Protein-based materials are an important area of research for various reasons. Natural protein materials such as spider silk have mechanical properties which compare favourably to artificial or inorganic materials, and in addition are biodegradable and can be produced from easily available feedstocks. It is also possible to produce materials that incorporate the functionality of a natural protein, such as ligand-binding or catalysis of reactions, thus allowing this functionality to be used in the solid rather than solution phase. Two particularly interesting components for protein-based materials are amyloid fibrils and tandem repeat proteins. Amyloid fibrils are exceptionally strong, tough, highly-ordered structures that self-assemble from a wide range of simple building blocks. Meanwhile, tandem repeat proteins are a class of proteins that act as scaffolds to mediate protein-protein interactions and are known to act as elastic springs. Unlike globular proteins, tandem repeat proteins can be designed to bind specific ligands, and their ligand-binding properties and stability can be tuned separately. This work details the synthesis and characterisation of repeat protein and amyloid fibril components for a “smart” hydrogel, the production of these gels, and their characterisation using a microfluidic method that I developed. Although amyloid fibrils have previously been decorated with functional proteins, hitherto, this has usually been done by assembling the fibrils from already-functionalised components. This approach limits the functionality to species that can survive the harsh conditions of amyloid aggregation and do not disturb fibril assembly. Therefore, a method was developed to produce amyloid fibrils that displayed an alkyne functionality on their surface to allow functional proteins or other species to be attached after assembly. This involved the design and synthesis (using solid-phase peptide chemistry) of a peptide based on the previously known TTR105-115 peptide (derived from the amyloidogenic Transthyretin protein). These fibrils were characterised by AFM and TEM and it was then shown that the assembled fibrils could be functionalised using an azide-alkyne “click” reaction. The reaction was shown to work with a variety of ligands including proteins, which were found to retain their structure and function after crosslinking to the fibril. The fibrils with ligands attached were characterised by a variety of methods including LCMS (liquid chromatography-mass spectrometry) and super-resolution optical microscopy. Next, repeat proteins were produced recombinantly containing non-natural azido amino acids at their termini. Incorporation of non-natural amino acids was carried out using a number of different methods including amber codon suppression and methionine replacement. Micron-sized hydrogels were then formed from microfluidic-generated droplets by covalently crosslinking the alkyne-functionalised fibrils with the azide-functionalised repeat proteins. The initial experiments to show proof of principle were carried out with consensus-designed repeat proteins, but repeat proteins based on natural sequences were also used to make hydrogels that could later be tested for potential uptake of peptides known to bind these proteins. These hydrogels could potentially be used for drug delivery or other applications in which a chemical response to a mechanical stimulus is desired. The mechanical properties of the hydrogels were measured using novel microfluidic devices, which were designed and fabricated using standard PDMS-based soft lithography.

