements arginine dans la séquence des peptides capables de pénétrer les cellules. De plus, une nouvelle méthode pour former des GUV à partir de systèmes complexes en seulement quelques minutes a été développée. Cette méthode est basée sur le gonflement d’un film de PVA sous une bicouche lipidique. Elle supprime la dégradation des molécules pendant la formation des GUV de lipides synthétiques, tels que des glycolipides et des phospholipides portant des groupements amides, où les méthodes traditionnelles ne réussissent pas à produire des vésicules non endommagées. / Giant Unilamellar Vesicles (GUVs) are a valuable tool to study lipid bilayer-biomolecule interactions in simplified cell-like model systems. In this work, a new method to efficiently form GUVs within minutes from more complex systems was developed. This method is based on swelling of a PVA-film under a lipid bilayer and minimizes damage of involved molecules during GUV formation. It also opens up many interesting perspectives for the formation of GUVs composed from new classes of synthetic lipids, such as glycolipids and amide-bearing phospholipids, where the traditional methods fail to efficiently produce “undamaged” vesicles. GUVs were also used for studying lipid-peptide interactions of a new class of elastin-like polypeptides functionalized with arginine-rich residues. It is shown that attractive interactions between lipids and peptides are suppressed by cargo-attachment and can be tuned by self-assembly into micelles and the arginine-amount of the cell penetrating residue.

La spectroscopie RMN 2D du deutérium en abondance naturelle (DAN) en utilisant les cristaux liquides chiraux comme solvant de RMN est une méthodologie efficace pour deux raisons: i) l’interaction quadrupolaire (interaction RMN sensible à l’ordre orientationnel des molécules et spécifique aux noyaux de spin I>1/2) n’est plus nulle en moyenne comme dans les liquides; ii) la chiralité du milieu permet de discriminer spectralement des énantiomères (molécule chirale) ou des directions énantiotopes (molécule prochirale). L'objectif principal de ce travail de Thèse était d'explorer le potentiel analytique de cette technique pour analyser le fractionnement isotopique naturel deutérium dans le cas d’ester d’acides gras saturés (AGS) et (poly)insaturés (AGI). Le but ultime de ce travail est de fournir de nouvelles informations (de nature stéréochimique) pour améliorer la compréhension de certains mécanismes enzymatiques impliqués au cours de leur biosynthèse. Dans ce contexte, nous avons montré que l'utilisation de solutions organiques orientées de polypeptides (mésophases chirales) utilisant des co-solvants polaires comme le DMF ou la pyridine (Py) fournissait les meilleurs résultats en termes d’(énantio)discrimination spectrale (nombre de sites et amplitude) sur la base des déplacements chimiques et des éclatements quadrupolaires du deutérium. Ainsi, la mésophase chirale (PBLG/Py) a été utilisée avec succès pour analyser les AGI comme le linoléate de méthyle (C18), pour lequel nous avons pu mesurer l'excès énantio-isotopomèrique (eei) au niveau de chaque site méthylène de la molécule. Pour la première fois, d'importantes informations relatives à la stéréochimie des mécanismes enzymatiques, inaccessibles par la méthode conventionnelle SNIF-NMR®, ont été déterminées. La même méthodologie impliquant la mésophase achirale (PBG/Py) et chirale (PBLG/Py) s’est révélée également être un excellent outil pour l'analyse des SAFA sous leur forme libre (C14 à C18) ou sous leur forme de triglycérides (3*C4 et 3*C14). L'attribution des doublets 2H détectés sur les spectres RMN 2D anisotropes des SAFA a été confirmée par l'élaboration d'un modèle théorique capable de prédire le comportement orientationnel de ces molécules flexibles. Comme exemple illustratif, la méthode a été appliquée pour étudier les mécanismes enzymatiques convertissant le linoléate en vernoléate dans deux plantes différentes. Enfin, pour améliorer la compréhension des interactions «soluté-polypeptide» impliquées dans les mécanismes d'orientation et d’énantio-discrimination, nous avons étudié par RMN 2D DAN, l'évolution des paramètres d'ordre (matrice de Saupe) de deux molécules prochirales, rigides et apolaires dissoutes dans une mésophase chirale préparée en mélangeant deux polypeptides de même stéréochimie (L), mais dont la nature chimique des chaînes latérales est différente, en fonction de leur proportion massique respective. / Natural abundance deuterium (NAD) 2D-NMR spectroscopy using (a)chiral liquid crystals as NMR solvent is a powerful method for two reasons: i) the quadrupolar interaction (an order-sensitive NMR interaction specific to spins I > ½) is not averaged to zero anymore as in liquids; ii) the chirality of medium allows enantiomers (chiral molecule) or enantiotopic directions (prochiral molecule) to be spectrally discriminated. The main objective of this Thesis work was to explore the analytical potential of this technique to analyze the natural distribution of deuterium in the case of esters of saturated fatty acids (SAFA) and (poly)unsaturated fatty acids (PUFA). The ultimate goal of this research is to collect new (stereochemical) information to improve the understanding of some enzymatic mechanisms involved during their biosynthesis. In this context, we have shown that the use of oriented solutions of polypeptide (chiral mesophases) using polar co-solvents such as DMF or pyridine (py) provided better results in terms of spectral (enantio)discrimination (number of sites and magnitude) on the the basis of chemical shifts and deuterium quadrupole splittings. Thus, the chiral mesophase (PBLG/py) has been applied successfully to analyse PUFA’s like the methyl linoleate (C18) for which we could measure the enantio-isotopomeric excess (eie) at each methylen site of the molecule. For the first time, important information related to the stereochemistry of enzymatic mechanisms, inaccessible by the conventional method SNIF-NMR®, have been determined. The same methodology using achiral (PBG / py) and chiral (PBG / py) mesophases, revealed also to be an excellent tool for analysing SAFA in their free form (C14 to C18), or in triglyceride forms (3*C4 and 3*C14). The assignment of 2H doublets detected on 2D-NMR spectra of SAFA’s was confirmed by developing a theoretical model predicting the orientational ordering behavior of these flexible molecules. As illustrative exemple, the method was applied for investigating the enzymatic mechanisms converting the linoleate into vernoleate in two different plants. Finally, to improve the understanding of “solute/polypeptide" interactions involved in the orientation and enantiodiscrimination mechanisms, we have investigated by NAD 2D-NMR the evolution of the order parameters (Saupe matrix) of two apolar, rigid prochiral molecules dissolved in a chiral mesophase made by mixing two polypeptides having the same stereochemistry (L) but different type of side chains, versus their respective mass proportion.

Cette étude concerne le développement de nanoparticules hybrides offrant de nouvelles stratégies pour la thérapie et le diagnostic médical. Elles sont constituées d’un cœur magnétique jouant le rôle d’agent de contraste pour l’IRM et d’inducteur de chaleur par hyperthermie, d’une couronne de polymère thermosensible permettant d’encapsuler des principes actifs et de peptides de reconnaissance biologique. Une grande partie de l’étude a consisté à étudier les processus d'adsorption de copolymères poly(éther)-b-poly(L-lysine) de composition variable sur les particules magnétiques et à comprendre le rôle de la conformation des chaînes polymère à la surface des particules sur la stabilité des colloïdes en milieu physiologique. Un agent antitumoral a été encapsulé puis libéré de façon contrôlée sous l’effet d’un champ magnétique alternatif en exploitant le caractère thermosensible des blocs polyéthers. Des séquences peptidiques ciblant les zones d’inflammation de la barrière hémato-encéphalique ont été greffées sur les copolymères. L’efficacité du ciblage a été validée par IRM et fluorescence sur un modèle animal démontrant ainsi la multifonctionnalité des nanoparticules. / This work deals with the development of hybrid nanoparticles that could offer new strategies for therapy and diagnostic. These are based on a magnetic core which can play the role of contrast agent for MRI as well as heat inductor in AC magnetic field. This inorganic core is surrounded by a thermo-responsive polymeric brush that controls the loading and the release of drugs, and can be functionalized by specific ligands ensuring the targeting specificity. A large part of this work consists in studying the adsorption mechanism of poly(ether)-b-poly(L-lysine) based block copolymers onto magnetic particle and to better understand the influence of the polymer chain conformation at particles surface on the colloidal stability under physiological conditions. An anticancer drug has been loaded and released in a controlled manner under alternative magnetic field by taking advantage from the thermosensitivity of the polyether block. Targeting peptides specific of inflammation sites at the blood brain barrier have been grafted onto copolymers. The targeting specificity has been demonstrated by MRI and fluorescence imaging in rats attesting the multifunctionality of such nanoparticles.