Dissertations / Theses on the topic 'Protein misfolding and aggregation'
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BROGGINI, LUCA. "MOLECULAR DETERMINANTS UNDERLYING PROTEIN MISFOLDING AND AGGREGATION." Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/831967.
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Proteins have evolved to adopt distinctive and well-defined functional states under physiological conditions, either as monomers or complexes. The achievement of a three-dimensional structure allows proteins to exert their physiological functions. Nevertheless, when proteins lose – or fail to acquire – their spatial organization, they can convert into aggregated species that can be harmful to the organism.
Conformational diseases gather all those pathologies characterized by the misfolding and aggregation of proteins. Indeed, while the formation and deposition of proteinaceous aggregates can be toxic to cells, the lack of active folded protein disrupts normal physiological pathways.
Although considerable progresses have been made in the recent years, to date conformational diseases are still incurable. Indeed, the incomplete understanding of the causes guiding protein misfolding and aggregation prevents the development of efficient treatments. At the same time, the complexity and the diversity of the processes leading to the formation of aggregated species make the task extremely challenging.
This PhD project was developed to provide a more comprehensive overview of the molecular bases underlying the conversion of soluble and functional states into aggregated and potentially toxic species.
To reach such aims, we applied an integrative approach on two model systems, neuroserpin (NS) and beta-2 microglobulin (2m). In particular, we combined a series of biophysical, biochemical and structural techniques to study these two proteins which have been largely used as model systems for serpin polymerization and amyloid formation, respectively.
We found that protein misfolding and aggregation processes depend on several molecular properties, including primary sequence, denatured state compactness, thermal stability, ability to form oligomers under physiological conditions, and the presence of post-translation modifications.
The data presented in this PhD thesis add valuable information to depict the complex framework of protein misfolding and aggregation.
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Buell, Alexander Kai. "On the kinetics of protein misfolding and aggregation." Thesis, University of Cambridge, 2011. https://www.repository.cam.ac.uk/handle/1810/270324.
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Protein (mis)folding into highly ordered, fibrillar structures, amyloid fibrils, is a hallmark of several, mainly neurodegenerative, disorders. The mechanism of this supra-molecular self-assembly reaction, as well as its relationship to protein folding are not well understood. In particular, the molecular origin of the metastability of the soluble state of proteins with respect to the aggregated states has not been clearly established. In this dissertation, it is demonstrated, that highly accurate kinetic experiments, using a novel biosensing method, can yield fundamental insight into the dynamics of proteins in the region of the free energy landscape corresponding to protein aggregation. First, a section on Method development describes the extension and elaboration of the previously established kinetic assay relying on quartz crystal microbalance measurements for the study of amyloid fibril elongation (Chapter 3). This methodology is then applied in order to study in great detail the origin of the various contributions to the free energy barriers separating the soluble state of a protein from its aggregated state. In particular, the relative importance of residual structure, hydrophobicity (Chapter 4) and electrostatic interactions (Chapter 5) for the total free energy of activation are discussed. In the last part of this thesis (Chapter 6), it is demonstrated that this biosensing method can also be used to study the binding of small molecules to amyloid fibrils, a very useful feature in the framework of the quest for potential inhibitors of amyloid formation. In addition, it is shown that Thioflavin T, to-date the most frequently employed fluorescent label molecule for bulk solution kinetic studies, can in the presence of potential amyloid inhibitor candidates be highly unreliable as a means to quantify the effect of the inhibitor on amyloid formation kinetics. In summary, the work in this thesis contributes to both the fundamental and the applied aspects of the field of protein aggregation.
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Todorova, Nevena, and Nevena Todorova@rmit edu au. "Molecular modelling of peptide folding, misfolding and aggregation phenomena." RMIT University. Applied Science, 2009. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20091130.111240.
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In this thesis we present computer modelling studies that were implemented to investigate protein behavior in various environments causing their folding, unfolding and aggregation. Applications related to two important proteins - insulin and apolipoprotein C-II (ApoC-II) are presented. The use of atomistic simulation methodologies based on empirical force fields has enhanced our understanding of many physical processes governing protein structure and dynamics. However, the force fields used in classical modelling studies are often designed for a particular class of proteins and rely on continuous improvement and validation by comparison of simulations with experimental data. In Chapter 4 we present a comprehensive comparison of five popular force fields for simulation of insulin. The effect of each force field on the conformational evolution and structural properties of the protein is analysed in detail and compared with available experimental data. A fundamental phenomenon in nature is the ability of proteins to fold ab initio to their functional native conformation, also known as their biologically active state. Due to the heterogeneity and dimensionality of the systems involved, it is necessary to employ methodologies capable of accelerating rare events, specifically, configurational changes that involve the crossing of large free energy barriers. In Chapter 5, using the recently developed method BE-META we were able to identify the structural transitions and possible folding pathways of insulin. Another interesting phenomenon is the misfolding of proteins causing their aggregation, that may lead to formation of either amorphous compounds or structures of elongated-unbranched morphology known as amyloid fibrils. The deposition of amyloid fibrils in the human body may cause many debilitating diseases such as Alzheimer's and variant Creutzfeldt-Jakob diseases, thus making this field of research important and urgent. The human plasma protein apoC-II serves important roles in lipid transport, and it has been shown to form amyloid-like aggregates in solution. We have performed computational studies to investigate the effect of mutations, such as Met oxidation and the residue substitutions to hydrophobic Val and hydrophilic Gln, on dynamics of apoC-II(60-70) peptide. The conformation features relevant to the amyloidogenic propensities of the peptide were identified and presented in Chapter 6. The involvement of lipids at the various stages of development of amyloid diseases is becoming more evident in recent research efforts. In particular, micellar and sub-micellar concentrations have showed to have different effect on fibril growth and kinetics of native apoC-II and derived peptides. In Chapter 7 we investigated the influences of phospholipids at various concentrations on the structure of apoC-II(60-70) using MD and umbrella sampling methods. The molecular mechanisms of lipid effects on the peptide conformation and dynamics were identified. In Chapter 8 preliminary results on the structural stability of pre-formed oligomeric composites of apoC-II(60-70) peptide of different sizes and arrangements were also presented. The effects of mutation (oxidised Met, Met60Val and Met60Gln) on the most stable cluster was also investigated. To conclude, several ideas for continuation of research in the protein folding and aggregation field are discussed in the Future Work section of this thesis.
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Devlin, Glyn L. "The mechanisms of serpin misfolding and its inhibition." Monash University, Dept. of Biochemistry and Molecular Biology, 2003. http://arrow.monash.edu.au/hdl/1959.1/9469.
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Borgia, Madeleine Bridget Windsor. "Studies of the aggregation and misfolding of titin Ig-like domains." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609256.
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Freer, Rosie. "Molecular origins of tissue vulnerability to aberrant aggregation in protein misfolding diseases." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/275420.
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Neurodegenerative disorders, including Alzheimer’s disease (AD) and Parkinson’s disease (PD), are increasingly common in our ageing society, are remain incurable. A major obstacle encountered by researchers in their attempts to find effective therapies is represented by the current lack of understanding of the molecular origins of these disorders. It is becoming clear that, although the aggregation of specific proteins, including amyloid β (Aβ) and tau in AD and α-synuclein in PD, hallmark these disorders, such behaviour is a consequence of a wider, system-level disruption of protein homeostasis. In order to identify the genetic factors contributing to such a disruption, the transcriptional changes that occur during neurodegenerative disease progression have received considerable scientific attention in recent years. In our approach, we considered another hallmark of these diseases - their characteristic patterns of spreading across the brain - to identify the nature of the transcriptional signature which underlies tissue vulnerability to protein aggregation. By understanding why tissues succumb in their characteristic sequential pattern in neurodegenerative diseases, and why some tissues remain almost completely resistant throughout, we hoped to obtain insight into the molecular origins of these disorders. Our results show that the AD progression can be predicted from a transcriptional signature in healthy brains related to the protein aggregation homeostasis of Aβ, tau, and the wider proteome. We highlight a relationship between a specific subproteome at high risk of aggregation (formed by supersaturated proteins), and the vulnerability to neurodegenerative diseases. We thus identify an AD-specific supersaturated set of proteins - termed the metastable subproteome, whose expression in normal brains recapitulates the staging of AD, with more vulnerable tissues having higher metastable subproteome expression. We find evidence of these vulnerability signatures transcending the tissue level of interrogation, with cellular and subcellular analysis also showing elevated levels of proteins known and predicted to predispose the aberrant aggregation of Aβ and tau. These results characterise the key protein homeostasis pathways in the inception and progression of AD, and establish an approach with the potential to be applied to other protein misfolding diseases, in the brain and beyond.
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Xu, Mingming. "Discovery of inhibitors against a-synuclein aggregation." Thesis, Griffith University, 2020. http://hdl.handle.net/10072/392373.
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Abnormal protein aggregation has been linked to many neurodegenerative diseases, including Parkinson’s disease (PD). The main pathological hallmark of PD is the formation of Lewy bodies and Lewy neurites, both containing the pre-synaptic protein α-synuclein (α-syn). Native α-syn, under normal conditions, exists in a soluble unfolded state but undergoes misfolding and aggregation into toxic aggregates under pathological conditions. Toxic α-syn species can cause oxidative stress, membrane penetration, synaptic and mitochondrial dysfunction, leading to neuronal death and eventually neurodegeneration. Currently, early diagnosis and treatments targeting PD pathogenesis are urgently needed. Given its critical role in PD, α-syn is an attractive target for the development of both diagnostic tools and effective therapeutics.