Dans l’épiderme, les mélanocytes participent à la protection de la peau contre les rayons ionisants du soleil en synthétisant un pigment, la mélanine, dans des compartiments apparentés aux lysosomes appelés melanosomes. La mélanogenèse est un processus séquentiel initié par la production de fibres amyloïdes dont la composante principale est la protéine PMEL. Ces fibres séquestrent la mélanine et permettent l’élimination d’intermédiaires toxiques produits lors de sa synthèse. La mélanogenèse et le phénotype pigmenté sont affectés lorsque le processus de formation des fibres est altéré. Les fibres résultent du clivage de PMEL dans les endosomes précurseurs des mélanosomes mais les protéases impliquées dans ce processus restent peu ou pas caractérisées. Afin de mieux comprendre les mécanismes de formation des fibres amyloïdes dérivées de PMEL, j’ai étudié le rôle de deux protéases : les Bêta-sécrétases BACE1 et BACE2. En combinant des techniques de biochimie, d’immunocytochimie et d’imagerie photonique et électronique, j’ai montré que la perte de l’expression de Bace2 in vivo (souris KO BACE2) ou sa déplétion (siRNA) dans une lignée de mélanocytes inhibent le clivage amyloïdogénique de PMEL et affectent à la fois la formation de fibres de PMEL dans les mélanosomes et la pigmentation. J’ai pu notamment reproduire in vitro le clivage spécifique de PMEL en utilisant une forme recombinante de BACE2. En parallèle, j’ai également étudié le rôle de BACE1 dans la mélanogenèse. Mes résultats indiquent que BACE1, bien que n’étant pas impliquée dans le clivage de PMEL, régulerait la maturation des mélanosomes précoces in vivo et in cellulo, en modulant les contacts entre mélanosomes et réticulum endoplasmique (RE). Dans les mélanocytes, BACE1 est présente dans le RE et interagit avec des protéines impliquées dans les contacts RE-endosomes. Ces contacts seraient cruciaux pour le transfert de molécules nécessaires à la maturation des mélanosomes. L’ensemble de ces résultats démontre un rôle pour chacune des Bêta-sécrétases dans le processus de mélanogenèse, levant le voile sur des processus clés liés à la biogenèse des mélanosomes. Par ailleurs, les fibres de PMEL constituant le modèle le plus abouti de l’amyloïdogenèse physiologique chez les mammifères, ces études pourraient à plus long terme aider à la compréhension de la formation des fibres amyloïdes pathologiques ; notamment dans la maladie d’Alzheimer où l’amyloïdogenèse d’APP est très similaire à celle de PMEL. / In the epidermis, melanocytes synthetize a pigment called melanin, in lysosome-related-organelles called melanosomes, in order to protect the skin against the ionizing radiations of the sun. Melanogenesis is a sequential process initiated by the formation of amyloid fibrils whose principal component is the protein PMEL. Those fibrils sequester the melanin pigment and allow the removal of toxic intermediates formed during its synthesis. Melanogenesis and the pigmented phenotype are affected when the process of fibrils formation is altered. Fibrils come from the processing of PMEL in endosome precursors of melanosomes but the proteases implicated in this process are not well characterized. In order to better understand the mechanisms implicated in the formation of the PMEL amyloid fibrils, I studied the role of two proteases: the Beta-secretases BACE1 and BACE2. Using a combination of biochemical, immunocytochemical methods and photonic and electronic imaging, I have shown that the loss of Bace2 expression in vivo (BACE2 KO mice) or its depletion (siRNA), in a melanocyte cell line, inhibit the amyloidogenic processing of PMEL and affect both the formation of the PMEL fibrils in melanosomes and pigmentation. I could reproduce in vitro the specific cleavage of PMEL by using a recombinant form of BACE2. In parallel, I have also studied the role of BACE1 in melanogenesis. My results indicate that BACE1, even though it is not implicated in PMEL processing, could regulate the maturation of early melanosomes in vivo and in cellulo, by modulating the contacts between melanosomes and endoplasmic reticulum (ER). In melanocytes, BACE1 is present in the ER and interacts with proteins implicated in ER-endosomes contacts. Those contacts would be crucial for the transfer of molecules that are necessary for melanosome maturation. All together those results demonstrate the role of both Beta-secretases in melanogenesis, and reveal key processes involved in melanosome biogenesis. Moreover, because PMEL fibrils are the most completed model of physiological amyloidogenesis in mammals, theses studies could help in the future the understanding of the formation of pathological amyloid fibrils; in particular in the Alzheimer’s disease where the amyloidogenesis of APP is very similar to the one of PMEL.

The thesis deals with the testing of amyloidogenicity of various carbon nanoparticles and polymers. The first part of the thesis provides the theoretical background of amyloidoses, a group of diseases in which proteins are stored in the insoluble form of amyloid. In addition, the theoretical part also deals with a general overview of nanomaterials and the most important methods. Several types of nanomaterials were tested within the thesis, so the part Results and Discussion was divided into two subchapters: 1) Carbon nanospecies and amyloid fibril formation, and 2) Polysaccharides, glycogen modifications and amyloid fibril formation. The first subchapter concerns the testing of four types of carbon nanoparticles (single-walled carbon nanotubes (SWNT), fullerenes (C60), carbon quantum dots (CDs) and nanodiamonds (NDs)). These materials were tested on a model system hen egg white lysozyme (HEWL). Using fluorescence measurements and transmission electron microscopy (TEM), the nanoparticles were ranked from the most to the least amyloidogenic as follows: NDs> control> C60> CDs> SWNT. The second subchapter deals with the effect of selected polysaccharides (glycogen (GG), mannan (MAN), phytoglycogen (PG)) and modified GG on amyloid fibril formation. These materials were tested on the HEWL model system,...

