Dissertations / Theses on the topic 'Misfolding disease'
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1
Guest, William Clay. "Template-directed protein misfolding in neurodegenerative disease." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/41990.
Full textAbstract:
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.
2
Lane, Fiona Mary. "Defining mechanisms of neurodegeneration associated with protein misfolding diseases." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/19542.
Full textAbstract:
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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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.
Full textAbstract:
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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Cristofani, R. M. "PROTEIN MISFOLDING IN KENNEDY¿S DISEASE AND IN RELATED MOTOR NEURON DISEASES (MNDS)." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/339901.
Full textAbstract:
Motor neuron diseases, like spinobulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS) are characterized by the presence of inclusions or aggregates of proteinaceous materials. In SBMA, inclusions are formed by testosterone dependent aggregates of mutant androgen receptor (AR) with an elongated polyglutamine tract (ARpolyQ), while in ALS inclusions contain several aggregated proteins including TDP43, ubiquilin, optineurin. Exceptions are familial ALS forms linked to superoxide dismutase 1 (SOD1) mutations, to mutated TDP43 and to C9ORF72 poly-dipeptides (DPRs), in which aggregates are mainly composed of mutant SOD1, mutant TDP43 or DPRs, respectively. In general, protein aggregation is due to generation of aberrant protein conformations (misfolding) combined to a failure, in neuronal cells, of the protein quality control (PQC) system, which may be insufficient to correctly remove the misfolded proteins. In other target tissue, such as the muscles, a different physiological PQC regulation may be helpful to remove misfolded proteins related to MNDs.
The PQC system requires the activities of chaperones, degradative systems ubiquitin- proteasome (UPS) and autophagy. After misfolded protein recognition by chaperones, the dynein motor complex plays a crucial role to efficiently remove these species via autophagy, transporting them to autophagosome and assisting autophagosome- lysosome fusion.
In this thesis, I have investigated the implications of protein misfolding in SBMA and in ALS. Taking advantage of a comparative analysis of misfolded proteins response in skeletal muscle and in spinal cord of SMBA mice, we proved that autophagy is dramatically perturbed in muscles. Indeed, we found the up-regulation of most autophagic markers (Beclin-1, ATG10, p62/SQSTM1, LC3). In addition, the chaperon small Heat Shock Protein B8 (HSPB8) and its co-chaperone BCL2-Associated Athanogene 3 (BAG3), required for autophagy, were robustly up-regulated together with other specific HSPB8 interactors (HSPB2 and HSPB3). Interestingly, the BAG3:BAG1 ratio, increased in muscle, suggesting preferential misfolded proteins routing to autophagy rather than to proteasome. Misfolded proteins, recognized by HSPB8-BAG3 complex, are actively transport by dynein to MTOC to be inserted in autophagosome and degraded by autophagy,
Then, we analysed the role of dynein mediate transport in the autophagic removal of misfolded proteins. In immortalized motoneuronal NSC34 cells, we found that the reduction of dynein protein levels, obtained using a specific siRNA, resulted in autophagy inhibition and in unexpected testosterone dependent ARpolyQ aggregates reduction. Also, we found that pharmacological dynein inhibition, with erythro-9-(2- Hydroxy-3-nonyl) adenine hydrochloride (EHNA), in NSC34 cells expressing ARpolyQ, mutant SOD1, truncated TDP43 form or C9ORF72 DPRs, induced a great reduction of mutant protein aggregates, even in presence of an autophagy inhibitor (3-MA), but not of a proteasome inhibitor (MG132). By performing fractionation studies we found that EHNA increased the ARpolyQ levels in PBS and Triton-X100 fractions. Surprisingly, we found that ENHA effects were paralleled by an increased expression of BAG1, a co- chaperone which routes misfolded proteins to UPS, but not of BAG3 suggesting the prevalence of UPS functions. Indeed, when dynein activity was blocked, BAG3:BAG1 ratio was decreased, thus in favour of BAG1 expression, suggesting the involvement of the pro-degradative activity of BAG1 on ARpolyQ aggregates. Collectively, these data show that mutant ARpolyQ induces a potent autophagic response in muscle cells. This may be useful to evaluate the SBMA progression.
In parallel, dynein blockage perturbs autophagy and modifies the response of PQC system to misfolded protein. This results in reduced aggregation of MNDs-related misfolded proteins, a phenomenon that may occurs via an increase in their solubility and the induction of UPS functions.
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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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Sajjad, Muhammad Umar. "Regulation of the redox homeostasis during polyglutamine misfolding in Huntington's Disease." Thesis, University of Southampton, 2010. https://eprints.soton.ac.uk/168315/.