This thesis consists of a series of published and unpublished papers. In Chapter 1, which was published as a review, the progress towards discovering imaging probes and aggregation inhibitors for α-syn was summarized. Since a key property of such required therapeutic agents is specific binding to the target protein, relevant strategies and techniques in the discovery of α-syn-targeted drugs are discussed. As my PhD project aimed to screen small molecules capable of binding to α-syn specifically and then discover new α-syn aggregation inhibitors from the screened structures, relevant techniques were discussed at the end of Chapter 1. Mass spectrometry was chosen to discover specific α-syn binding molecules as this technique allows rapid detection of direct interactions between molecules and proteins. The materials and methods that were used in the included publications, were summarized in detail in Chapter 2.
To provide sufficient protein for our study, the in-house α-syn having equally good quality as the commercial protein, was successfully generated in Chapter 3. Also, high yield of pure protein can be acquired from medium scale of bacteria culture, saving plenty of time and money for preparing proteins for large-scale screening. The protein expression and purification was a part of the supplementary data in the publication included in Chapter 4, where an automated screening system based on the connection of a mass spectrometer and the auto-sampler from a high performance liquid chromatograph was successfully established. This system allows computer-controlled sample loading and data acquisition with high stability and reproducibility. We first discovered a new inhibitor by screening over 4,300 pure molecules. The new compound, 3-[(3-methoxyphenyl)carbamoyl]-7-[(E)-2-phenylethenyl]-4,7- dihydropyrazolo [1,5-a]pyrimidine-5-carboxylic acid, not only significantly inhibited the misfolding and aggregation of α-syn, protected neuroblastoma cells from α-syn toxicity, but also has a more specific binding site compared with positive controls. The capability of the MS-based screening was further extended to the discovery of active components from natural products (manuscript in submission). A total of 29 marine fractions from our collaborators, were tested by MS and a new cholesterol derivative with significant inhibition of α-syn aggregation, was discovered and isolated from the active fraction. This MS-guided isolation of active components from natural products can also be applied to investigating traditional Chinese medicines with known therapeutic effects.
Post-translational modifications (PTMs) of α-syn, especially enzymatic glycosylation with N-acetylglucosamine (GlcNAc) onto the proteins hydroxylated amino acid residues, have been reported to affect the pathogenic self-assembly of α-syn. As such, manipulation of the proteins’ O-GlcNAcylation statuses has been proposed to offer a therapeutic route toward addressing PD. In Chapter 5, small peptides with different sequences and modification sites were synthesized by our collaborators. In the thioflavin-T assay, which is a golden standard for measuring α-syn aggregation, two peptides with O-GlcNAcylation at the serine site exhibited significant inhibition. Therefore, small glycopeptides that couple the protective effects of O-GlcNAc with the selectivity of recognition sequences may prove useful tools to modulate α-syn aggregation (manuscript under review). Other sources of compounds including new analogs of anle138b, which is a well-studied α-syn aggregation modulator, were evaluated. Two derivatives of anle138b exerted promising effects on the aggregation of α-syn. Interestingly, these synthesized compounds and peptides did not form protein-ligand complexes in the mass spectra, indicating that these molecules, unlike the compounds we discovered in Chapter 4, may interact with α-syn aggregates instead of α-syn monomers. In the last chapter, general conclusions of the thesis were made and future directions were also discussed.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
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Belfiore, Ramona. "Protein Misfolding and Aggregation in Neurodegeneration: In Vitro And In Vivo Study Cases." Doctoral thesis, Università di Catania, 2018. http://hdl.handle.net/10761/4178.
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Neurodegenerative diseases are nowadays increasing in incidence and widely distributed around the world. Despite those disorders show very different symptoms and morbidity, intracellular and extracellular protein misfolding and accumulation appears as a common pathological pathway. In the present thesis work I analyzed two cases of toxic protein deposition involved in ALS and AD. First, I looked at SOD1-G93A mutant protein, whose neuronal deposit is associated to familial and sporadic ALS. The mitochondrial porin VDAC1 has been proposed as a binding target of SOD1 mutant forms to mitochondria. By affinity studies we found that VDAC1 protein specifically binds SOD1-G93A but not wild type SOD1. Notably, it is known that the N-Terminal end of Hexokinase 1 (N-HK1) interacts with VDAC1: thus, we produced a synthetic peptide corresponding to the first 11 aa of human HK1 and tested its action as a potential interfering molecule between VDAC1/SOD1-G93A bond. Both in a protein-protein interaction and in a protein mitochondrial interaction we obtained a decrease of VDAC1/SOD1-G93A binding with respect of the increased N-HK1 peptide concentration. Summarizing, SOD1-G93A binds VDAC1 and impairs HK1 binding and our results suggest for N-HK1 peptide a neuroprotective potential in ALS patients.
The second part of my thesis work was focused on Amyloid and tau protein accumulation in 3xTg-AD mice. In order to study neuropathology and cognitive deficits in (AD), several transgenic models of AD have been identified. Accumulation of Abeta and fibrillary tangles as well as impairments in working and learning memory are age-related hallmark of AD pathology. To produce a progressive characterization of Abeta and tau pathology in 3xTg-AD mice we aged female mice at 2, 6, 12 and 20 months of age. We tested mice in a behavioral assay named Morris Water Maze (MWM) and we used in vitro biochemical assays, to observe Abeta soluble and insoluble fraction as well as tau phosphorylation in both cortex and hippocampus of 3xTg-AD mice. Our data on MWM demonstrate a progressive impairment in learning with a strongly significant difference between 3xTg-AD mice and controls, from 6 months of age. Notably, we also found a progressive increase in both soluble and insoluble Abeta40 and Abeta42, an age dependent tau hyperphosphorylation at specific AD linked phospho-sites, and an intense glial reactivity. Overall, our data confirm that female 3xTg-AD mice consistently show AD-like pathology, therefore this transgenic mouse model can be used as an extremely powerful tool to investigate pathogenic mechanisms underlying Alzheimer s disease.
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Binger, Katrina Jean. "The reversibility of amyloid fibril formation." Connect to thesis, 2009. http://repository.unimelb.edu.au/10187/4912.
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The aggregation of misfolded proteins into amyloid fibrils is implicated in the pathogenesis of several human degenerative diseases, including Alzheimer’s, Parkinson’s and Type II diabetes. Links between the deposition of amyloid fibrils and the progression of these diseases are poorly understood, with much of the current research focused on monomer misfolding and subsequent assembly of oligomers and mature fibrils. This project examines the formation of human apolipoprotein (apo) C-II amyloid fibrils, with a focus on the stability and reversibility of amyloid fibril assembly.The initial stages of the project were to develop a model for apoC-II amyloid fibril formation. This was achieved by analysis of the concentration dependent kinetics of apoC-II amyloid fibril formation, and correlation of these data with the final size distribution of the fibrils, determined by sedimentation velocity experiments. On the basis of these studies, a new reversible model for apoC-II amyloid fibril formation is proposed that includes fibril breaking and re-joining as integral parts of the assembly mechanism. The model was tested by rigorous experimentation, with antibody-labelling transmission electron microscopy providing direct evidence for spontaneous fibril breaking and re-joining.
The development of this model for apoC-II fibril assembly provided the foundation for experiments to investigate factors that promote, inhibit or reverse amyloid fibril formation. Factors that were considered include a molecular chaperone protein, αB-crystallin, and a chemical modification, methionine oxidation. Investigations on the effect of αB-crystallin revealed that the inhibition of apoC-II fibril formation occurs by two distinct mechanisms: transient interaction with monomer preventing oligomerisation, and binding to mature fibrils, which inhibits fibril elongation. Studies on the effect of methionine oxidation on apoC-II fibril formation showed that both the assembly and stability of the fibrils was affected by this modification. ApoC-II contains two methionine residues (Met-9 and Met-60), and upon oxidation of these residues fibril formation was inhibited. In addition, the treatment of pre-formed fibrils with hydrogen peroxide caused dissociation of the fibrils via the oxidation of Met-60, located with the fibril core structural region. The final chapter details the development of antibodies that specifically recognise the conformation of apoC-II amyloid fibrils, which provide the foundation for future studies to examine the role that apoC-II amyloid fibrils play in disease.
Overall, this thesis reveals the dynamic and reversible nature of amyloid fibril formation. New insight is also obtained of the general stability of amyloid fibrils and the processes that may regulate their formation, persistence and disease pathogenesis in vivo.
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Daidone, Isabella. "Folding, misfolding and aggregation of proteins and peptides: a molecular dynamics study." Doctoral thesis, La Sapienza, 2005. http://hdl.handle.net/11573/917339.
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Flagmeier, Patrick. "An interdisciplinary approach to studying mechanistic, structural and toxic features of protein aggregates associated with neurodegenerative disorders." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/274344.
