Dissertationen zum Thema „Β-amyloid structures“

Polymere Nanopartikel sind ein vielversprechender Ansatz für die Diagnose und Therapie von Krankheiten. Sie ermöglichen den Einsatz von schwerlöslichen oder instabilen Wirkstoffen. Ein weiterer Vorteil ist die Möglichkeit das Targetings, durch gezielte Modifikationen des Nanopartikels wird der Wirkstoff zum Zielort transportiert und kann dort in der gewünschten Form freigesetzt werden; dadurch könnten bei erhöhter Wirksamkeit die Nebenwirkungen von Medikamenten reduziert werden. Ziel dieser Arbeit war die Untersuchung von physikalischen und biochemischen Eigenschaften von Nanopartikeln bestehend aus einem abbaustabilen Polystyren- Kern und einer biologisch abbaubaren Schale aus Polybutylcyanoacrylat. Es werden Methoden beschrieben, um die Größe, Struktur und den Abbau dieser Wirkstoffträger zu untersuchen. Die untersuchten Nanopartikel zeigen RAYLEIGH-Streuung, sowohl Größe als auch Abbau können durch Messung des Absorptionsspektrums bestimmt werden. Weiterhin konnten diese Eigenschaften mit Hilfe von dynamischer und statischer Lichtstreuung sowie Neutronenkleinwinkelstreuung untersucht werden. Bei letzterer Methode konnte gezeigt werden, dass die Schale größtenteils abgebaut werden kann, während der Kern intakt bleibt. In einem weiteren Teil der Arbeit wurde die Überwindung der Blut-Hirn-Schranke durch polymere Nanopartikel untersucht. Dabei wurde der fluoreszierende Thioflavine als Modellwirkstoffe eingesetzt. Das Durchdringen der Blut-Hirn-Schranke konnte nur mit Nanopartikeln erreicht werden, an deren Oberfläche ein Apolipoprotein E-Peptid gekoppelt war. Es konnte gezeigt werden, das die Nanopartikelschale im Gehirn abgebaut wird, der Wirkstoff freigesetzt wird und an Amyloid β, einem Marker der Alzheimer-Krankheit, bindet.

Cette thèse a exploré l'intégration de moteurs moléculaires synthétiques photoactivables dans des réseaux de gels supramoléculaires. L'objectif principal était d'obtenir un mouvement macroscopique réversible en exploitant à la fois la rotation unidirectionnelle des moteurs moléculaires et la nature réversible des interactions supramoléculaires. Des moteurs moléculaires hautement fonctionnalisés ont été synthétisés et intégrés comme unités de réticulation dans des réseaux de gel supramoléculaire de peptides de diphénylalanine et de poly(γ-benzyl-L-glutamate) et d'oligonucléotides d'ADN. L'activation de la rotation unidirectionnelle des moteurs moléculaires par la lumière a permis de produire un travail nanomécanique suffisant pour perturber les interactions supramoléculaires dans les réseaux de gel à base de peptides, ce qui entraîne la contraction ou la fonte du gel à l'échelle macroscopique. Grâce aux interactions supramoléculaires réversibles, le matériau gélifié initial a pu être récupéré dans l'obscurité, soit spontanément, soit par l'application d'un stimulus thermique. Les systèmes étudiés dans cette thèse représentent une nouvelle classe de matériaux fonctionnant dans des conditions dissipatives hors équilibre, promettant des applications dans divers domaines tels que la biologie, la médecine et la science des matériaux
This thesis explored the integration of light-driven synthetic molecular motors in supramolecular gel networks. The main goal was to achieve reversible macroscopic motion by exploiting both the unidirectional rotation of molecular motors and the reversible nature of supramolecular interactions. Highly functionalized molecular motors have been synthesized and integrated as crosslinking units in supramolecular gel networks of diphenylalanine and poly(γ- benzyl-L-glutamate) peptides, as well as DNA oligonucleotides. Activation of the unidirectional rotation of molecular motors by light, allowed the production of nanomechanical work which is sufficient to disrupt supramolecular interactions in peptide-based gel networks leading to contraction or melting of the gel material at the macroscopic scale. Thanks to the reversible supramolecular interactions, the initial gel material was recovered in the dark, either spontaneously or by applying a thermal stimulus. The systems studied in this thesis represent a novel class of materials operating in dissipative out-of-equilibrium conditions, holding promise of applications in various fields such as biology, medicine and material science