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Huntington's Disease (HD) is one of many neurodegenerative diseases that are associated with protein misfolding, aggregation and oxidative stress. While several changes in the redox homeostasis have been shown to occur in HD animal models and HD brains, the formal relationships between intracellular protein misfolding that occurs in HD, redox dysregulation and cellular toxicity are unknown. Therefore, several cellular models of intracellular polyglutamine (polyQ) protein misfolding were established for mechanistic studies. Various in vitro transient and stable cell expression systems expressing an N-terminal fragment of huntingtin (htt) (httExon 1, httEx1) with/or without a polyQ expansion and fused to fluorescent proteins were characterized. Mutant httEx1 (mhttEx1) constructs expressed in both neuronal and non-neuronal cell lines produced early polyQ aggregates and intracellular inclusion bodies (IBs) followed by cell toxicity that increased over time in time-course experiments. Using oxidation-sensitive probes, reactive oxygen species (ROS) were measured in polyQ-expressing cells using single, live-cell imaging analysis by confocal microscopy or population assays in order to explore the relationship between polyQ aggregation, ROS production and cellular toxicity. This study highlighted an early increase in ROS due to the expression of aggregation-prone mhttEx1 in both transient and stable cellular systems that coincided with polyQ aggregation, but preceded cell death. Suppression of ROS and toxicity was achieved by two antioxidant compounds (L-NAC and Trolox). Moreover, the use of MitoQ (Coenzyme Q10 covalently attached to triphenylphosphonium cation (TPP+)) at nanomolar concentrations abrogated the increased ROS due to mhttEx1 suggesting a mitochondrial origin of ROS. Given that molecular chaperones regulate the folding/misfolding of proteins and are involved in the regulation of the cellular redox homeostasis, the role of the redoxactivatable chaperone DJ-1 in HD was investigated. Protein expression analysis in HD cell models, a rodent model of HD and human HD brain samples showed an up-regulation of DJ-1 protein expression compared to control samples. Oxidation of DJ-1 was also elevated in the human HD cortex. To test for a functional role of DJ-1 elevation and oxidation in HD, DJ-1 was overexpressed with wild-type or mhttEx1 in cell lines and mouse primary astrocytes. Overexpression of DJ-1 accelerated mhttEx1 aggregation and toxicity both of which could be suppressed by exposure of cells to mild oxidants suggesting that DJ-1, when redox-activated to a chaperone, modulates polyQ aggregation and toxicity. This hypothesis was tested by overexpression of mhttEx1 with a DJ-1 mutant lacking a critical redox activatable cysteine (Cys106). The C106S-DJ-1 mutant lost its ability to reduce polyQ aggregation and toxicity under oxidising conditions upon co-expression with mhttEx1 suggesting that DJ-1 indeed functions as a modulator of polyQ misfolding and toxicity. Together this work suggests that ROS may be produced during polyQ aggregation and is involved in cellular toxicity. This study also shows that DJ-1 regulates both, polyQ aggregation and toxicity in cell models and given the increased DJ-1 expression in vitro and in vivo (human HD), this protein could be a potential target for HD therapy.
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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.
Full textAbstract:
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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Trist, Benjamin. "Superoxide dismutase 1 in the aetiology of Parkinson’s disease." Thesis, The University of Sydney, 2019. http://hdl.handle.net/2123/20579.
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Parkinson’s disease is the most common neurodegenerative movement disorder worldwide, and is the fastest growing neurological disease ahead of Alzheimer’s disease. Characteristic motor dysfunction results from the selective death of dopamine neurons in the substantia nigra pars compacta, however the aetiology of this neuron loss remains unknown. As such, current treatments help to alleviate Parkinsonian motor symptoms, but none are able to slow or halt the rate of dopaminergic neuron loss. A greater understanding of the molecular pathways leading to dopamine neuron death will accelerate the development of disease-modifying treatments that slow or halt neurodegeneration in this disorder. This thesis focusses on three focal points within the parkinsonian degenerative cascade; oxidative stress, copper dyshomeostasis, and protein misfolding, and aims to introduce the antioxidant copper-binding protein, superoxide dismutase 1 (SOD1), as an important nexus between these key pathologies. I describe, for the first time, misfolding and dysfunction of SOD1 localized to degenerating brain regions in Parkinson’s disease, which is significantly associated with both Lewy proteinopathy and neuron death in these regions. Importantly, I provide evidence that the development of this pathology precedes nigral dopamine neuron loss, and is therefore likely to be a causative factor in, rather than a result of, neurodegeneration in Parkinson’s disease. The sole use of post-mortem tissues within this study ensured any identified biochemical changes accurately reflected endogenous changes occurring within PD and ALS patients; a major criticism of current model systems aiming to recapitulate PD and ALS pathology. My work proposes SOD1 may constitute a novel target for therapeutic interventions aiming to slow the rate of dopaminergic neuron loss in Parkinson’s disease. Novel SOD1 proteinopathy in the Parkinson’s disease brain bears remarkable similarities to neurotoxic SOD1 proteinopathy in a proportion of familial amyotrophic lateral sclerosis (fALS) patients, suggesting similar pathways to neuron death in both disorders. The absence of SOD1 gene mutations in Parkinson’s disease patients exhibiting substantial SOD1 proteinopathy strengthens data from SOD1-fALS research demonstrating that non-genetic factors play key roles in SOD1 misfolding and dysfunction, especially biometal dyshomeostasis and oxidative stress. I demonstrate that these factors may also underlie the misfolding of soluble wild-type SOD1 protein in the vulnerable ventral spinal cord in non-SOD1-fALS and sALS patients, and propose that SOD1 toxicity arises in these patients irrespective of the formation of large insoluble misfolded SOD1 deposits. Importantly, shared pathways to neurodegeneration in Parkinson’s disease and ALS identified within this thesis highlight the potential for the translation of therapeutic approaches targeting SOD1, already in clinical trials for ALS, into disease-modifying therapies for Parkinson’s disease.