Light chain (AL) amyloidosis is a disease that occurs due to the presence of a small plasma-cell clone, which produces amyloidogenic light chains. These chains can misfold and aggregate, leading to the deposition of amyloid fibrils in tissues. If left untreated or if treatment is ineffective, this can result in irreversible organ dysfunction and eventual death. Current therapeutic treatments generally target and remove the clonal plasma cell population responsible for secreting full-length light chains which is not always effective or safe, however, a different approach to halt pathological LC misfolding would be to inhibit the amyloidogenesis cascade at its starting point. Small molecules have been identified that have the ability to bind to highly conserved residues in the interface between heavy and light chains which can be used to potentially impede the process of amyloid fibril deposition before the native FL LC can misfold or undergo proteolysis to form amyloid fibrils. To test whether small-molecule kinetic stabilizers are effective in stabilizing light chains, we measured the ability of the small molecule to bind to LCs, and the ability of light chains to aggregate and unfold in the absence and presence of small-molecule. Our findings suggest that the binding of stabilizers to the interface between variable domains of the LC dimer can increase equilibrium stability and decrease the rate of aggregation, thereby delaying the onset of amyloid formation.

No description available.

In the face of global warming and shrinking resources of fossil fuels the interest in solar energy has increased in recent years. However, the low energy and cost efficiency of current solar cells has up to this date hindered solar energy from playing a major role on the energy market. Photon upconversion is the process in which light of low energy is converted to high energy photons. Lately, this phenomenon has attracted renewed interest and ongoing research in this field mainly focuses on solar energy applications, solar cells in particular. The aim of this study was to investigate and evaluate amyloid fibrils as nanotemplates for an upconversion system based on the dyes platinum octaetylporphyrin (PtOEP) and 9,10- diphenylanthracene (DPA). This well-known pair of organic dyes upconverts light in the visible spectrum through a mechanism known as sensitized triplet-triplet annihilation. Amyloid fibrils are β-sheet rich protein fibril structures, formed by self-assembly of peptides. Amyloid fibrils were prepared from whey protein isolate using heat and acidic solutions. Dyes were incorporated according to a wellestablished technique, in which dyes are grinded together with the protein in solid state prior to fibrillization. Photophysical properties of pure fibrils and dye-incorporated fibrils were studied using UV-VIS spectroscopy and fluorescence spectroscopy. Atomic force microscopy was further employed to confirm the presence of amyloid fibrils as well as to study fibril structure. Results indicate that amyloid fibrils may not be the optimal host material for the upconversion system PtOEP/DPA. It was found that the absorption and emission spectra of this system overlap to a great deal with that of the fibrils. Though no upconverted emission clearly generated by the dye system was recorded, anti-Stokes emission was indeed observed. Interestingly, this emission appears to be strongly enhanced by the presence of dyes. It is suggested that this emission may be attributed to the protein residues rather than the amyloid structure. Future studies are encouraged to further investigate these remarkable findings. / Intresset för solceller har ökat under de senaste åren, till stor del tillföljd av den globala uppvärmningen och de sinande oljeresurserna. Dagens solceller har dock problem med låg energi- och kostnadseffektivitet, vilket gör att solenergin än så länge har svårt att hävda sig på energimarknaden. Photon upconversion är ett fotofysikaliskt fenomen där fotoner med låg energi omvandlas till fotoner med hög energi. Den senaste tiden har denna process fått förnyat intresse och forskningen inom området har ökat, inte minst med sikte på att integrera processen i solceller och därmed öka dess effektivitet. Målet med denna studie var att undersöka huruvida amyloidfibriller kan användas som stomme för ett photon upconversion-system baserat på platinum-oktaetylporfyrin (PtOEP) och 9,10-difenylantracen (DPA). Dessa två organiska färgämnen är ett välkänt par som konverterar synligt ljus med låg frekvens till mer hög frekvent ljus i det synliga spektrumet, via en mekanism som kallas sensitized triplet-triplet annihilation. Amyloidfibriller är proteinbaserade fiberstrukturer med hög andel β-flak, vilka bildas genom självassociation av peptider. I denna studie skapades amyloidfibriller av vassleprotein genom upphettning i sur lösning. Färgämnena inkorporerades enligt en välbeprövad metod där proteinet mortlas tillsammans med färgämnena i fast tillstånd, innan fibrilleringsprocessen påbörjas. De fotofysikaliska egenskaperna hos fibriller med och utan färgämnen analyserade med UV-VIS samt fluorescensspektroskopi. Atomkraftsmikroskopi användes för att bekräfta att fibriller fanns i proven, samt för att studera dess struktur. De erhållna resultaten antyder att amyloidfibriller inte är ett optimalt material för systemet PtOEP/DPA, delvis på grund av att absorptions- och emissionsspektrumet för systemet överlappar med fibrillernas egna spektrum. Anti-Stokes emission detekterades, men denna är med stor sannolikhet inte orsakad av färgämnena. Dock noterades, intressant nog, att denna emission ökar betydligt i närvaro av färgämnena. En möjlighet är att denna emission är kopplad till monomerer i proteinet snarare än till fibrillstrukturen, eftersom emission observerades hos både nativt och fibrillerat protein. Framtida studier uppmuntras att vidare undersöka dessa effekter.