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The misfolding and aggregation of proteins is closely associated with more than fifty human disorders, including Alzheimer's and Parkinson's diseases, all of which are currently incurable and many represent a major threat to human life. The mechanism of protein aggregation is subject to extensive studies. The damaging effects associated with protein aggregation have been attributed to amyloidogenic species that are present during the misfolding process. In particular, oligomeric species are, however, intrinsically difficult to study as a consequence of their low abundance and highly heterogeneous nature. The first chapter of my thesis gives an introduction into the field of protein folding and misfolding with a focus on the study of protein aggregation, and toxic effects relevant to human disorders. The second chapter of my thesis describes the development of a methodology that enables the study of aggregate induced lipid bilayer permeability, possibly the most general mechanism of protein aggregate toxicity. Surface-tethered lipid vesicles functioning as optochemical probes sensitive to membrane integrity are imaged using total internal reflection microscopy. It is shown that oligomeric species of the 42-residue form of the Aβ peptide (Aβ42) are responsible for the membrane disruption. The methodology can be applied to the study of other proteins such as α-synuclein and tau, and the ability of antibodies and chaperones to counteract the aggregate induced lipid bilayer permeability can be assessed. Furthermore, lipid bilayer permeability induced by aggregates formed in human induced pluripotent stem cells can be studied. The third chapter presents a new approach for the measurement of protein aggregation kinetics by following the development of the lipid bilayer permeability over the course of the aggregation process of Aβ42. The aggregation kinetics can be modulated with molecular chaperones and pre-formed seed fibrils, which allows secondary nucleation to be identified as the process that drives the formation of species responsible for the lipid bilayer permeability. The fourth chapter describes the development of a three-pronged strategy to study the mechanism of α-synuclein amyloid formation. The aggregation is studied in the presence of lipid vesicles or pre-formed fibrils at neutral or acidic pH of the solution. The influence of single-point mutations on the aggregation of α-synuclein is described. Furthermore, the strategy is applied to the characterisation of the ability of antibodies and small molecules to inhibit the aggregation, and thus has the potential for the development of therapeutical agents. The work presented in the fifth chapter characterises the amyloid fibril populations formed by α-synuclein and mutational variants associated with familial Parkinson's disease. X-ray crystallography, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy and atomic force microscopy have all been applied to the analysis of these amyloid fibrils. Finally, the sixth chapter summarises the results described in this thesis and points out future opportunities in the context of fundamental and translational studies related to the research area of protein misfolding disorders.
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Wiberg, Henning. "Analytical Approaches to Neurodegenerative Disease Protein Aggregation." Licentiate thesis, KTH, Analytisk kemi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-34027.
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Lapinska, Urszula. "Microfluidics and chemical kinetics to analyse protein interactions, aggregation, and physicochemical properties." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/284929.
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Proteins play a major role in living systems and present a wide spectrum of functionalities. Many different types of proteins are involved into biological processes, such as the catalysis of biochemical reactions, cellular membrane transport, immune system response and DNA replication. However, some proteins and peptides might become harmful to living organisms; for example, their abnormal aggregation causes neurodegenerative disorders including Alzheimer disease (AD). One of the causes of AD is the presence of amyloid beta peptides Aβ(1-42), Aβ(1-40), which self-assemble into insoluble fibrils and plaques, which surround neuronal cells impeding synapsis. The number of AD patients is increasing, but a cure has not been founded yet. Therefore, it is crucial to investigate the mechanisms underlying amyloid aggregation and screening for compounds able to prevent this irreversible process. Microfluidics permits characterising the physicochemical properties of proteins, investigate their aggregation and study their interactions with other molecules. Chemical kinetics allows studying the microscopic events occurring during protein self-assembly. The combination of these two techniques provides a powerful tool for the identification of compounds inhibiting the aggregation process. In this thesis by using microfluidics, chemical kinetics and other biophysical assays, I have investigated the proteins isoelectric point (pI) and the inhibition of aberrant Aβ(1-42) self-assembly process. Firstly, I describe the development of a microfluidic platform allowing for the measurement of the protein pI, in a gradient-free manner. This approach overcomes a fundamental limitation of convectional techniques that is the achievement of a stable and well-controlled pH gradient. Secondly, I investigate the inhibiting effect of llama nanobodies on Aβ(1-42) aggregation. The findings from this study show that nanobodies target monomeric species with high affinity whereas interactions with fibril surfaces are weak. Finally, I discuss the use of other compounds inhibiting specific nucleation stages. These include the chaperones clusterin and brichos, as well as soot and pure carbon nanoparticles. Importantly, the addition of both chaperones to Aβ(1-42) solutions has an additive inhibitory effect on aggregation. My findings will improve the characterization of the physicochemical properties of proteins as well as providing promising candidates for the inhibition of specific stages of amyloid beta aggregation opening the way to possible cures for AD disease.
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CHOUDHARY, DHAWAL. "Studio a livello di singola molecola del folding, misfolding e aggregazione di proteine e dell’attività chaperonica della HSPB8." Doctoral thesis, Università degli studi di Modena e Reggio Emilia, 2020. http://hdl.handle.net/11380/1199862.
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Negli ultimi decenni le pinze ottiche si sono rivelate una tecnica sperimentale estremamente efficace per eseguire studi di spettroscopia di forza a livello di singola molecola. In particolare, un’applicazione delle pinze ottiche che sta avendo una rilevanza biomedica sempre più importante è quella relativa allo studio dei processi di ripiegamento corretto (folding), non corretto (misfolding) e dell’aggregazione di proteine. Di forte rilevanza biomedica è anche la possibilità offerta dalle pinze ottiche di caratterizzare in grande dettaglio i meccanismi molecolari che mediano le interazioni tra due o più biomolecole, come ad esempio tra uno chaperone molecolare e il suo substrato. La rilevanza medica di questi studi deriva dal fatto che l'errato ripiegamento e l'aggregazione delle proteine sono processi deleteri, spesso associati a neurodegenerazione. Gli chaperoni molecolari si sono evoluti come strumento molecolare per combattere sia il misfolding che l’aggregazione proteica. Un funzionamento non corretto degli chaperoni molecolari spesso causa perdita di proteostasi e l’insorgenza di varie patologie umane. Il lavoro descritto in questa tesi spiega in maniera dettagliata l’approccio sperimentale utilizzato per utilizzare le pinze ottiche per lo studio del folding, misfolding e aggregazione di proteine. In particolare in questa tesi vengono descritti: i) i risultati di esperimenti mirati alla elucidazione del processo di ripiegamento corretto e non del sensore al calcio NCS-1 (Neuronal Calcium Sensor 1; ii) l'approccio sperimentale adottato per descrivere la dinamica strutturale e funzionale di vari chaperoni molecolari utilizzando le pinze ottiche e la microscopia a forza atomica; iii) recenti sviluppi tecnici che hanno ampliato le possibili applicazioni delle pinze ottiche in campo biologico; iv) i risultati di esperimenti mirati a far luce sui meccanismi molecolari che mediano l’attività chaperonica dello chaperone molecolare HSPB8. In quest’ultimi esperimenti abbiamo manipolato meccanicamente monomeri e tetrameri della Maltose Binding Protein (MBP) e analizzato i loro processi di folding, misfolding e aggregazione in presenza e assenza del HSPB8 wild-type e del suo mutante HSPB8-K141E. I nostri risultati dimostrano una forte attività antiaggregante (holdase activity) della HSPB8 che riduce significativamente l'aggregazione delle molecole di MBP e un’attività antiaggregante molto ridotta del mutante HSPB8-K141E. Inoltre, i nostri studi rivelano una inaspettata attività pro-folding (foldase activity) sia della forma mutata che di quella wild-type della HSPB8. Questi dati sperimentali evidenziano nuovi meccanismi di interazione tra HSPB8 e i suoi substrati e suggeriscono un ruolo fisiologico più complesso per questo chaperone molecolare di quanto precedentemente ipotizzato.Optical tweezers have evolved as an exemplary Single Molecule Force Spectroscopy (SMFS) technique over the past three decades. A distinct and bio medically relevant application of Optical Tweezers is their ability to observe directly at single molecule level the folding, misfolding and aggregation of protein molecules. Additionally the dynamic approach of Optical Tweezer setup also allows for the isolated study of interactions between two or more biomolecules, such as chaperone-protein interactions, in real time. The medical relevance of such studies stems from the fact that misfolding and aggregation of proteins are deleterious processes and have been linked to many neurodegenerative disorders. While molecular chaperones have evolved as an evolutionarily conserved sword and shield mechanism against such deleterious processes, wherein their holdase action acts as a shield preventing further aggregation of misfolded protein species and their foldase action acts as a sword and actively assists misfolded structure to regains their natively folded state. The dysfunction of this chaperone activity is also cytotoxic and can lead to loss of proteostasis. The present thesis dwells deeper in this specific application of Optical tweezer. The thesis will elaborate upon how optical tweezers can extract the mechanistic details of the folding and misfolding of protein molecules by reviewing the experiments performed on NCS-1 (Neuronal Calcium Sensor 1). It will also discuss the experimental approach taken by SMFS techniques like Optical Tweezers and AFM (Atomic Force Microscopy) to study the structural and functional dynamics of molecular chaperones. Furthermore, the thesis will explore the recent developments in Optical Tweezers and their biological applications. Finally, I describe the results of experiments we have carried out on the maltose binding protein to elucidate the mechanism of action of the chaperone HSPB8. We have mechanically denatured homotetramers of MBP as well as single MBP molecules and analyzed their folding and aggregation processes in the presence and absence of wild-type HSPB8 and its mutant form HSPB8-K141E/N. Our results reveal a strong holdase activity of wild type HSPB8, which either prevents completely the aggregation of denatured MBP molecules or allows the substrate to form only small and mechanically weak aggregates while this holdase activity is significantly suppressed in the mutant. Moreover, and importantly, a careful analysis of the data also discloses an unexpected foldase activity of both wild type and mutated forms of HSPB8, which guides the folding process of denatured MBP molecules into their native states. Our findings highlight new mechanisms of interaction between HSPB8 and its substrates and suggest a more complex physiological role for this chaperone than previously assumed.
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Villar, i. Piqué Anna. "Characterization of intracellular protein aggregates." Doctoral thesis, Universitat Autònoma de Barcelona, 2013. http://hdl.handle.net/10803/120241.