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Kundra, Rishika. "Homeostasis of metastable proteins in Alzheimer's disease." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/268485.
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Alzheimer’s disease (AD) is the most common cause of dementia, affecting almost 40 million people worldwide, and it is predicted that this number will rise to nearly 150 million by 2050. It results not only in enormous distress for affected individuals and carers but also a substantial economic burden on society. Although more than 100 years have passed since its discovery, no cure for AD exists, despite enormous efforts in basic and clinical research over the past few decades, due to limited understanding of its underlying mechanisms. Neurodegenerative disorders, of which AD is an example, are highly complex disorders characterized by extensive neuronal dysfunction associated with the misfolding and aggregation of a specific set of proteins, including amyloid plaques and neurofibrillary tangles in AD. One promising avenue for progress in the field is to improve our understanding of the mechanisms by which cellular dysfunction arises from the initial protein aggregation events. The studies described in the thesis are based on the recent finding that a large number of proteins are inherently supersaturated, being expressed at concentrations higher than their solubilities, and constituting a metastable subproteome potentially susceptible to aggregation. These studies illustrate the dependence of aggregation prone metastable proteins on the cellular degradation machineries. They also study the role of metastable proteins and their homeostasis complement in the vulnerability of various body and brain tissues to protein aggregation diseases. Using extensive sequencing data and network based systems biology approaches, they elucidate how fundamental physicochemical properties of an individual or group of proteins relate to their biological function or dysfunction.
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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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Sörgjerd, Karin. "Molecular Aspects of Transthyretin Amyloid Disease." Doctoral thesis, Linköpings universitet, Biokemi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-12566.
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This thesis was made to get a deeper understanding of how chaperones interact with unstable, aggregation prone, misfolded proteins involved in human disease. Over the last two decades, there has been much focus on misfolding diseases within the fields of biochemistry and molecular biotechnology research. It has become obvious that proteins that misfold (as a consequence of a mutation or outer factors), are the cause of many diseases. Molecular chaperones are proteins that have been defined as agents that help other proteins to fold correctly and to prevent aggregation. Their role in the misfolding disease process has been the subject for this thesis. Transthyretin (TTR) is a protein found in human plasma and in cerebrospinal fluid. It works as a transport protein, transporting thyroxin and holo-retinol binding protein. The structure of TTR consists of four identical subunits connected through hydrogen bonds and hydrophobic interactions. Over 100 point mutations in the TTR gene are associated with amyloidosis often involving peripheral neurodegeneration (familial amyloidotic polyneuropathy (FAP)). Amyloidosis represents a group of diseases leading to extra cellular deposition of fibrillar protein known as amyloid. We used human SH-SY5Y neuroblastoma cells as a model for neurodegeneration. Various conformers of TTR were incubated with the cells for different amounts of time. The experiments showed that early prefibrillar oligomers of TTR induced apoptosis when neuroblastoma cells were exposed to these species whereas mature fibrils were not cytotoxic. We also found increased expression of the molecular chaperone BiP in cells challenged with TTR oligomers. Point mutations destabilize TTR and result in monomers that are unstable and prone to aggregate. TTR D18G is naturally occurring and the most destabilized TTR mutant found to date. It leads to central nervous system (CNS) amyloidosis. The CNS phenotype is rare for TTR amyloid disease. Most proteins associated with amyloid disease are secreted proteins and secreted proteins must pass the quality control check within the endoplasmic reticulum (ER). BiP is a Hsp70 molecular chaperone situated in the ER. BiP is one of the most important components of the quality control system in the cell. We have used TTR D18G as a model for understanding how an extremely aggregation prone protein is handled by BiP. We have shown that BiP can selectively capture TTR D18G during co-expression in both E. coli and during over expression in human 293T cells and collects the mutant in oligomeric states. We have also shown that degradation of TTR D18G in human 293T cells occurs slower in presence of BiP, that BiP is present in amyloid deposition in human brain and mitigates cytotoxicity of TTR D18G oligomers.Denna avhandling handlar om proteiner. Särskilt de som inte fungerar som de ska utan har blivit vad man kallar ”felveckade”. Anledningen till att proteiner veckas fel beror ofta (men inte alltid) på mutationer i arvsmassan. Felveckade proteiner kan leda till sjukdomar hos människor och djur (man brukar tala om amyloidsjukdomar), ofta av neurologisk karaktär. Exempel på amyloidsjukdomar är polyneuropati, där perifera nervsystemet är drabbat, vilket leder till begränsad rörelseförmåga och senare till förlamning; och Alzheimer´s sjukdom, där centrala nervsystemet är drabbat och leder till begränsad tankeförmåga och minnesförluster. Studierna som presenteras i denna avhandling har gått ut på att få en bättre förståelse för hur felveckade proteiner interagerar med det som vi har naturligt i cellerna och som fungerar som skyddande, hjälpande proteiner, så kallade chaperoner. Transtyretin (TTR) är ett protein som cirkulerar i blodet och transporterar tyroxin (som är ett hormon som bland annat har betydelse för ämnesomsättningen) samt retinol-bindande protein (vitamin A). I TTR genen har man funnit över 100 punktmutationer, vilka har kopplats samman med amyloidsjukdomar, bland annat ”Skellefteåsjukan”. Mutationer i TTR genen leder ofta till att proteinet blir instabilt vilket leder till upplösning av TTR tetrameren till monomerer. Dessa monomerer kan därefter sammanfogas på nytt men denna gång på ett sätt som är farligt för organismen. I denna avhandling har fokus legat på en mutation som kallas TTR D18G, vilken har identifierats i olika delar av världen och leder till en dödlig form av amyloidos i centrala nervsystemet. Det chaperon som vi har studerat benämns BiP och är beläget i en cellkomponent som kallas för det endoplasmatiska retiklet (ER). I ER finns cellens kontrollsystem i vilket det ses till att felveckade proteiner inte släpps ut utan istället bryts ned. Denna avhandling har visat att BiP kan fånga upp TTR D18G inuti celler och där samla mutanten i lösliga partiklar som i detta fall är ofarliga för cellen. Avhandligen har också visat att nedbrytningen av TTR D18G sker mycket långsammare när BiP finns i riklig mängd.
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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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Guilliams, Tim Thomas. "Nanobodies as tools to gain insights into [alpha]-synuclein misfolding in Parkinson's disease." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608094.
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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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Weise, A. "Structural investigations of intrinsically disordered polypeptides : biosynthesis on the ribosome and protein misfolding disease." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1472901/.
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Intrinsically disordered proteins constitute a significant proportion of our proteome and are emerging targets of modern structural and molecular biology through their involvement in a range of functions crucial for the survival of the cell. Their malfunction and aggregation is known in many cases to lead to devastating pathologies including Parkinson’s disease (α-synuclein, αSyn) and amyloid lateral sclerosis (TAR DNA- binding protein, TDP-43). NMR spectroscopy, is a high resolution structural technique that is especially well suited to provide residue-specific information on flexible and unstructured systems, and which is used to investigate the structural and dynamic aspects of αSyn and TDP-43. TDP-43, is a eukaryotic, 440-residue, multi-domain protein with a predicted disordered C-terminal tail (CTD274-414) that initiates the aggregation of TDP-43 and harbours the disease-associated mutations. This thesis describes the development of an expression and purification strategy and the NMR investigations of the structure and dynamics of the CTD274-414 region both in isolation and as it exists within living cells. Using these methods we have initially obtained the complete protein backbone assignment of the CTD274-414 under 8 M urea conditions, which indicated a mostly disordered conformation, serving now as a valuable template to determine the secondary structure propensity of this domain within E. coli lysate. In addition, the aggregation behaviour of the CTD274-414 was investigated using biophysical techniques, which revealed that while the CTD274-414 aggregates appear to have fibrillar morphology, the overall aggregation properties suggest that CTD274-414 does not form typical β-amyloids. The understanding provided by structural biology over recent years of the PD-associated protein αSyn has enabled detailed insights into the conformational dynamics of several forms of this ’chameleon’ protein. Here, a detailed study of the biosynthesis of αSyn using NMR spectroscopic investigations of ribosome-bound nascent chain complexes (RNCs) is presented. By considering the length-dependent emergence, the effects of charge and also the interaction of αSyn RNCs with the TF chaperone, detailed insights of the interactions arising from the interplay between TF, the ribosome and nascent polypeptide chains were gained. These results were used to create a structural model beginning to reveal the specific manner in which disordered proteins interact with the biosynthesis machinery.