No / ß2-microglobulin (ß2m) forms amyloid fibrils that deposit in the musculo-skeletal system in patients undergoing long-term hemodialysis. How ß2m self-assembles in vivo is not understood, since the monomeric wild-type protein is incapable of forming fibrils in isolation in vitro at neutral pH, while elongation of fibril-seeds made from recombinant protein has only been achieved at low pH or at neutral pH in the presence of detergents or cosolvents. Here we describe a systematic study of the effect of 11 physiologically relevant factors on ß2m fibrillogenesis at pH 7.0 without denaturants. By comparing the results obtained for the wild-type protein with those of two variants (¿N6 and V37A), the role of protein stability in fibrillogenesis is explored. We show that ¿N6 forms low yields of amyloid-like fibrils at pH 7.0 in the absence of seeds, suggesting that this species could initiate fibrillogenesis in vivo. By contrast, high yields of amyloid-like fibrils are observed for all proteins when assembly is seeded with fibril-seeds formed from recombinant protein at pH 2.5 stabilized by the addition of heparin, serum amyloid P component (SAP), apolipoprotein E (apoE), uremic serum, or synovial fluid. The results suggest that the conditions within the synovium facilitate fibrillogenesis of ß2m and show that different physiological factors may act synergistically to promote fibril formation. By comparing the behavior of wild-type ß2m with that of ¿N6 and V37A, we show that the physiologically relevant factors enhance fibrillogenesis by stabilizing fibril-seeds, thereby allowing fibril extension by rare assembly competent species formed by local unfolding of native monomers.

No description available.