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Durant les últimes dècades, l'agregació de proteïnes ha esdevingut un tema d’investigació molt dinàmic que s'estén transversalment per diferents camps de recerca, incloent la bioquímica, la biotecnologia, la biomedicina i la nanotecnologia. D'una banda, l'acumulació de proteïnes en dipòsits amiloids insolubles constitueix una característica comuna de molts trastorns humans, coneguts com a malalties conformacionals. D'altra banda, des d'un punt de vista biotecnològic, l’agregació proteica representa un obstacle habitual en la producció de proteïnes recombinants, que en general s'acumulen en forma de cossos d’inclusió intracel·lulars. Malgrat que els cossos d’inclusió han estat tradicionalment considerats partícules desestructurades i amb molt poc interès, nombroses evidències indiquen que aquests agregats contenen estructura de tipus amiloid, la qual cosa aplana el camí per a emprar-los en l'estudi de l’agregació amiloid.
La tesi que aquí es presenta recapitula la feina pertanyent a una sèrie de publicacions relatives a l’agregació amiloid de proteïnes en l'espai intracel·lular. En tres d'aquests treballs, s'utilitzen tres models cel·lulars diferents filogenèticament distants (bacteris, llevats i plantes) per abordar l’estudi de la formació de dipòsits proteics amb l'objectiu de caracteritzar-los i analitzar-ne el seu impacte en el metabolisme cel·lular. En una publicació complementària, explotem els agregats bacterians per tal de desenvolupar un assaig de screening in vitro per trobar moduladors de l’agregació amiloid. Finalment, també s'inclouen dues obres de revisió sobre la deposició de proteïnes en bacteris i el paper d’aquest organisme com a model per a l'estudi de l'agregació amiloid.
Les dades obtingudes de tots aquests estudis indiquen que l'agregació en conformació amiloid és una propietat genèrica dels polipèptids i un fenomen omnipresent a la natura. No obstant, l'aparició d'aquests dipòsits en l'entorn cel·lular pot anar acompanyada d'un cert grau de toxicitat. Aquí, analitzem l'efecte d'envelliment promogut pels cossos d'inclusió intracel·lulars en cèl·lules bacterianes. A més, descrivim com les cèl·lules procariotes i eucariotes estan dotades d'una poderosa maquinària de qualitat proteica que permet fer front a aquesta situació perjudicial. Addicionalment, la caracterització en profunditat dels agregats proteics en bacteris i del seu procés de formació permet utilitzar-los com a eina en la recerca d'inhibidors de l'agregació amiloid amb potencial interès biomèdic i farmacèutic.
En general doncs, aquesta tesi aprofundeix en l'estudi de l'agregació amiloid de proteïnes i amplia el coneixement per a emprar organismes simples com models cel·lulars rellevants.During the last decades, protein aggregation has become a dynamic research topic extending across distinct investigation fields, including biochemistry, biotechnology, biomedicine and nanotechnology. On one side, the accumulation of proteins into insoluble amyloid deposits constitutes a common hallmark of many human disorders, known as conformational diseases. On the other side, from a biotechnological point of view, protein deposition is regarded as a usual hindrance in the production of recombinant proteins, which generally assemble into intracellular inclusion bodies. Although inclusion bodies were traditionally considered unstructured particles lacking of interest, the increasing number of evidences indicating that these aggregates contain amyloid-like structure pave the way for employing them in the study of amyloid deposition. The thesis presented here recapitulates the work belonging to a set of publications concerning amyloid protein aggregation in the intracellular space. In three of these works, we use three distinct cellular models phylogenetically distant (bacteria, yeast and plants) to address the formation of protein deposits with the aim to characterize them and to analyze their impact in the cellular metabolism. In a complementary publication, we exploit bacterial aggregates to develop an in vitro screening assay for amyloid modulators. Finally, we also include two revision works about protein deposition in bacteria and its role as model in the study of amyloid aggregation. The data collected from these studies indicate that aggregation into amyloid structures is a general property of polypeptides and a ubiquitous phenomenon in Nature. However, the apparition of these deposits in the cellular environment can be accompanied by a certain degree of toxicity. Here, we analyze the aging effect promoted by intracellular inclusion bodies in bacterial cells. In addition, we report how both prokaryotic and eukaryotic cells are endowed with a powerful protein quality machinery to challenge this damaging scenario. Moreover, the deep characterization of bacterial protein aggregates and their formation process permits their use as a tool in the searching for amyloid aggregation inhibitors with putative biomedical and pharmaceutical interest. Overall, this thesis delves into the study of amyloid protein aggregation and adds insights to employ simple organisms as relevant cellular models.
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Gragg, Megan Ellen. "Mutant Rhodopsins in Autosomal Dominant Retinitis Pigmentosa Display Variable Aggregation Properties." Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1522935340252319.
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Vecchi, Giulia. "Proteomics studies of protein homeostasis and aggregation in ageing and neurodegeneration." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/273348.
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Upon ageing, a progressive disruption of protein homeostasis often leads to extensive protein aggregation and neurodegeneration. It is therefore important to study at the proteome level the origins and consequences of such disruption, which so far have remained elusive. Addressing this problem has recently become possible by major advances in mass spectrometry-based (MS) proteomics, which allows the identifications and quantification of thousands of proteins in a variety of biological samples. In the first part of this thesis, I analyse proteome-wide MS data for the nematode worm C. elegans upon ageing, in wild type (WT), long-lived and short-lived mutant strains. By comparing the total abundance and the soluble abundance for nearly 4000 proteins, I provide extensive evidence that proteins are expressed in adult worms at levels close to their solubility limits. With the use of sequence-based prediction tools, I then identify specific physico-chemical properties associated with this age-related protein homeostasis impairment. The results that I obtained reveal that the total intracellular protein content remains constant, in spite of the fact that the proteome undergoes wide remodeling upon ageing, resulting into severe protein homeostasis disruption and widespread protein aggregation. These results suggest a protein-dependent decrease in solubility associated with the protein homeostasis failure. In the second part of the thesis, I determine and classify potential interactions of misfolded protein oligomers with other proteins. This phenomenon is widely believed to give rise to cytotoxicity, although the mechanisms by which this happens are not fully understood. To address this question, I process and analyse MS data from structurally different oligomers (toxic type A and nontoxic type B) of the protein HypF-N, incubated in vitro with proteins extracted from murine cell cultures. I find that more than 2500 proteins are pulled down with the misfolded oligomers. These results indicate that the two types of oligomers interact with the same pool of proteins and differ only in the degree of binding. Functional annotation analysis on the groups reveals a preference of the oligomers to bind proteins in specific biological pathways and categories, including in particular mitochondrial membrane proteins, RNA-binding proteins and molecular chaperones. Overall, in this study I complement the powerful and high-throughput experimental approach of MS proteomics with bioinformatics analyses and prediction algorithms to define the physical, chemical and biological features of protein homeostasis disruption upon ageing and the interactome of misfolded oligomers.
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Roostaee, Alireza. "Importance of dimerization in aggregation and neurotoxicity of Prion and [alpha]-Synuclein in prion and Parkinson's diseases." Thèse, Université de Sherbrooke, 2012. http://hdl.handle.net/11143/6650.