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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.
Full textAbstract:
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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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.
Full textAbstract:
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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Cardova, Alzbeta. "Modelling prion-induced neurodegeneration in PrP transgenic Drosophila." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/290412.
Full textAbstract:
The aim of my thesis was to develop and characterise PrP transgenic Drosophila melanogaster of various genotypes to study the process of prion-induced neurodegeneration in this model. Prion diseases are caused by the occurrence of an abnormally-folded form of PrP (PrPSc) protein that arises either from the environment as an acquired disease, from mutation in the PrP-coding gene as a genetic disease or sporadically from causes unknown. The PrPSc then recruits PrPC, the normal form of PrP, that is ubiquitously present in the mammalian CNS and triggers neurotoxicity and neurodegeneration that is transmissible between individuals of the same or even different species. All prion diseases are currently incurable, fatal and the mechanism of prion-induced neurodegeneration remains to be discovered. In this thesis, Drosophila transgenic for ovine (chromosome 3 and dual PrP transgenic flies), hamster, humanised murine, human and cervid PrP were characterised for expression and biochemical properties. The ultimate goal of my thesis was investigation of cell-to-cell spread of misfolded PrP in Drosophila CNS. To achieve this, a mutant form of PrP that is thought to misfold was co-expressed with the normal form PrPC that served as a substrate in the same dual PrP-transgenic fly. The process was modelled using hamster, humanised murine or ovine PrP transgenes that carry human mutations associated with the spontaneous onset of transmissible neurodegeneration in the natural host. Various approaches towards independent spatial expression of PrP in Drosophila were exploited here in both single and dual PrP expressing flies. Moreover, the ability to initiate misfolding and the impact of this on the fly phenotype was investigated. Both apparent misfolding and phenotypic changes were seen in different fly models suggesting the models were successful. To this extent, PrP transgenic Drosophila were developed to allow for relatively rapid modelling of mammalian prion disease in this invertebrate organism.
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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.
Full textAbstract:
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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Barria, Matus Marcelo Alejandro. "Modelling human prion replication in cell-free systems." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/10025.
Full textAbstract:
One of the key molecular events in the transmissible spongiform encephalopathies or prion diseases is the conformational conversion of the cellular prion protein PrPC into the misfolded and pathogenic isoform, PrPSc. Prion diseases are fatal neurodegenerative conditions affecting humans and other animal species, which present with diverse clinical and neuropathological phenotypes. In humans, prion diseases can occur as sporadic, familial or acquired forms. Sporadic Creutzfeldt–Jakob disease (sCJD) accounts for the majority of cases. The current classification system of human prion diseases recognizes several distinct clinico-pathological entities including sCJD, variant Creutzfeldt-Jakob disease (vCJD), Gerstmann–Straussler–Scheinker syndrome, fatal familial insomnia and variably protease-sensitive proteinopathy. Prion protein gene (PRNP) mutations and polymorphisms, and PrPSc types have a profound effect on these clinico-pathological phenotypes. Prion diseases of sheep and goats, cattle, and cervids are all actual animal health problems and present potential risks to human health. Thus far the only known zoonotic prion disease is bovine spongiform encephalopathy, which has resulted in vCJD in humans. The recognition of new forms of prion diseases in animal and humans has generated increased awareness of the animal and public health risks associated with prion disease. However the mechanisms involved in prion replication, transmission, and neurodegeneration remain poorly understood. This thesis uses in vitro PrP conversion assays (protein misfolding cyclic amplification and real time quaking-induced conversion) to model different aspects of human prion replication: Molecular susceptibility, genetic compatibility, spontaneous formation and the effect of molecules that might enhance or prevent conversion were each investigated in order to obtain a better understanding of the molecular mechanism of the prion replication. I have addressed the hypothesis that the major determinant factors in prion disease pathogenesis (PRNP genetics, PrPSc types and species barriers) are intrinsic to the prion protein conversion process and their effects can be faithfully recapitulated by in vitro conversion assays. The results shows that in vitro conversion assays used in this thesis can model the combined effects of different PrP type and genotypes, can replicate aspects of cross-species transmission potential and provide information about molecular barrier to zoonotic transmission, can model de novo PrPSc formation, and can assess the potential impact of chaperones on conversion of the human prion protein. In summary, this work provides evidence that the origin, propagation and transmission of prions can be meaningfully investigated in cell-free systems.
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MODA, FABIO. "Engineered adeno associated-viruses expressing anti-prp molecules and polyelectrolyte gold nanoparticles as new therapeutic strategies for prion diseases in mouse models." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2010. http://hdl.handle.net/10281/19196.