The inherited form of primary open angle glaucoma, a disorder characterized by increased intraocular pressure and retina degeneration, is linked to mutations in the olfactomedin (OLF) domain of the myocilin gene. Disease-causing myocilin variants accumulate within trabecular meshwork cells instead of being secreted to the trabecular extracellular matrix thought to regulate aqueous humor flow and control intraocular pressure. Like other diseases of protein misfolding, we hypothesize myocilin toxicity originates from defects in protein biophysical properties. In this thesis, the first preparative recombinant high-yield expression and purification system for the C-terminal OLF domain of myocilin (myoc-OLF) is described. To determine the relative stability of wild-type (WT) and mutant OLF domains, a fluorescence thermal stability assay was adapted to provide the first direct evidence that mutated OLF is folded but less thermally stable than WT. In addition, mutant myocilin can be stabilized by chemical chaperones. Together, this work provides the first quantitative demonstration of compromised stability among identified OLF variants and placing myocilin glaucoma in the context of other complex diseases of protein misfolding. Subsequent investigations into the biophysical properties of WT myoc-OLF provide insight into its structure and function. In particular, myoc-OLF is stable in the presence of glycosaminoglycans (GAGs), as well as over a wide pH range in buffers with functional groups reminiscent of such GAGs. Myoc-OLF contains significant â-sheet and â-turn secondary structure as revealed by circular dichroism analysis. At neutral pH, thermal melts indicate a highly cooperative transition with a melting temperature of ~55°C. A compact core structural domain of OLF was identified by limited proteolysis and consists of approximately residues 238-461, which retains the single disulfide bond and is as stable as the full myoc-OLF construct. This construct also is capable of generating 3D crystals for structure determination. This data, presented in Chapter 3, inform new testable hypotheses for interactions with specific trabecular extracellular matrix components. To gain further insight into the biological function of myoc-OLF, a facile fluorescence chemical stability assay was designed to identify possible ligands and drug candidates. In the assay described in Chapter 4, the target protein is initially destabilized with a chemical denaturant and is tested for re-stabilization upon the addition of small molecules. The assay requires no prior knowledge of the structure and/or function of the target protein, and it is amendable to high-throughput screening. Application of the assay using a library of 1,280 compounds revealed 14 possible ligands and drug candidates for myoc-OLF that may also generate insights into myoc-OLF function. Due to the high â-sheet content of monomeric myoc-OLF and presence of an aggregated species upon myoc-OLF purification, the ability of myoc-OLF to form amyloid fibrils was suspected and verified. The fibril forming region was confirmed to reside in the OLF domain of myocilin. Kinetic analyses of fibril formation reveal a self-propagating process common to amyloid. The presence of an aggregated species was confirmed in cells transfected with WT myocilin, but to a greater extent in cells transfected with P370L mutant myocilin. Both cell lines stained positive for amyloid. Taken together, these results provide further insights into the structure of myocilin and suggest a new hypothesis for glaucoma pathogenesis. Finally, in a related study, small molecule drug candidates were investigated to treat acid â-glucosidase (GCase), the deficient lysosomal enzyme in Gaucher disease, another protein conformational disorder. Three new GCase active-site directed 3,4,5,6-tetrahydroxylazepane inhibitors were synthesized that exhibit half inhibitory concentrations (IC50) in the low millimolar to low micromolar range. Although the compounds thermally stabilize GCase at pH 7.4, only one of the synthesized analogs exhibits chaperoning activity under typical assay conditions. This successful pharmacological chaperone is also one in which GCase is in its proposed active conformation as revealed by X-ray crystallography. Probing the plasticity of the active-site of GCase offers additional insight into possible molecular determinants for an effective small molecule therapy for GD.

Les protéines amyloïdes sont impliquées dans les maladies neurodégénératives comme Alzheimer, Parkinson et les maladies à prions et forment des structures complexes, les fibres amyloïdes. Le mécanisme de formation de ces fibres est un processus complexe qui implique plusieurs espèces d’agrégats intermédiaires. Parmi ces espèces, des petits agrégats, les oligomères, sont reconnus comme étant l’espèce amyloïde toxique, mais leur mécanisme de toxicité et d’agrégation sont mal compris. Cette thèse présente les résultats d’une étude numérique des premières étapes d’oligomérisation d’un peptide modèle GNNQQNY, issu d’une protéine prion, pour des systèmes allant du trimère au 50-mère, par le biais de simulations de dynamique moléculaire couplée au potentiel gros-grain OPEP. Nous trouvons que le mécanisme d’agrégation du peptide GNNQQNY suit un processus complexe de nucléation, tel qu’observé expérimentalement pour plusieurs protéines amyloïdes. Nous observons aussi que plusieurs chemins de formation sont accessibles à l’échelle du 20-mère et du 50-mère, ce qui confère aux structures un certain degré de polymorphisme et nous sommes capable de reproduire, dans nos simulations, des oligomères protofibrillaires qui présentent des caractéristiques structurelles observées expérimentalement chez les fibres amyloïdes. / Amyloid proteins are involved in neurodegenerative diseases such as Alzheimer’s, Parkinson’s and prion diseases and form complex structures called amyloid fibrils. The fibril formation mechanism is a complex process, which involves several intermediary species. Among these species, small early aggregates, called oligomers, are thought to be the toxic amyloid species but their toxicity and aggregation mechanisms are poorly understood. This thesis aims at presenting the results of a numerical study of the first oligomerization steps of the model peptide GNNQQNY, from a prion protein, for system sizes ranging from the trimer to the 50-mer, via molecular dynamics simulations using the OPEP coarse-grained potential. We find that GNNQQNY’s assembly follows a complex nucleation process, as observed experimentally for numerous amyloid proteins. We also observe that the 20-mer and 50-mer systems form polymorphic structures that are the byproducts of different formation pathways. We further report the spontaneous formation of protofibrillar oligomers with structural characteristics typical of experimentally determined amyloid fibril structures.