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Abstract: Neurodegenerative diseases are associated with progressive loss of structure or function of neurons which results in cell death. Recent evidence indicate that all neurodegenerative disorders, sporadic or transmissible, may have a common pathological mechanism at the molecular level. This common feature consists of protein aggregation and accumulation of harmful aggregates in neuronal cells resulting in cellular apoptosis and neurotoxicity. Neurodegenerative diseases can affect abstract thinking, skilled movements, emotional feelings, cognition, memory and other abilities. This diverse group of diseases includes Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), prion diseases or transmissible spongiform encephalopathies (TSEs) and amyotrophic lateral sclerosis. In my project I worked on the molecular mechanism of protein aggregation, propagation and neurotoxicity in Parkinson's disease and prion disease. Prion disease and PD are associated with misfolding and aggregation of PrPc and a-Synuclein (a-Syn), respectively. Despite being two important neurodegenerative disorders, molecular mechanisms of a-Syn or PrPC aggregation and amyloidogenesis are still unclear in PD and prion disease. Furthermore, the toxic protein species in PD have not been characterized yet. In this study we characterize the mechanism of a-Syn and PrPc misfolding in a physiological-like cell free condition in the absence of a-Syn aggregates, PrPc ggregated isoform (Pre's), denaturants or acidic environment. A number of studies indicate that dimerization of PrPc or a-Syn may be a key step in the aggregation process. To test this hypothesis we verified if enforced dimerization of PrPc or a-Syn may induce a conformational change reminiscent of the conversion of PrPc or a-Syn to PrPR' or a-Syn aggregates, respectively. We used a well-described inducible dimerization strategy where a dimerizing domain called FK506-binding protein (Fv) was fused to PrPc or a-Syn in order to produce chimeric proteins Fv-PrP and a-SynF'''. A divalent ligand AP20187 was used to induce protein dimerization. Addition of AP20187 to recombinant Fv-PrP in physiological-like conditions resulted in a rapid conformational change characterized by an increase in beta-sheet (13-Sheet) structure and simultaneous aggregation of the proteins. However, non-dimerized PrP formed 13-Sheet conformation in very slower rates. In the presence of AP20187, we also report a rapid random coil into 13-sheet conformational transformation of a-SynF" within 24 h, whereas wild type a-Syn showed 24 h delay to achieve P-sheet structure after 48 h. Electron microscopy experiments demonstrated that dimerization induced amyloid fibril formation after 48 h for both Fv-PrP and a-Syr?", whereas in the absence of dimerizing ligand AP20187, PrP or a-Syn converted into amyloid fibrils after 3 days or even later. Dimerization-induced Fv-PrP aggregates were partially resistant to PK digestion which is a characteristics of the naturally occurring PrPR'. The rates of amyloidogenesis in the presence of dimerization was also characterized by Thioflavin T (ThT) fluorescence probing. Whereas the stable structure of Fv-PrP showed no ThT binding for over 60 h of incubation at 37°C, the addition of AP20187 to Fv-PrP resulted in a time-dependent increase in ThT binding. As for a-SynR, dimerization accelerated the rate of ThT binding and amyloid formation comparing to the slower amyloidogenesis rate of wild type a-Syn in the absence of dimerizer AP20187. The impact of dimerization on a-Syn aggregation was further determined by Fluorescence ANS probing, indicating a higher affinity of dimerization-induced a-SynF" aggregates for binding to ANS comparing to wild type a-Syn aggregates. These results indicate that dimerization increases the aggregation and amyloidogenesis processes for Fv-PrP and a-SynF". Both Fv-PrP and a-SynF" amyloids were successfully propagated in vitro by protein misfolding amplification (PMCA) cycle. These results ar in agreement with the theory that all protein aggregates in neurodegenerative diseases propagate with the same molecular mechanism. Neurotoxicity of recombinant Fv-PrP and a-SynF" aggregates was determined in cellulo and in vivo, respectively. Aggregates of Fv-PrP were toxic to cultured cells whilst soluble Fv-PrP and amyloid fibres were harmless to the cells. When injected to the mice brain, both a-Syni" and a-Syn pre-fibrillar aggregates internalized cells and induced neurotoxicity in the hippocampus of wild-type mice. These recombinant toxic aggregates further converted into non-toxic amyloids which were successfully amplified by PMCA method, providing the first evidence for the in vitro propagation of synthetic a-Syn aggregates. These results suggest an important role for protein dimerization in aggregation and amyloidogenesis, and therefore, in the pathology of PD and prion disease. The similarities between aggregation, amyloidogenesis and toxicity of PrPC and ct-Syn provide further evidence on the existance of a prion-like mechanism in all neurodegenerative disorders. // Résumé: Les maladies neurodégénératives sont associées à la perte progressive des propriétés structurales ou fonctionnelles des neurones, ce qui engendre la mort des cellules. De récentes études indiquent que tous les désordres neurodégénératifs, sporadiques ou transmissibles, peuvent avoir un mécanisme pathologique commun au niveau moléculaire. Ce dispositif commun se compose de l'agrégation de protéines, de la propagation des agrégats, et de l'accumulation d’agrégats toxiques dans les cellules neuronales, menant à l'apoptose et à la neurotoxicité cellulaire. Les maladies neurodégénératives peuvent affecter la pensée abstraite, les mouvements habiles, les sentiments émotifs, la connaissance, la Mémoire et d'autres capacités cognitives. Ce groupe divers de maladies inclut la maladie d'Alzheimer (AD), de Parkinson (PD), de Huntington (HD), les maladies à prions ou encéphalopathies spongiformes transmissibles (TSEs) et la sclérose latérale amyotrophique (ALS). [symboles non conformes]
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Salazar, Vázquez Lilian Shadai. "How protein misfolding can lead to cellular dysfunction and disease : the case of islet amyloid polypeptide involved in type 2 diabetes mellitus." Thesis, Sorbonne université, 2019. https://accesdistant.sorbonne-universite.fr/login?url=http://theses-intra.upmc.fr/modules/resources/download/theses/2019SORUS371.pdf.
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Pour avoir une fonction biologique, une protéine se replie dans une structure spécifique. La cellule contrôle le repliement correct des protéines et dispose de mécanismes pour détecter et éliminer les protéines mal repliées. Néanmoins, certaines protéines évitent ce processus de contrôle. Les protéines amyloïdes sont des protéines mal repliées qui forment un type caractéristique de fibrilles amyloïdes allongées; en fonction de la séquence protéique et du site de dépôt de l'amyloïde, ils sont liés à différentes maladies. Le polypeptide amyloïde d'îlot (IAPP), un peptide de 37 acides aminés coproduit et co-sécrété avec l'insuline par les cellules β, est impliqué dans le diabète de type 2 et appartient à ce groupe de protéines amyloïdes. Les fibrilles sont formées dans l'îlot pancréatique. Cependant, les conditions dans lesquelles les fibres sont formées et leur cytotoxicité dans d'autres cellules sont encore inconnues. Nous montrons ici que les peptides flanquants de l'IAPP humain, produits au cours de la maturation de l'hIAPP, des extrémités N-terminale et C-terminale ne sont pas amyloïdogènes et que la toxicité de l'IAPP humain dans différentes lignées cellulaires. Nous trouvons par des tests biophysiques ThT, TEM et CD que les résidus N-terminaux et C-terminaux de l'IAPP ne forment pas de fibrilles en solution, dans les vésicules artificielles ou dans les cellules et ne modifient pas l'effet toxique de l'hIAPP. Pour la toxicité de hIAPP, nous utilisons les lignées Ins-1 (cellules β), SHSY5 (neuronales), F442A et 3T3L1 (adipeuses), mhAT3F (hépatique) et C2C12 (muscle). Nous observons la formation de fibrilles dans toutes les lignées cellulaires, mais la toxicité n’est pas directement liée à la présence de fibrilles. Nous nous attendons à ce que notre test soit le point de départ d'autres études in vitro sur différentes lignées cellulaires. En outre, l’inhibition de la fibrille de l’IAPP pourrait être une cible pour le développement de médicaments anti-amyloïdesTo have a biological function, a protein folds into a specific structure. The cell controls the correct folding of the proteins and has mechanisms to detect and eliminate misfolded proteins; nevertheless some proteins achieve to avoid this control process. Amyloid proteins are misfolded proteins that form a characteristic type of elongated amyloid fibril; depending on the protein sequence and the site of amyloid deposition they are related to different human diseases. Islet amyloid polypeptide (IAPP) a 37 amino acid peptide co-produced and co-secreted with insulin by β-cells, is involve in type 2 diabetes disease and belongs to this group of amyloid proteins. The fibrils are formed in the pancreatic islet. However the conditions under which the fibers are formed and their cytotoxicity in other cells are still unknown. Here we show that the human IAPP flanking peptides, produced during hIAPP maturation, N-terminal and C-terminal are not amyloidogenic and the toxicity of human IAPP in different cell lines. We find through biophysical assays ThT, TEM and CD that N-terminal and C-terminal residues of IAPP do not form fibrils in solution, in artificial vesicles or in cells and do not modify hIAPP toxic effect. For the toxicity of hIAPP we use Ins-1 (β-cells), SHSY5 (neuronal), F442A and 3T3L1 (adipose), mhAT3F (hepatic) and C2C12 (muscle) lines. We observe fibril formation in all cell lines, however the toxicity do not related directly with the presence of fibril. We anticipate our assay to be a starting point for more in vitro studies in different cells lines. Furthermore, IAPP fibril inhibition could be a target for anti-amyloid drug development
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Robinson, Philip John. "The folding, misfolding and aggregation of prions." Thesis, University of Warwick, 2009. http://wrap.warwick.ac.uk/2792/.
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Prion diseases are a group of fatal neurodegenerative disorders that include Creutzfeldt-Jakob Disease (CJD), Bovine Spongiform Encephalopathy (BSE) and scrapie, which are all associated with the misfolding of the cellular form of the prion protein, PrPC, into the disease associated isoform, PrPSc. This thesis investigates two properties of PrP that may influence the misfolding process; (i) the normal folding mechanism of PrP and (ii) the interactions of PrP with lipid membranes. Firstly, equilibrium folding experiments investigate whether the folding pathway of PrP is influenced by a disease modulating mutation, Q167R, which confers disease resistance. The unfolding of PrPWt is compared to PrPQ167R by monitoring fluorescence and circular dichroism of folding sensitive tryptophan mutants. The results show that the mutation significantly destabilises the protein, which can be rationalised from high resolution structures of PrP. Furthermore, comparison of the folding of mouse and hamster PrP highlights dramatic differences between their folding pathways, which may contribute to the species barrier that is observed in prion disease transmission. The second part of the thesis studies the influence of membrane environments on prion conversion. Firstly, the interaction between PrP and lipid membranes composed of POPC (a zwitterionic phospholipid) and POPS (an anionic phospholipid), are investigated through fluorescence, circular dichroism and centrifugation binding assays. The results show that PrP interacts peripherally with POPC membranes, without significant changes in protein structure. In contrast, high affinity binding to POPS membranes, results in membrane penetration and an increase in β-sheet structure. Furthermore, cryo-electron microscopy reveals that the PrPmembrane interaction disrupts the native vesicle structure and results in the formation of membrane junctions. Finally the morphology and mechanism of growth of prion aggregates on supported lipid bilayers are studied through atomic force microscopy, which shows how the phospholipid content of membranes directs prions down alternative aggregation pathways.
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Subramaniam, Vinod. "Biophysics of protein misfolding." Enschede : University of Twente [Host], 2007. http://doc.utwente.nl/58042.
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Almstedt, Karin. "Protein Misfolding in Human Diseases." Doctoral thesis, Linköpings universitet, Biokemi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-21077.