In questa tesi sono stati affrontati tre progetti indipendenti, accomunati dall’uso della modellistica molecolare e in particolare della dinamica molecolare. Nel primo progetto è stato studiato il dominio Sec14-PH della neurofibromina (NF1). I domini sec14 sono stati scoperti in numerose proteine dai procarioti all’uomo come scambiatori di lipidi tra membrane, per mezzo di una tasca la cui apertura è legata al movimento di una specifica alpha-elica (elica lid). La struttura cristallina del dominio Sec14 di NF1 (sia del wild type sia di mutanti associati all’insorgenza della patologia neurofibromatosi) ha rivelato la sua particolarità di essere strutturalmente accoppiato ad un dominio PH che interagisce fortemente con l’elica lid tramite un suo loop (detto lid-lock loop). Su questa base è stato formulato un meccanismo di apertura della tasca del Sec14 che coinvolgerebbe un movimento concertato del lid-lock loop, ma questo movimento non è mai stato osservato o dimostrato. Guidati da dati sperimentali sulla denaturazione termica del Sec14-PH di NF1, sia del wild type sia di alcuni mutanti, diverse simulazioni ad alta temperatura sono state effettuare per comparare la dinamica del dominio wild type con un mutante patologico associato all’insorgenza della patologia neurofibromatosi. Con le nostre simulazioni è stato possibile proporre un meccanismo di funzionamento dell’apertura dell’elica lid e fornire delle basi strutturali e dinamiche dell’insorgenza della patologia nel caso del mutante specifico studiato. Nel secondo progetto è stato affrontato lo studio di una proteina chiamata EtfAB che catalizza un processo recentemente scoperto noto come biforcazione elettronica basata sulle flavine. Questo meccanismo è sfruttato solo da alcuni microrganismi anerobici come terza via di accoppiamento energetico e finora si conoscono quattro famiglie di proteine, evolutivamente non correlate, in grado di catalizzarlo. Una di queste è EtfAB, della quale non è chiaro come possa avvenire il trasferimento elettronico tra le due molecole di FAD ad essa legate. Infatti, la distanza tra questi due FAD osservata nella struttura cristalline di EtfAB è di 18 Å, mentre si ritiene più plausibile che i trasferimenti elettronici in biologia non avvengano a distanze maggiori di 14 Å. Per questo è stato suggerito un possibile meccanismo che potrebbe avvicinare le due molecole di FAD. Usando la dinamica molecolare è stato possibile testare, e smentire, il meccanismo proposto. Inoltre, con il Density Functional Theory (DFT), è stato possibile fornire un’interpretazione ad alcuni dati spettroscopici riguardo il possibile trasferimento elettronico tra le due molecole di FAD. Nel terzo progetto, ho collaborato con il Prof. Luca Bertini ad un progetto sulla produzione e propagazione di alcune specie reattive dell’ossigeno (ROS) nel contesto del peptide amiloide beta coinvolto nella patogenesi dell’Alzheimer. Nell’ambito dell’ipotesi amiloide sull’insorgenza della patologia di Alzheimer, un ruolo importante è stato attribuito ai danni causati dai ROS, prodotti da un complesso metallico all’interno del peptide amiloide stesso, in particolare dal radicale ossidrilico (OH.-). Tuttavia, i dettagli su come questi radicali propaghino e reagiscano non sono ancora stati chiariti. Mentre i calcoli DFT del Prof. Bertini affrontavano le capacità ossidative del radicale ossidrilico e i possibili prodotti di reazione nel contesto del peptide amiloide beta, con i miei calcoli di dinamica molecolari è stata fornita una panoramica su quali possibili bersagli del radicale ossidrilico, coordinato allo ione Cu del complesso, possano effettivamente reagire entrando in contatto con il radicale ossidrilico a causa dei moti dinamici del peptide.