Full textAbstract:
The prion diseases are neurodegenerative disorders of humans and animals that are
sporadic or inherited in origin and can be transmitted. Despite remarkable differences in phenotypic expression, these disorders share a similar pathogenic mechanism, i.e. a posttranslational modification of the prion protein from a normal cellular isoform (PrPC) to insoluble and protease-resistant disease-specific species (termed PrPSc). PrPSc accumulates in the brain and, according to prion hypothesis, is responsible for the propagation of the pathologic process and transmissibility of the disease, by converting PrPC into a likeness of itself. In a model of prion replication, direct interaction between
PrPSc template and the endogenous PrPC is proposed to drive the formation of nascent
infectious prions. For these reasons therapies to prevent prion diseases can be targeted towards the selective binding of PrPC or PrPSc and the process of conversion. Many compounds have been proposed as potential therapies in the treatment of prion diseases. With the development of novel gene delivery system and nanomedicine, it has been possible to design innovative in vitro therapies effective in cure chronically prion infected cells. ScFvD18, an antibody fragment composed by the variable regions of the heavy and light chains, already resulted in efficient clearing PrPSc in prion infected cells. Fo this reason, ScFvD18 was engineered in Adeno-Associated Viral vectors (AAVs) serotype 9
(AAV9-ScFvD18) and inoculated into the brain of prion infected mice to assess its effectiveness in modify disease progression. Also polyelectrolyte covered gold nanoparticles (AuNPs) are excellent therapeutic compounds due to the intrinsic properties as being non-toxic, inert to most chemical reactions and easy to prepare. In vitro experiments showed that even picomolar amount of AuNPs with layer-wise deposition of
oppositely charged synthetic polyelectrolytes, such as polyallylamine hydrochloride (PAH)and polystyrenesulfonate (PSS), were able to hamper the accumulation of PrPSc in cell culture. The efficacy of these nanogold particles was further assessed in prion infected
mice.
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Björk, Linnea. "Synthesis of proteophenes that can be utilized as fluorescent ligands for biological targets." Thesis, Linköpings universitet, Kemi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-160185.
Full textAbstract:
Small fluorescent probes are important tools when studying protein aggregates involved in different neurodegenerative diseases, such as Alzheimer’s disease. Luminescent conjugated oligothiophenes have been developed and shown to be excellent ligands when studying morphology among amyloids, due to their conjugated thiophene backbone that provides them with unique photophysical properties. This kind of probes are being developed successively to enhance the specificity of their biological targets. In this project, luminescent conjugated oligothiophenes functionalized with amino acids, so called proteophenes, have been synthesized to investigate their optical properties. Since amino acids are chiral molecules, the possibility of induced chirality to the thiophene backbone was examined, as well as the proteophenes ability to work as amyloidospecific ligands for the study of protein aggregates. The synthesis of four different proteophenes are presented in this report, along with analysis results of their photophysical properties.
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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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CICARDI, MARIA ELENA. "ROLE OF THE PROTEIN QUALITY CONTOL SYSTEM IN MOTOR NEURON DISEASES: THE CASE OF MUSCLE CELLS." Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/585905.
Full textAbstract:
Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA), are characterized by the progressive loss of motor neurons, and patients die for respiratory failure after the paralysis of voluntary muscle. Muscle system is, thus, highly affected but it is still unclear whether they play a role in disease onset or if they are a secondary target of toxicity. In my PhD period, I analyzed the behaviour in muscle cells of misfolded protein causing neurodegenerative diseases. I found that misfolded protein aggregates also in muscle cells. As a second step I studied the activation of the protein quality control system and its role at basal level in presence of misfolded proteins. I found that autophagy and proteasome are differently involved in the degradation of misfolded proteins in both muscle and motor neurons. Nevertheless, increasing the degradation of misfolded proteins by overexpressing key molecular chaperone or treating cells with autophagy inhibitors rescue the formation of aggregates in both models of disease.
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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.
Full textAbstract:
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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Costanzo, Maddalena. "Mechanism of spreading of prion and polyglutamine aggregates and role of the cellular prion protein in Huntington’s disease." Thesis, Paris 11, 2012. http://www.theses.fr/2012PA112186/document.