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There are several diseases well known that are due to aberrant protein folding. These types of diseases can be divided into three main categories: Loss-of-function diseases Gain-of-toxic-function diseases Infectious misfolding diseases Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to inherited mutations. The rare disease marble brain disease (MBD) also known as carbonic anhydrase II deficiency syndrome (CADS) can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. We have over the past 10-15 years studied the folding, misfolding and aggregation of the enzyme human carbonic anhydrase II. In summary our HCA II folding studies have shown that the protein folds via an intermediate of molten-globule type, which lacks enzyme activity and the molten globule state of HCA II is prone to aggregation. One mutation associated with MBD entails the His107Tyr (H107Y) substitution. We have demonstrated that the H107Y mutation is a remarkably destabilizing mutation influencing the folding behavior of HCA II. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II towards populating ensembles of intermediates of molten globule type under physiological conditions. The enormously destabilizing effects of the H107Y mutation is not due to loss of specific interactions of H107 with residue E117, instead it is caused by long range sterical destabilizing effects of the bulky tyrosine residue. We also showed that the folding equilibrium can be shifted towards the native state by binding of the small-molecule drug acetazolamide, and we present a small molecule inhibitor assessment with select sulfonamide inhibitors of varying potency to investigate the effectiveness of these molecules to inhibit the misfolding of HCA II H107Y. We also demonstrate that high concentration of the activator compound L-His increases the enzyme activity of the mutant but without stabilizing the folded protein. The infectious misfolding diseases is the smallest group of misfolding diseases. The only protein known to have the ability to be infectious is the prion protein. The human prion diseases Kuru, Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob are characterized by depositions of amyloid plaque from misfolded prion protein (HuPrP) in various regions of the brain depending on disease. Amyloidogenesis of HuPrP is hence strongly correlated with prion disease. Our results show that amyloid formation of recHuPrP90-231 can be achieved starting from the native protein under gentle conditions without addition of denaturant or altered pH. The process is efficiently catalyzed by addition of preformed recHuPrP90-231 amyloid seeds. It is plausible that amyloid seeding reflect the mechanism of transmissibility of prion diseases. Elucidating the mechanism of PrP amyloidogenesis is therefore of interest for strategic prevention of prion infection.
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Li, W. "Investigation and manipulation of SOD1 mutant misfolding, aggregation and seeding." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/1558299/.
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The presence of ubiquitylated protein aggregates in neurons and surrounding cells is considered one of the hallmarks of neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). Since the identification of SOD1 as the first causative gene in 1993, extensive research has been carried out to investigate the role Cu/Zn superoxide dismutase-1 (SOD1) aggregation plays in ALS pathogenesis. Recently, it has been reported that SOD1 inclusions could propagate in a prion-like manner, by seeding the aggregation of soluble functional proteins and transmitting aggregation to neighbouring cells. HSJ1 (DnaJB2) is a chaperone that can reduce protein aggregation in several neurodegenerative disease models; such as, Huntington’s disease and Parkinson’s disease. HSJ1a overexpression has also been shown to improve motor neuron survival in an animal model of SOD1-ALS. In this study, I tested the hypothesis that HSJ1 could alter SOD1-aggregation and seeding in vitro and in cell models. I developed an in vitro system with purified proteins to explore the tendency of SOD1 wild type (SOD1WT) and mutants (SOD1MT) to aggregate and seed further aggregation. The results showed that SOD1WT is resistant to seeding unless the protein is destabilized and partially unfolded. Purified HSJ1 could reduce SOD1 aggregation. I then developed transient and inducible cell models to investigate the effects of HSJ1 on SOD1 aggregate formation and expansion. In cells, HSJ1 interacted preferentially with SOD1MT, and could reduce SOD1 inclusion formation, and disassembled pre-existing SOD1 inclusions. Using an inducible stable cell line expressing HSJ1a, unfolded protein response (UPR) markers were modestly reduced after ER stress, suggesting HSJ1a expression could potentially reduce disease-related intracellular stress. Collectively, these findings shed light on HSJ1 as a potential candidate targeting misfolded and aggregated SOD1 for future investigation.
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Schmittschmitt, Jason Peter. "Biophysical characterization of protein folding and misfolding." Texas A&M University, 2003. http://hdl.handle.net/1969.1/46.
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The HPr proteins were characterized as folding by a two-state folding mechanism. Here, we present a comparison of the equilibrium and kinetic folding for the HPr protein from Bacillus subtilis, E coli and a key variant from these proteins. For the wild-type protein we find that GHX is greater than GUDC, suggesting that the HPr does not fold by a simple two-state mechanism. This discrepancy is revealed by testing the two-state nature of the folding reaction of HPr with mutation. We show that removing a single charge side chain (Asp 69) converts the HPr protein back to a simple two-state mechanism.
Ribonuclease Sa and two charge-reversal variants can be converted into amyloidin vitro by the addition of 2,2,2-triflouroethanol (TFE). We report here amyloid fibril formation for these proteins as a function of pH. The pH at maximal fibril formation correlates with the pH dependence of protein solubility, but not with stability, for these variants. Additionally, we show that the pH at maximal fibril formation for a number of ivwell-characterized proteins is near the pI, where the protein is expected to be the least soluble. This suggests that protein solubility is an important determinant of fibril formation.
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Guest, William Clay. "Template-directed protein misfolding in neurodegenerative disease." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/41990.
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Protein misfolding diseases represent a large burden to human health for which only symptomatic
treatment is generally available. These diseases, such as Creutzfeldt-Jakob disease, amyotrophic
lateral sclerosis, and the systemic amyloidoses, are characterized by conversion of globular, nativelyfolded
proteins into pathologic β-sheet rich protein aggregates deposited in affected tissues. Understanding
the thermodynamic and kinetic details of protein misfolding on a molecular level depends
on accurately appraising the free energies of the folded, partially unfolded intermediate,
and misfolded protein conformers. There are multiple energetic and entropic contributions to the
total free energy, including nonpolar, electrostatic, solvation, and configurational terms. To accurately
assess the electrostatic contribution, a method to calculate the spatially-varying dielectric
constant in a protein/water system was developed using a generalization of Kirkwood Frohlich theory
along with brief all-atom molecular dynamics simulations. This method was combined with
previously validated models for nonpolar solvation and configurational entropy in an algorithm to
calculate the free energy change on partial unfolding of contiguous protein subsequences. Results
were compared with those from a minimal, topologically-based Gō model and direct calculation
of free energies by steered all-atom molecular dynamics simulations. This algorithm was applied
to understand the early steps in the misfolding mechanism for β₂-microglobulin, prion protein,
and superoxide dismutase 1 (SOD1). It was hypothesized that SOD1 misfolding may follow a
template-directed mechanism like that discovered previously for prion protein, so misfolding of
SOD1 was induced in cell culture by transfection with mutant SOD1 constructs and observed to
stably propagate intracellularly and intercellularly much like an infectious prion. A defined minimal
assay with recombinant SOD protein demonstrated the sufficiency of mutant SOD1 alone
to trigger wtSOD1 misfolding, reminiscent of the “protein-only” hypothesis of prion spread. Finally,
protein misfolding as a feature of disease may extend beyond neurodegeneration and amyloid
formation to cancer, in which derangement of protein folding quality control may lead to antibodyrecognizable
misfolded protein present selectively on cancer cell surfaces. The evidence for this
hypothesis and possible therapeutic targets are discussed as a future direction.
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Suk, Ji Young. "Molecular consequences of protein misfolding mutations in FBN1." Thesis, University of Oxford, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270282.
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Roboti, Peristera. "Disease-related misfolding of the myelin proteolipid protein." Thesis, University of Manchester, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.493943.
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A wide range of mutations in the myelin integral plasma membrane proteolipid protein (PLP) are associated with dysmyelinating diseases of varying severity, and whilst missense mutations in PLP transmembrane domains cause severe disease few such mutants result in a mild phenotype. The molecular pathology of such diseases has generally been attributed to endoplasmic reticulum (ER) retention of misfolded ing in the induction of ER stress. However, the cellular mechanism(s) that control the observed phenotypic variations have not yet been elucidated. The work documented in this thesis established that the cellular fate of three distinct transmembrane missense mutants of PLP is differentially regulated by the ER quality control process upon stable inducible expression in HeLa cells.
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Nerelius, Charlotte. "Protein misfolding and amyloid formation : strategies for prevention /." Uppsala : Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, 2009. http://epsilon.slu.se/200941.pdf.
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Locker, C. Rebecca. "Minimalist models of proteins : misfolding and folding affinity." Diss., Georgia Institute of Technology, 2003. http://hdl.handle.net/1853/27573.
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Pokrishevsky, Edward. "Induction of wild-type SOD1 misfolding, aggregation and its cell-to-cell propagation." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/61079.
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Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration and loss of motor neurons that appears to spread through the neuroaxis in a spatiotemporally restricted manner. Misfolded Cu/Zn superoxide dismutase (SOD1) has been detected in all ALS patients, despite SOD1 mutations accounting for only 2% of total cases, while the presence of inclusions containing pathological TAR-DNA binding protein-43 (TDP-43) represent a hallmark of all non-SOD1/FUS familial ALS. We previously reported that TDP-43 and FUS can trigger misfolding of human wild-type SOD1 (HuWtSOD1) in living cells, however the mechanisms and consequences are unknown. Here, we used immunocytochemistry, immunoprecipitation and cell viability studies to demonstrate that TDP-43 or FUS-induced misfolded HuWtSOD1 can propagate from cell-to-cell via conditioned media, and seed cytotoxic misfolding of endogenous HuWtSOD1 in the recipient cells in a prion-like fashion. Knockdown of SOD1 using siRNA in recipient cells, or incubation of conditioned media with misfolded SOD1-specific antibodies, inhibits intercellular transmission, indicating that HuWtSOD1 is an obligate seed and substrate of propagated misfolding. Furthermore, we developed several chimeric SOD1-GFP proteins that we validated to aggregate in the presence of pathological SOD1 or TDP-43 seed. We used this assay, along with immunofluorescence, live-cell microscopy and flow cytometry studies, to show that intermolecular conversion of SOD1 by pathological TDP-43 is mediated by tryptophan residues in both proteins. Furthermore, we used the reporter proteins to show that human spinal cord extracts prepared from familial, but not sporadic, ALS patients can trigger SOD1 aggregation in cultured cells. Finally, we used this system to show that small molecules, akin to 5-fluorouridine, can block this intermolecular kindling of SOD1 aggregation, and demonstrated that our assay can be used as a high-throughput tool for screening drugs against induced SOD1 aggregation. Altogether, our studies indicate that pathological TDP-43 and FUS may exert motor neuron pathology in ALS through the initiation of tryptophan-dependent propagated SOD1 misfolding. Furthermore, it is key to recognize that elucidation of the pathogenic role of a simple structural motif in ALS may provide a framework for understanding other neurodegenerative diseases in which propagated protein misfolding is shown to occur.Medicine, Faculty of
Experimental Medicine, Division of
Medicine, Department of
Graduate
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Lane, Fiona Mary. "Defining mechanisms of neurodegeneration associated with protein misfolding diseases." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/19542.