In this thesis, three independent projects were addressed, sharing the computational approach based on molecular modeling and in particular molecular dynamics. In the first project, the Sec14-PH domain of neurofibromin (NF1) was investigated. The Sec14 domains have been identified in many different proteins, from prokaryotes to humans, serving as exchangers of lipid molecules between membranes, by means of a pocket whose opening is allowed by the motion of a specific alpha-helix (called lid helix). The crystal structure of the NF1-Sec14 domain (of both the wild type and some mutants associated with the onset of neurofibromatosis pathology) has revealed its peculiarity of being structurally coupled to a PH domain that strongly interacts with the lid helix through a long loop (called lid-lock loop). On this basis, a mechanism for the opening of the Sec14 lipid pocket was formulated which would involve a concerted movement of the lid-lock loop, but this movement has actually never been shown. Guided by available experimental data on the thermal denaturation of Sec14-PH domain of NF1, both on the wild type and some neurofibromin-related mutants, several simulations at high temperature were carried out to compare the dynamics of the wild type domain with a pathological mutant associated with the onset of neurofibromatosis. Our simulations lead us to suggest an opening mechanism for the lid helix and provide a hypothesis for the structural and dynamic basis of the onset of the disease in the case of the specific mutant. The second project addressed the study of a protein called EtfAB which catalyzes a recently discovered process known as Flavin-Based Electron Bifurcation (FBEB). This mechanism is only exploited by some anaerobic microorganisms as a third way of energy coupling. So far, four unrelated protein families are known that are able to catalyze FBEB. Among these, EtfAB, catalyzes the electron transfer between the two FAD molecules bound to it. Surprisingly, the distance between these two FADs, as observed in the crystal structure of EtfAB, is 18 Å, whereas biological electron transfer is considered more likely to occur at a maximal distance of 14 Å. To explain this, a possible mechanism has been suggested that could bring the two FAD molecules closer together. Using molecular dynamics, it was possible to test, and discard, the proposed mechanism. Furthermore, with the Density Functional Theory (DFT), it was possible to provide an interpretation to some spectroscopic data regarding the possible electron transfer between the two FAD molecules. In the third project, I collaborated with Prof. Luca Bertini on a project on the production and propagation of some reactive oxygen species (ROS) in the context of the amyloid-beta peptide involved in the pathogenesis of Alzheimer's. In the amyloid hypothesis on the onset of Alzheimer's disease, an important role has been attributed to the damage caused by ROS, produced by a metal ion coordinated to the amyloid peptide itself, in particular by the hydroxyl radical (OH.-). However, the details of how these radicals propagate and react have not yet been clarified. While Prof. Bertini's DFT calculations addressed the oxidative capacities of the hydroxyl radical and the possible reaction products in the context of the amyloid-beta peptide, my molecular dynamics simulations provided an overview on which possible targets of the hydroxyl radical, coordinated to the ion Cu of the complex, could actually react with the hydroxyl radical due to the dynamic motions of the peptide.

La maladie d’Alzheimer (MA) est un désordre neuro-dégénératif chronique fatal et la forme la plus répandue des démences chez l’adulte qui touchent plus de 28 millions de personnes dans le monde. En absence de traitement pour les démences neurodégénérative dont la maladie d’Alzheimer, le coût de celles-ci est estimé à 1 trillion d’USD en 2018 ce qui représente des enjeux économiques et sociétaux majeurs au niveau national et mondial. La MA est une forme d’amylose qui est caractérisée par l’agrégation du peptide amyloïde beta (Aβ) dans le cerveau des patients. Le facteur de risque génétique principal de la forme tardive (après 65 ans) de cette maladie est l’isoforme E4 de l’apolipoprotéine E (apoE) qui intervient dans le transport et le métabolisme des lipides et interagit avec le peptide Aβ. La modulation de la structure des isoformes d’apoE et de leur interaction avec l’Aβ apparaît comme une cible prometteuse dans la conception rationnelle de thérapies de la maladie. Celle-ci nécessite néanmoins une compréhension approfondie des propriétés structurales et dynamiques des deux partenaires moléculaires. Dans le cadre de cette thèse, nous avons étudié la structure de trois isoformes (E2, E3 et E4) de l’apoE par différentes techniques de biologie structurale et principalement par la réticulation chimique couplée à la spectrométrie de masse (CXMS) quantitative et par la bioinformatique structurale. Ces données complémentées par la spectroscopie infrarouge ont permis de construire des modèles structuraux de