Full textAbstract:
La pathogénèse de la plupart des maladies neurodégénératives incluant les maladies transmissibles comme les encéphalopathies à prion, les maladies génétiques de type maladie de Huntington et les maladies sporadiques comme les maladies d’Alzheimer et de Parkinson est directement liée à la formation d’agrégats protéiques fibrillaires. Pendant de nombreuses années, le concept de dissémination et d’infectivité de ces agrégats a été réservé aux maladies à prion. Cependant, de récents résultats montrent que ces protéines amyloidiques extracellulaires (β-amyloïde) comme intracellulaires (α-synucléine, tau, huntingtin) sont capables de bouger (et possiblement de se répliquer) d’une zone à l’autre du cerveau à la façon des prions (Brundin et al., 2010; Jucker and Walker, 2011; Aguzzi and Rajendran, 2009). Récemment une nouveau lien a été établie entre prions et différentes protéinopathies à agrégats. Il a été suggéré que le prion cellulaire, PrPC, dont la forme pathologique (PrPSc) est responsable des maladies à prion, pourrait servir de médiateur dans la toxicité de la protéine β-amyloïde impliquée dans la maladie d’Alzheimer comme dans d’autres conformations-β, indépendamment de la propagation des prions infectieux (revue de Biasini et al., 2012). Malgré une intense recherche sur les maladies neurodégénératives à prion ou non, de nombreuses questions restent ouvertes à la fois au niveau du mécanisme de dissémination des agrégats protéiques que du mécanisme de toxicité. Dans la première partie de ma thèse, j’ai contribué à étudier le rôle de cellules dendritiques (DCs) dans la dissémination de l’infection à prion aux neurones. J’ai démontré que le transfert de PrPSc des cellules dendritiques infectées par un homogénat de cerveau infecté par du prion vers les neurones était dû à contact direct entre ces cellules et a pour résultat la transmission de l’infectivité aux neurones en co-culture. Ces résultats confirment le possible rôle des cellules dendritiques dans la propagation du prion de la périphérie vers le système nerveux central. J’ai aussi trouvé un potentiel mécanisme de transfert de PrPSc des cellules dendritiques aux neurones via des nanotubes (TNTs) et exclu l’implication de la sécrétion de PrPSc dans notre système. Dans la seconde partie de ma thèse, j’ai étudié les mécanismes de dissémination et de toxicité des agrégats protéiques huntingtin et le possible rôle de PrPC dans ces évènements. J’ai démontré que les agrégats Htt sont transférés entre les lignées de cellules neuronales et les neurones primaires et qu’un contact direct cellule à cellule est requis. De même, j’ai montré l’implication des TNTs dans ce transfert et l’agrégation des Htt sauvages endogènes dans les neurones primaires, probablement en suivant le transfert des agrégats Htt. La dernière partie de mes résultats montre que PrPC est impliqué dans la propagation de la toxicité induite par les Htt mutants dans des neurones primaires en cultureThe pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathies, inherited disorders like Huntington’s disease, and sporadic diseases like Alzheimer’s and Parkinson’s diseases, appear to be directly linked to the formation of fibrillar protein aggregates. For many years, the concept of aggregate spreading and infectivity has been confined to prion diseases. However, recent evidence indicate that both extracellular (e.g. amyloid-β) and intracellular (α- synuclein, tau, huntingtin) amyloidogenic protein are able to move (and possibly replicate) within the brains of affected individuals, thereby contributing to the spread of pathology in a prion-like manner (Brundin et al., 2010; Jucker and Walker, 2011; Aguzzi and Rajendran, 2009). Recently another intriguing connection has been made between prions and other aggregation proteinopathies, as it was suggested that the cellular prion protein, PrPC, whose pathological counterpart is responsible for prion diseases, possibly mediates the toxicity of Aβ, the pathogenic protein in Alzheimer’s disease, and of other β- conformers independently of the propagation of infectious prions (reviewed in Biasini et al., 2012). However, despite the intense research, many questions in prion and non-prion neurodegenerative diseases are still open regarding both the mechanism of protein aggregate spreading and the mechanism of toxicity. In the first part of my thesis, I contributed to investigate the role of DCs (dendritic cells) in the spreading of prion infection to neuronal cells. I demonstrated that the transfer of PrPSc from DCs (loaded with prion infected brain homogenate) to primary neurons was triggered by direct cell–cell contact and resulted in transmission of infectivity to the co-cultured neurons. These data confirm the possible role of DCs in prion spreading from the periphery to the nervous system. I also provided a plausible transfer mechanism of PrPSc through tunneling nanotubes (TNTs) shown to connect DCs to primary neurons and excluded the involvement of PrPSc secretion in our system. In the second part of my thesis, I investigated the mechanisms of the spreading and toxicity of Htt aggregates and the possible role of PrPC in these events. I demonstrated that Htt aggregates transfer between neuronal cells and primary neurons and that cell-cell contact is required. I also showed the involvement of TNTs in the transfer and reported the aggregation of endogenous wild-type Htt in primary neurons, possibly following the transfer of Htt aggregates. Finally, the last part of my results provides evidences that PrPC is involved in the spreading of the toxicity mediated by mutant Htt in primary neuronal cultures
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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.
Full textAbstract:
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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Spagnolli, Giovanni. "Folding, Misfolding and Therapeutics in Prion Diseases." Doctoral thesis, Università degli studi di Trento, 2021. http://hdl.handle.net/11572/308935.