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Protein misfolding diseases (PMDs) are a broad group of disorders including Alzheimer’s, Parkinson’s and prion diseases. They are characterised by the presence of aggregated, misfolded host proteins which are thought to cause cell death. Prion diseases are associated with misfolded prion protein (PrPSc), which has a tendency to form fibrillar aggregates. By contrast, Alzheimer’s disease (AD) is associated with misfolded amyloid beta (Aβ), which aggregates to form characteristic Aβ plaques. A feature which is common across PMDs is that small assemblies (oligomers) of the misfolded proteins are thought to be the important neurotoxic species, and it has been proposed that there may be a shared mechanism leading to cell death across PMDs caused by oligomers. In this study, the toxicity of different misfolded forms of recombinant PrP (recPrP) and recombinant Aβ (recAβ) and the mechanisms leading to cell death were investigated using a primary cell culture model. In addition, the importance of the disulphide bond in recPrP in relation to oligomer formation was explored using size exclusion chromatography and mass spectrometry, the toxicity of the different resulting oligomer populations were also investigated. Both recPrP oligomers and fibrils were shown to cause toxicity to mouse primary cortical neurons. Interestingly, oligomers were shown to cause apoptotic cell death, while the fibrils did not, suggesting the activation of different pathways. By contrast, recAβ fibrils were shown to be non-toxic to cortical neurons, Aβ oligomers, however, were shown to cause toxicity. Similar to recPrP, my data showed that it is likely that recAβ 1-42 oligomers also cause apoptosis. However, by contrast this seemed to be caused by excitotoxicity, which was not found to be the case for recPrP. Additionally, I have shown that the presence or absence of the disulphide bond in PrP has a profound effect on the size of oligomers which form. RecPrP lacking a disulphide bond leads to the formation of larger oligomers which are highly toxic to primary neurons. Findings from this study suggest that structural properties such as the disulphide bond in PrP can affect the size and toxicity of oligomers, furthermore, whilst oligomers have been shown to be important in both AD and prion diseases, they may not trigger the same pathways leading to cell death.
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Lang, Lisa. "SOD1 Aggregation : Relevance of thermodynamic stability." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-139943.
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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the upper and lower motor neurons causing muscle atrophy and paralysis followed by death. Aggregates containing superoxide dismutase (SOD1) are found as pathological hallmark in diseased ALS patients. Consequently ALS is regarded as a protein misfolding disorder like Alzheimer’s disease and Parkinson’s disease. So far, little is known about the cause and mechanism behind SOD1 aggregation but the inherent property of all polypeptide chains to form stable aggregated structures indicates that the protein misfolding diseases share a common mechanism. Our results show that SOD1 aggregation starts from the globally unfolded state, since fibrillation is fastest at full occupancy of denatured protein induced either by chemical denaturation or mutation. Even so, the fibrillation rate shows a surprisingly weak dependence on the concentration of globally unfolded SOD1 indicating fibril fragmentation as the dominant mechanism for aggregate formation. This is further supported by the observation that the SOD1 sample has to be mechanically agitated for fibrillation to occur. Interestingly, we observe a similar SOD1 aggregation behaviour in vivo, where the survival times of ALS transgenic mice correlates with mutant stability, and aggregate growth depends weekly on the concentration of unfolded monomer. Additionally, in-cell NMR measurements reveal that in live cells the thermodynamic equilibrium is shifted towards the unfolded state of SOD1, which is also more fully extended than in vitro. This suggests that the globally unfolded aggregation competent protein is more abundant in the crowded environment in vivo than dilute in vitro conditions. Finally, antibody analysis of aggregates from ALS transgenic mice reveals the existence of aggregate strains involving different parts of the protein depending on mutation, which may offer an explanation for the various disease phenotypes observed in ALS. Altogether these findings provide important clues for understanding SOD1 aggregation with implications for ALS, as well as other protein misfolding diseases.
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Leinartaité, Lina. "Zinc in folding and misfolding of SOD1 : Implications for ALS." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-107543.
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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease causing degeneration of upper and lower motor neurons. Most ALS cases are sporadic; only 6% are associated with mutations in Cu, Zn superoxide dismutase (SOD1). It is believed, however, that sporadic and familiar forms of ALS share a common mechanism, where SOD1 plays an important role: SOD1 knockout mice do not develop ALS, whereas the overexpression of human SOD1 in mice produces ALS-like symptoms. Increasing evidence suggest that the SOD1 structure gains cytotoxic properties, but detailed description of the toxic species is missing. This thesis work is focused on understanding how structural and dynamic properties of SOD1 change along its folding free-energy landscape and indicates the structural hot-spots from where the cytotoxic species may originate. Thus, binding of the zinc controls folding, stability and turnover of SOD1: (i) miscoordination of Zn2+ by the Cu-ligands speeds up folding of the SOD1 core structure, however, it stabilizes SOD1 in a state where both active-site loops IV and VII are unfolded, (ii) coordination of Zn2+ in the Zn-site, induces the folding of loop VII and stabilizes the native and functional fold of both active-site loops and (iii) the tremendous stability gain due to Zn-site metallation corresponds to a folded state’s lifetime of > 100 years, thus the cellular lifetime of SOD1 is likely controlled by Zn2+ release, which again is coupled to opening of active-site loops. Hence the active-site loops IV and VII stand out as critical and floppy parts of the SOD1 structure. Moreover, a number of ALS-associated mutations, benign to apo-SOD1 stability, are shown here to affect integrity of active-site loops in holo-SOD1, which, in turn, increases population of SOD1 species with these loops disorganized. Finally, the close relation between SOD1 and Zn2+ can also act in the reverse direction: a perturbed folding free-energy landscape of SOD1 can disturb Zn2+ homeostasis.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.
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Scheinost, Johanna C. "A cholesterol oxidative metabolite and its role in protein misfolding." Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.504523.
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Griffiths-Jones, Samuel R. "Peptide models for protein beta-sheets." Thesis, University of Nottingham, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364650.
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Münch, Christian. "Initiation and propagation of mutant superoxide dismutase 1 misfolding." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609791.
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Nuhu, Mariam. "Protein-protein interactions and aggregation in biotherapeutics." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/proteinprotein-interactions-and-aggregation-in-biotherapeutics(1dba3d89-1474-486c-9eb9-6e21b4616dd9).html.
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Protein aggregation is a frequently cited problem during the development of liquid protein formulations, which is especially problematic since each protein exhibits different aggregation behaviour. Aggregation can be controlled by judicious choice of solution conditions, such as salt and buffer type and concentration, pH, and small molecule additives. However, finding conditions is still a trial and error process. In order to improve formulation development, a fundamental understanding of how excipients impact upon protein aggregation would significantly contribute to the development of stable protein therapeutics. The underlying mechanisms that control effects of excipients on protein behaviour are poorly understood. This dissertation is directed at understanding how excipients alter the conformational and colloidal stability of proteins and the link to aggregation. This knowledge can be used for finding novel ways of either predicting or preventing/inhibiting protein aggregation. Experiments using static and dynamic light scattering, intrinsic fluorescence, turbidity and electrophoretic light scattering were conducted to study the effect of solution conditions such as pH, salt type and concentration on protein aggregation behaviour for three model systems: lysozyme, insulin and a monoclonal antibody. Emphasis is placed on understanding the effects of solution additives on protein-protein interactions and the link to aggregation. This understanding has allowed the rational development of stable formulations with novel additives, such as arginine containing dipeptides and polycations.
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Outeiro, Tiago Fleming de Oliveira. "Yeast as a model organism to study diseases of protein misfolding." Doctoral thesis, Universidade do Porto. Reitoria, 2004. http://hdl.handle.net/10216/10638.
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O'Connor, Matthew. "Ruminant prion disease detection and characterisation using protein misfolding cyclic amplification." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/41599/.