l’apoE2, E3 et E4. Nous avons mis en évidence l’interaction des domaines N- et C-terminal et la présence de multiples conformations de l’apoE chez les trois isoformes. Nos données suggèrent un équilibre entre deux principales conformations de l’apoE dont la population relative diffère entre les trois isoformes. Nous proposons les interfaces à cibler dans le cadre de thérapie visant à moduler les propriétés structurales des isoformes de l’apoE.Nous avons également mis en évidence que chaque isoforme d’apoE (E2, E3 et E4) interfère avec l’agrégation d’Aβ. Le peptide interagit avec les deux domaines N- et C- terminal de l’apoE. L’étude quantitative de l’interaction par CXMS a révélé des différences entre les cross-links formés en présence des isoformes. La modélisation du complexe apoE-Aβ a permis de mettre en évidence les interfaces impliquées dans l’interaction. Celle-ci possède une composante hydrophobe et électrostatique qui diffère chez les isoformes d’apoE. Nous proposons un mécanisme de l’interaction apoE-Aβ qui est initié par les propriétés hydrophobes des deux partenaires et qui est stabilisé par la suite via des contacts électrostatiques. Par ailleurs, une étude permettant d’explorer le potentiel de la nouvelle chimie de réticulation des résidues acides de protéine dans des applications en protéomique structurale a été effectuée.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Mass spectrometry has in recent years been established as the standard method for protein identification and characterization in proteomics with excellent intrinsic sensitivity and specificity. Fourier transform ion cyclotron resonance is the mass spectrometric technique that provides the highest resolving power and mass accuracy, increasing the amount of information that can be obtained from complex samples. This thesis concerns how useful information on proteins of interest can be extracted from mass spectrometric data on different levels of protein structure and how to obtain this data experimentally. It was shown that it is possible to analyze complex mixtures of protein tryptic digests by direct infusion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and identify abundant proteins by peptide mass fingerprinting. Coupling on-line methods such as liquid chromatography and capillary electrophoresis increased the number of proteins that could be identified in human body fluids. Protein identification was also improved by novel statistical methods utilizing prediction of chromatographic behavior and the non-randomness of enzymatic digestion. To identify proteins by short sequence tags, electron capture dissociation was implemented, improved and finally coupled on-line to liquid chromatography for the first time. The combined techniques can be used to sequence large proteins de novo or to localize and characterize any labile post-translational modification. New computer algorithms for the automated analysis of isotope exchange mass spectra were developed to facilitate the study of protein structural dynamics. The non-covalent interaction between HIV-inhibitory peptides and the oligomerization of amyloid β-peptides were investigated, reporting several new findings with possible relevance for development of anti-HIV drug therapies and understanding of fundamental mechanisms in Alzheimer’s disease.

Polymere Nanopartikel sind ein vielversprechender Ansatz für die Diagnose und Therapie von Krankheiten. Sie ermöglichen den Einsatz von schwerlöslichen oder instabilen Wirkstoffen. Ein weiterer Vorteil ist die Möglichkeit das Targetings, durch gezielte Modifikationen des Nanopartikels wird der Wirkstoff zum Zielort transportiert und kann dort in der gewünschten Form freigesetzt werden; dadurch könnten bei erhöhter Wirksamkeit die Nebenwirkungen von Medikamenten reduziert werden. Ziel dieser Arbeit war die Untersuchung von physikalischen und biochemischen Eigenschaften von Nanopartikeln bestehend aus einem abbaustabilen Polystyren- Kern und einer biologisch abbaubaren Schale aus Polybutylcyanoacrylat. Es werden Methoden beschrieben, um die Größe, Struktur und den Abbau dieser Wirkstoffträger zu untersuchen. Die untersuchten Nanopartikel zeigen RAYLEIGH-Streuung, sowohl Größe als auch Abbau können durch Messung des Absorptionsspektrums bestimmt werden. Weiterhin konnten diese Eigenschaften mit Hilfe von dynamischer und statischer Lichtstreuung sowie Neutronenkleinwinkelstreuung untersucht werden. Bei letzterer Methode konnte gezeigt werden, dass die Schale größtenteils abgebaut werden kann, während der Kern intakt bleibt. In einem weiteren Teil der Arbeit wurde die Überwindung der Blut-Hirn-Schranke durch polymere Nanopartikel untersucht. Dabei wurde der fluoreszierende Thioflavine als Modellwirkstoffe eingesetzt. Das Durchdringen der Blut-Hirn-Schranke konnte nur mit Nanopartikeln erreicht werden, an deren Oberfläche ein Apolipoprotein E-Peptid gekoppelt war. Es konnte gezeigt werden, das die Nanopartikelschale im Gehirn abgebaut wird, der Wirkstoff freigesetzt wird und an Amyloid β, einem Marker der Alzheimer-Krankheit, bindet.

碩士
國立陽明大學
生化暨分子生物研究所
104
Alzheimer’s disease (AD) is the most common cause of dementia in elderly people. It is a chronic neurodegenerative disease. Currently, the hypothesis for the pathogenic mechanism of AD is amyloid cascade hypothesis. It states that the aggregation of β-amyloid (Aβ) is the primary cause of AD. Aβ contains 39-42 residues amino acid. It was a proteolytic product derived fromβ-amyloid precursor protein (βAPP). The aggregation process of Aβ involves conformational changes and self-assembly, indicating that the structural property plays an important role in Aβ aggregation. Previous studies also reported that the aggregation of Aβ was linked to its interaction with cell membrane-like enviroment. The interaction between Aβ and cell membrane might alter the structural property and conformational stability of Aβ. Some studies suppoted the view that cell membrane environment might inhibit the aggregation of Aβ, whereas others thought that cell membrane enviroments would promote the aggregation of Aβ. In this study, we used negatively charged LMPG micelles and familial Alzheimer’s disease-linked Iowa-type (Aβ40(D23N)) as model systems for investigating the Aβ-cell membrane interaction mechanism, and applied nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, thioflavine-T (Th-T) fluorometric assay and transmission electron microscopy (TEM) to characterize the interaction between Aβ and membrane lipid. By compairing the structure and aggregation behavior of Aβ40(D23N) in aqueous solution and LMPG micelles environment, we obtained that LMPG micelles would interact with Aβ40(D23N), leading to an increase of the α-helical propensity of Aβ40(D23N) and an inhibition of Aβ40(D23N) aggregation. Structural analysis showed that Aβ40(D23N) interacted with LMPG micelles through two short α-helical regions, L17VFFAENVGS26 and K28GAIIGLM35. These results may provide the information about the mechanism of Aβ-cell membrane interaction.

碩士
國立中正大學
分子生物研究所
94
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the progressive deposition of the aggregated amyloid β-peptide (Aβ) on the cell surfaces and in the walls of cerebral blood vessels. Aβ is a 39-43 amino acid polypeptide derived from proteolytic cleavages of the β-amyloid precursor protein (APP) by β- and γ-secretase. There are three groups of mutation sites in APP found in AD patients. These mutation sites locate near the ?, β- and γ-secretase cutting sites. It is believed that mutations near the β- and γ-secretase cutting sites induce an increase in cutting rates by both secretases, in turn enhancing the formation rate of Aβ. On the contrary, mutations near ?secretase cutting sites may result in a decrease in the cutting rate of ?secretase, but relatively increasing the cutting rate of β-secretase, in turn leading to an increase of Aβ formation. However, there is another explanation for why these mutants cause the early-onset of AD. Mutations near β-secretase cutting site may affect the conformational change of Aβ peptide, resulting in an increase in their self-aggregation. Thus, we aimed to analyze the structure of mutants that may play important role in the molecular mechanism of aggregation. In this study, we successfully cloned, cultured and purified the isotope labeled wild-type and mutants Aβ peptides. The purified peptides were subjected to analysis by NMR. We believe understanding the structural and conformational differences between wild-type and mutants of Aβ peptides will shed light on the mechanism of aggregated Aβ-induced AD.

碩士
國立陽明大學
生命科學系暨基因體科學研究所
104
Alzheimer's disease (AD) a chronic neurodegenerative disease. It is the main cause of dementia in the elderly people. β-amyloid peptide (Aβ) is the main component of neuritic plaques which are a pathological hallmark of AD. β-amyloid peptide (Aβ) contains 39~42 amino acid residues. It was a proteolytic product derived from β-amyloid precursor protein (βAPP). Currently, the leading theory for explaining the etiology and pathogenesis of AD is the “Amyloid cascade hypothesis” which stated that Aβ aggregation resulted in brain cell death and dementia. Studies have shown that the aggregation of Aβ was linked to its interaction with cellular membranes. However, the underlying mechanism remains unclear. From a structural perspective, the interaction might induce conformational changes of Aβ resulting in an alteration of it’s aggregation behavior. To understand the role of cellular membranes in Aβ aggregation, the effects of lipids on the structure and aggregation behavior of Aβ were characterized by using thioflavine-T (Th-T) fluorometric assay, nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopies. An anionic lipid, lyso-myristoylphosphatidylglycerol (LMPG) and a genetic Aβ mutant, Dutch-type Aβ40 (Aβ40(E22Q)), were used in the present study. Structural studies indicated that LMPG micelles increased the α-helical content of Dutch-type Aβ40. The results of aggregation kinetics showed that LMPG micelles inhibited the aggregation of Dutch-type Aβ40. These results suggested that the interaction of Dutch-type Aβ40 with anionic lipids would increase its conformational stability leading to a reduction of its aggregation rate. These findings may help us gain an insight into the possible role of Aβ-lipid interaction in the process of Aβ aggregation.