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Prion diseases or transmissible spongiform encephalopathies (TSE) are characterised by the accumulation of a misfolded conformer (PrPSc) of a host encoded protein (PrPC). The misfolding event that leads to the formation PrPSc can be replicated in the in vitro amplification technique, protein misfolding cyclic amplification (PMCA). This thesis focuses on the application PMCA to study multiple aspects of prion misfolding in relation to ruminant prion diseases, specifically developing techniques to detect and characterise PrPSc in scrapie and BSE infections. Utilising recombinant hamster PrP (rPrP) as substrate in PMCA, multiple genotypes of scrapie were successfully amplified in an attempt to describe a quantifiable technique applicable to a wide range of scrapie isolates. Observations of non-specific protease resistant rPrP formation was investigated with modifications to the PMCA methodology, which ultimately proved unsuccessful in reducing non-specific protease resistant rPrP. Using brain PrPC as substrate, the quantitative PMCA technique was piloted with BSE to correlate in vitro replication efficiency with infectious titre in mouse bioassay, but no correlation was identified. Atypical forms of BSE occur primarily in older cattle, are asymptomatic and thought to be spontaneous diseases. None the less, infection models in rodents and primates have identified the zoonotic potential of H-type and L-type BSE. Therefore PMCA methods were developed which were able to successfully amplify both atypical forms of BSE. In particular, sensitive detection and discrimination from classical BSE was demonstrated for H-type BSE, which has not previously been amplified in PMCA. H-type BSE could be detected in 1x10¬-12 g brain material and was discriminated from classical BSE by increased protease sensitivity, relatively high molecular weight and antibody reactivity. Evidence exists for co-infection of TSE strains, yet scrapie and BSE co-infection in an ovine host remains unaddressed. To study the disease progression and tissue dissemination of co-infections a PMCA assay capable of specifically amplifying BSE PrPSc in the presence of excess scrapie was applied to artificially mixed brain homogenates containing BSE and scrapie, and compared to current statutory strain typing methods. The PMCA was found to have sensitivity and specificity of 100% in mixes containing 0.1% BSE and 99.9% scrapie brain material, which was more effective than conventional strain typing methods. The assay was then applied to the brain, spleen and lymph of scrapie and BSE experimental co-infections in two genotypes of sheep, and to animals which belonged to a flock with endemic natural scrapie and that also received experimental BSE infections. The PMCA data demonstrated that sheep with PRNP genotype ARQ/ARQ (at amino acid positions 134, 154 and 171) were resistant to BSE in a co-infection scenario. In sheep with PRNP genotype of VRQ/ARQ, mixed infections could occur, and animals with scrapie PrPSc only in the brain could harbour BSE PrPSc in peripheral tissues. Co-infection was also possible in sheep with natural scrapie infections. The assay was compared to conventional testing methods of western blotting, PrPd profiling and immunohistochemistry and displayed superior sensitivity in BSE detection. PMCA amplification of bovine BSE isolates in ovine substrates identified several instances in which the molecular characteristics of the PrPSc was scrapie-like in terms of molecular weight, antibody reactivity and glycoform profile, and in some cases PrPSc characteristic of BSE could no longer be recovered. This occurred in a genotype specific manner, ‘molecular switching’ was only apparent in ovine substrate VRQ/VRQ in accordance with previous findings. These results raise the possibility of such an event occurring in in vivo ovine BSE infections and the zoonotic potential of these scrapie like conformers are yet to be fully addressed.
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Outeiro, Tiago Fleming de Oliveira. "Yeast as a model organism to study diseases of protein misfolding." Tese, Universidade do Porto. Reitoria, 2004. http://hdl.handle.net/10216/10638.
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Kemplen, Katherine Rosemary. "Biophysical studies of folding and misfolding in tandem repeat proteins." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709235.
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Gurry, Thomas. "Order, disorder, and protein aggregation." Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/97347.
Full textAbstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2015.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 114-124).
Protein aggregation underlies a number of human diseases. Most notably, it occurs widely in neurodegenerative diseases, including Alzheimer's and Parkinson's. At the molecular level, neurotoxicity is thought to originate from toxic gains of function in multimeric aggregates of proteins that are otherwise predominantly monomeric and disordered, fluctuating between a very large number of structurally dissimilar states on nano- and microsecond timescales. These proteins, termed Intrinsically Disordered Proteins (IDPs), are notoriously difficult to probe using traditional biophysical techniques. In order to obtain structural information pertaining to the aggregation of IDPs, it is often necessary to develop computational and modeling tools, both to leverage the full extent of the experimental data, and to generate testable predictions for future experiments. In this thesis, I present three separate computational studies studying the formation of multimeric aggregates in IDPs, spanning different aspects of the aggregation process, from early nucleation events to fibril elongation. In the first study, I present a conformational ensemble of a-synuclein, the culprit protein of Parkinson's disease, constructed using a Variational Bayesian Weighting algorithm in combination with NMR data collected by our collaborators. We find that the data fit a description in which the protein predominantly exists as a disordered monomer but contains small quantities of multimeric states containing both helical and strand-rich conformations. In the second study, I focus on the process of amyloid fibril elongation in the Amyloid-[beta] (A[beta]) peptide of Alzheimer's disease. I compute the free energy surface associated with the fibril elongation reaction, and find that elongation of both A[beta]40 and A[beta]42 experimental fibril structures occurs on a downhill free energy pathway, proceeding via an obligate, fibril-associated hairpin intermediate. The fibril-associated hairpin is significantly more stable (relative to the fibrillar, elongated state) in A[beta]42 compared with A[beta]40, suggesting a potential clinical target of interest. Finally, I present lengthy, all-atom molecular simulations that suggest that nucleation of the minimum aggregating fragment of c-synuclein proceeds via a helical intermediate, requiring a structural conversion into a strand-rich nucleating species via a stochastic process of individual helices unfolding and self-associating via backbone hydrogen bonds.
by Thomas Gurry.
Ph. D.
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Knowles, Tuomas Pertti Jonathan. "Physical aspects in protein aggregation." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612258.
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White, Duncan Alexander. "Biophysical features of protein aggregation." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609696.
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Last, Alexander M. "Intermediates in protein folding and misfolding revealed by electrospray ionisation mass spectrometry." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393383.
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Lemin, Andrew James. "The unfolded protein response and HLA-B27 misfolding : implications for ankylosing spondylitis." Thesis, Durham University, 2010. http://etheses.dur.ac.uk/797/.
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The unfolded protein response (UPR) detects the presence of misfolded proteins in the endoplasmic reticulum (ER) and subsequently relieves ER stress by increasing the folding capacity of the ER. The secretory pathway substrate HLA-B27 is highly associated with the chronic inflammatory disease ankylosing spondylitis (AS) and has a tendency to misfold in the ER. Here, we show that overexpression of HLA-B27 and non-disease associated HLA-B7 in immortalised cell lines leads to heavy chain misoxidation, which is accompanied by upregulation of BiP and splicing of XBP1, a key step in the IRE1 pathway of the UPR which is increasingly being linked with intestinal inflammation. We also demonstrate that different cell lines respond to different ER stress stimuli in distinct ways. We establish that HT1080 cells inefficiently induce a UPR in response to tunicamycin and that this has consequences for cell survival. However, inefficient activation of the UPR in HT1080 cells can be overcome by secondary signals, since co-administration of the tyrosine kinase inhibitor genistein leads to activation of XBP1. Furthermore, we show that genistein can inhibit UPR induction of BiP in response to a range of ER stresses indicating that the cancer drug genistein can inhibit or activate the UPR depending on the environment and cell type. This has implications for inflammatory disease since regulation of the UPR is important in determining a cell’s tendency towards apoptosis.
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Bastidas, Oscar. "Computational Study of Protein-Protein Interactions in Misfolded States." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3521.
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Protein-protein interactions (PPI’s) play important roles in biological systems. In particular, intra-protein interactions help create and maintain correctly folded protein states and mutations that result in misfolded states may be associated with significant changes in PPI behavior. Six unrelated protein systems with known structure files, each consisting of a wild-type and mutant strain, were studied using the computational algorithm OpenContact©. OpenContact© is a simple tool that can be used to rapidly identify or map interactions “hot-spots” in a protein and was, consequently, used in this study as a starting point to examine the potential or possible role of PPI’s on the behavior of mutated, misfolded proteins. Specific results include the observations of single chain protein systems exhibiting mutant strains with significantly stronger inter-atomic interactions as well as a surprising gain of secondary structure in the mutant state. These observations stood in contrast to multi-chain systems (proteins with more than two constituent chains) that appeared to display stronger inter-atomic interactions for the wild-type strains. Results also indicated a potential classification scheme for intra-protein interaction behavior in mutated states based on several criteria. It is important to note, however, that observations on PPI behavior presented need to be verified across a greater number of systems than those studied here before any such trends can be concretely established.
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Barber, Michael. "Mechanisms and prevention of protein aggregation." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:924a4f23-a2d3-49af-b201-f29295bdf442.
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The deposition of amyloid in the central nervous system is associated with prevalent neurological disorders such as Alzheimer's and Parkinson's disease. This thesis studies the mechanisms and prevention of amyloid formation in vitro. We specifically focus on Parkinson's disease associated α-synuclein (α-syn). Using novel labeling methods we introduce NMR observable labels onto lysosomal protein glucocerebrosidase (GCase), a leading cause of Parkinson's disease. By introducing NMR active labels we are able to study GCase dynamics and screen potential drug therapeutics (chapter 3). Furthermore, we analyze the three way interaction between GCase, α-syn and lipids. We conclude that GCase is able to effectively chaperone α-syn under lysosomal conditions, both preventing amyloidogenesis and destabilizing mature amyloid fibrils (chapter 4). Additionally, a model chaperone-aggregate system is investigated to gain insight into the mechanisms of small heat shock protein chaperoning, and how such mechanisms prevent aggregation (chapter 5). Finally, a high resolution crystal structure of RNA editing enzyme Cid1 is presented, whilst not directly linked to aggregation, many of the techniques used in this thesis were first developed on Cid1 (chapter 7). Together, we utilize NMR, X-ray crystallography, electron microscopy and native mass spectrometry to elucidate aspects of protein aggregation mechanisms and prevention.
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Wain, Rachel. "Studies of protein folding and aggregation." Thesis, University of Oxford, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270186.
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Wilkins, Deborah K. "Studies of protein denaturation and aggregation." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325146.
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