The initial steps involved in the assembly of normally soluble proteins into amyloid fibrils remain unclear, yet over 20 human diseases are associated with proteins that aggregate in this manner. Protein surface modification is a potential means of mapping the interaction sites in early oligomers that precede amyloid formation. This dissertation focuses on the use of covalent labeling combined with mass spectrometry to elucidate the structural features of Cu(II)-induced β-2-microglobulin (β2m) amyloid formation. An improved covalent modification and MS-based approach for protein surface mapping has been developed to address the need for a reliable approach that ensures protein structural integrity during labeling experiments and provides readily detectable modifications. This approach involves measuring the kinetics of the modification reactions and allows any local perturbations caused by the covalent label to be readily identified and avoided. This MS-based method has been used to study human β2m, a monomeric protein that has been shown to aggregate into amyloid fibrils in dialysis patients leading to dialysis-related amyloidosis. Under conditions that lead to β2m amyloid formation, reactions of β2m with three complementary covalent labels have been used to identify the Cu(II) binding site, metal-induced conformational changes, and the oligomeric interfaces. Results confirm that Cu(II) binds to His31 and the N-terminal amine. Binding to these residues causes several structural changes in the N-terminal region and ABED β-sheet which likely enables formation of oligomeric intermediates. The covalent labeling data indicate that the pre-amyloid β2m dimer has an interface that involves the antiparallel arrangement of ABED sheets from two monomers. Moreover, our covalent labeling data allowed us to develop a model for the tetramer in which the interface is mediated by interactions between D strands of one dimer unit and the G strands of another dimer unit. Lastly, the selective covalent modification approach has been used to delineate the structural changes in β2m after interaction with Cu(II), Ni(II), and Zn(II) and their effect on its aggregation. Our covalent labeling data indicates that the unique effect of Cu(II) appears to be caused by the site at which the metal binds the protein and the conformational changes it induces.

The initial steps involved in the assembly of normally soluble proteins into amyloid fibrils remain unclear, yet over 20 human diseases are associated with proteins that aggregate in this manner. Protein surface modification is a potential means of mapping the interaction sites in early oligomers that precede amyloid formation. This dissertation focuses on the use of covalent labeling combined with mass spectrometry to elucidate the structural features of Cu(II)-induced β-2-microglobulin (β2m) amyloid formation. An improved covalent modification and MS-based approach for protein surface mapping has been developed to address the need for a reliable approach that ensures protein structural integrity during labeling experiments and provides readily detectable modifications. This approach involves measuring the kinetics of the modification reactions and allows any local perturbations caused by the covalent label to be readily identified and avoided. This MS-based method has been used to study human β2m, a monomeric protein that has been shown to aggregate into amyloid fibrils in dialysis patients leading to dialysis-related amyloidosis. Under conditions that lead to β2m amyloid formation, reactions of β2m with three complementary covalent labels have been used to identify the Cu(II) binding site, metal-induced conformational changes, and the oligomeric interfaces. Results confirm that Cu(II) binds to His31 and the N-terminal amine. Binding to these residues causes several structural changes in the N-terminal region and ABED β-sheet which likely enables formation of oligomeric intermediates. The covalent labeling data indicate that the pre-amyloid β2m dimer has an interface that involves the antiparallel arrangement of ABED sheets from two monomers. Moreover, our covalent labeling data allowed us to develop a model for the tetramer in which the interface is mediated by interactions between D strands of one dimer unit and the G strands of another dimer unit. Lastly, the selective covalent modification approach has been used to delineate the structural changes in β2m after interaction with Cu(II), Ni(II), and Zn(II) and their effect on its aggregation. Our covalent labeling data indicates that the unique effect of Cu(II) appears to be caused by the site at which the metal binds the protein and the conformational changes it induces.

碩士
國立中興大學
化學系所
96
Amyloid-β peptide (Aβ) is the principal constituent of plaques and fibrils associated with Alzheimer’s disease (AD) and is thought to be responsible for the neurotoxicity associated with the disease. Copper binding to Aβ has been hypothesized to play an important role in the neruotoxicity of Aβ. However, many properties of copper binding to Aβ have not been elucidated clearly, and the location of copper binding sites on Aβ is also in controversy. In this work, several possible models containing copper(II) coordinating with His residues of Aβ related to fibril formation were proposed, and the results provided a viewpoint to discuss formation of the fibril.

碩士
國立臺北科技大學
生物科技研究所
97
Several neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, are associated with amyloid fibrils formed by different polypeptides. Recently, the atomic structure of the amyloid-forming peptides GGVVIA and MVGGVV from the C-terminal hydrophobic segment of amyloid-β (Aβ) peptide has been determined and revealed a dry, tightly self-complementing structure between two β-sheets, termed as “steric zipper”. In this study, several all-atom molecular dynamics simulations with explicit water were conducted to investigate the structural stability and aggregation behavior of the GGVVIA and MVGGVV oligomers with various sizes. The results of both GGVVIA and MVGGVV oligomers showed that their structural stability increases with increasing the numbers of β-strands. Our results also showed that the two-sheet models exhibit higher structural stability than the one-sheet models, indicating that an extra β-sheet layer is necessary to stabilize the oligomers. We further suggested that the minimal nucleus seeds for GGVVIA and MVGGVV fibril formation could be SH2-ST2 and SH2-ST4 models, respectively. Our simulation results also revealed that the hydrophobic interactions between the adjacent β-strands within the same layer plays an important role in stabilizing both GGVVIA and MVGGVV oligomers. Between the two neighboring β-sheets, the hydrophobic steric zipper of GGVVIA formed via the side chains of V3, V4, and I5 plays a critical role in holding the hydrophobic steric zipper together. For the case of MVGGVV oligomers, the hydrophobic side chain of M1, V2, V5, and V6 also locks the hydrophobic steric zipper together between the two neighboring β-sheet layers. For GGVVIA oligomers, single glycine substitution at V3, V4, and I5 directly result in the loss of hydrophobic interactions between the adjacent β-strands and disrupt the hydrophobic steric zipper between these two β-sheets. Similarly, mutation simulations for MVGGVV showed that a single glycine residue substitution, M1G, V2G, V5G, and V6G, directly result in elongation between β-strands and disrupt the steric zipper between the two neighboring β-sheet layers. In summary, our simulation results provided detailed insights into understanding the aggregation behavior of the GGVVIA and MVGGVV oligomers in the atomic level. It may also be helpful for designing new inhibitors able to prevent the fibril formation of Aβ peptide.

Dissertação de mestrado, Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014
CRTAC (Cartilage Acidic Protein) firstly identified as a chondrocyte marker in humans and implicated in a number of diseases. This ancient protein is present from prokaryotes to vertebrates and the teleost are the only group that contain duplicates (CRTAC1/CRTAC2). The structure of CRTACs is poorly characterized and was the starting point of the present study. To establish the molecular and structural characterization of CRTAC, three recombinant proteins [human (h) CRTAC1 and sea bass (Dicentrarchus labrax, dl) CRTAC1 and CRTAC2] were over-expressed in E.coli as inclusion bodies and their identity was confirmed by mass spectrometry. The resulting refolded recombinant proteins were obtained with a productivity of 11,51, 8,4 and 11,9 mg of protein per gram of biomass for hCRTAC1, dlCRTAC1 and dlCRTAC2 respectively and approximately 23,48%, 9,09% and 33,46% of, hCRTAC1, dlCRTAC1 and dlCRTAC2 were lost as insoluble aggregates. Size exclusion chromatography revealed the presence of mostly soluble aggregated forms of piscine CRTACs and a mixture of aggregates and monomeric form for hCRTAC1. Spectroscopic studies of human CRTAC1 and sea bass CRTAC1/CRTAC2 showed that all proteins possess secondary and tertiary structure and are particularly rich in β- pleated sheet structure (≈40%), have ≈10,3% alpha-helix and the remainder is disordered. The thermal stability of CRTAC´s structure was assessed considering heating (from 20-90ºC) and freezing (-80ºC). CRTACs retain their native secondary and tertiary structures upon heating, however a slight loss of structure was observed for hCRTAC1 at 60ºC. Freezing induced loss of secondary structure and conformational changes more pronounced for hCRTAC1 and third cycle of dlCRTAC2 and less perceptible for dlCRTAC1. Amyloid formation by CRTACs was assessed in Thioflavin-T assays and a decrease in fluorescence was observed after incubation. In summary, CRTAC´s have propensity to form soluble aggregates that are highly thermostable and these structural properties are conserved from teleosts to humans.
A CRTAC (proteína acídica da cartilagem) é uma proteína da matriz extracelular, inicialmente identificada em humanos como marcador de condrócitos e que está também associada a várias doenças, como a esclerose múltipla. Esta proteína ancestral está presente desde os procariotas até aos vertebrados e os teleósteos são o único grupo que contém o gene duplicado (CRTAC1/CRTAC2). Ambas as proteínas apresentam estrutura similar e possuem na região N-terminal um domínio semelhante ao das cadeias α presente nas integrinas, mas diferem na região C-terminal pelo facto da CRTAC1 ter um domínio adicional descrito como um factor de crescimento epidérmico com ligação a iões Ca2+ . A estrutura conservada das CRTAC indica que estas proteínas podem ter uma função importante que ainda não foi identificada e o facto da sua estrutura estar pouco caracterizada foi o objecto deste estudo. Para o estudo de caracterização molecular e estrutural das CRTAC, três proteínas recombinantes [hCRTAC1 humana e CRTAC1 e 2 de robalo (Dicentrarchus labrax ,dl)] foram sobrexpressas como corpos de inclusão em E.coli e a sua identidade foi confirmada por espectroscopia de massa. Após a solubilização, a purificação e o “refolding”, a produtividade das proteínas recombinantes resultantes foi de, 11,89 para a hCRTAC1, 8,4 para a dlCRTAC1 e 11,9 para a dlCRTAC2, expressa em mg de proteína obtida por g de biomassa. Durante este processo, foram registadas perdas proteicas na forma de agregados insolúveis de aproximadamente 23,48% de hCRTAC1, 9,09% de dlCRTAC1 e 33,46% de dlCRTAC2. Análises de cromatografia de exclusão molecular, das proteínas CRTAC solubilizadas, revelaram a presença de agregados de elevado peso molecular da dlCRTAC1 e 2 de peixes e uma mistura de agregados e monómero da CRTAC1 humana. Os estudos espectroscópicos, demonstraram que, as proteínas recombinantes CRTAC têm estrutura secundária e terciária e que a sua estrutura secundária é particularmente rica em folha-β (≈40%), tendo apenas 10,3% de hélice-α e a restante estrutura é desordenada. Neste estudo foi avaliada a estabilidade térmica da estrutura das CRTAC, considerando o efeito do aquecimento (de 20º a 90ºC) e do congelamento em vários ciclos (-80ºC). Os resultados indicaram que, as CRTAC mantêm a sua estrutura secundária e terciária após o aquecimento, no entanto, no caso da CRTAC1 humana foi observada uma ligeira perda de estrutura a partir dos 60ºC. O efeito do congelamento induziu a uma perda de estrutura secundária e também a alterações conformacionais mais evidentes na CRTAC1 humana relativamente á dlCRTAC1 e dlCRTAC2 de peixes. A capacidade de formação de fibrilas amilóides pelas CRTAC, sob certas condições de incubação, foi avaliada utilizando ensaios com Tioflavina-T. Após a incubação foi observada uma diminuição da fluorescência e estes resultados sugerem que, as CRTAC´s podem facilmente formar agregados de grandes dimensões com propriedades não-amilóides que, podem ser degradados ou sair de solução após longos periodos de incubação. Admite-se a hipótese de que tais agregados possam ser estruturas funcionais. Resumindo, as proteínas CRTAC tendem a formar agregados solúveis que são altamente termoestáveis e estas propriedades estruturais são conservadas desde os teleósteos até aos humanos