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Prion diseases are rare neurodegenerative disorders affecting humans and other animals, caused by a proteinaceous infectious agent named prion. The pivotal event in these pathologies is the conversion of PrPC, a physiologically expressed protein of poorly characterized function, into a misfolded conformer, named PrPSc, which is capable of replicating its conformationally-encoded information by inducing the conversion of its physiological counterpart. The aggregates resulting from this misfolding process accumulate in the central nervous system of affected organisms leading to neuronal death. Prion diseases are always fatal and no therapy is currently available. The lack of an effective therapeutic strategy to tackle such conditions is the result of the poor available information regarding many aspects of PrPSc, such as its structure, pathogenicity, and its replication mechanism. To complicate things further, PrPSc can appear as a set of distinct conformers, named strains, characterized by the capacity to evolve through modification and selection of their conformations, promoting resistance to treatments. In this work, we focus on two main aspects of prion biology, the elucidation of prion structure and propagation, and the development of a novel pharmacological strategy to tackle prion diseases. In both projects, we exploited the potential of integrative schemes combining computational methods and experimental data. Such approaches allowed us to build a plausible atomistic model of PrPSc and to propose a propagation mechanism describing the series of events underlying prion propagation. Moreover, the application of advanced computational schemes enabled us to identify a PrP folding intermediate displaying unique druggability properties. By exploiting the structural information of this protein conformer we identified a compound capable of acting as a pharmacological degrader for PrP by interfering with its folding pathway. Overall, this work highlights how the integration of computational and experimental methods is an extremely valuable scheme to answer complex biological questions, such as unraveling the mechanisms of protein misfolding and providing the tools to design pharmacological strategies for untreatable diseases.
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Ahmed, Abdullah. "Dévelopement d'une méthode bio-informatique pour la prédiction des régions amyloidogéniques dans les protéines." Phd thesis, Université Montpellier II - Sciences et Techniques du Languedoc, 2013. http://tel.archives-ouvertes.fr/tel-00998437.
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La formation d'agrégats protéiques insolubles et fibreux, appelés fibrilles amyloïdes, est impliquée dans une large variété de maladies humaines. Parmi elles, figurent entre autres, le diabète de type II, l'arthrite rhumatoïde et, notamment, les atteintes neurodégénératives débilitantes, telles que les maladies d'Alzheimer, de Parkinson ou encore de Huntington. Actuellement, il n'existe ni traitement, ni diagnostic précoce pour aucune de ces maladies.De nombreuses études ont montré que la capacité à former des fibrilles amyloïdes est une propriété inhérente à la chaîne polypeptidique. Ce constat a conduit au développement d'un certain nombre d'approches computationnelles permettant de prédire les propriétés amyloïdogéniques à partir de séquences d'amino-acides. Si ces méthodes s'avèrent très performantes vis à vis de courts peptides (~ 6 résidus), leur application à des séquences plus longues correspondant aux peptides et protéines en lien avec les maladies, engendre un nombre trop élevé de faux positifs. Le principal objectif de cette thèse consiste à développer une meilleure approche bioinformatique, capable de prédire les régions amyloïdogéniques à partir d'une séquence protéique. Récemment, l'utilisation de nouvelles techniques expérimentales a permis de mieux appréhender la structure des amyloïdes. Il est ainsi apparu que l'élément caractéristique de la majorité des fibrilles amyloïdes impliquées dans les maladies, était constitué d'une structure étagée (β-arcade), résultant de l'empilement de motifs " feuillet β - coude - feuillet b " appelés " β-arches ". Nous avons mis à profit cette particularité structurale pour créer une approche bioinformatique permettant de prédire les régions amyloïdogéniques d'une protéine à partir de l'information contenue dans sa séquence. Les résultats provenant de l'analyse des structures de type β-arcade, connues et modélisées, ont été compilés et traités à l'aide d'un algorithme écrit en langage Java, afin de créer le programme ArchCandy.L'application de ce programme à une sélection de séquences protéiques et peptidiques, connues pour leur lien avec les maladies, a permis de démontrer qu'il était en mesure de prédire correctement la majorité de ces séquences, de même que les séquences mutées impliquées dans les maladies familiales. Outre la prédiction de régions à haut potentiel amyloïde, ce programme suggère la conformation structurale adoptée par les fibrilles amyloïdes. Le séquençage de génomes entiers devenant toujours plus abordable, notre méthode offre une perspective de détermination individuelle des profils à risque, vis à vis de maladies neurodégénératives, liées à l'âge ou autres. Elle s'inscrit ainsi pleinement dans l'ère de la médecine personnalisée.
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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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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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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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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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Ruiz, Arlandis Gemma. "Binding and internalization of exogenous protein assemblies by mammalian cells." Thesis, Paris 11, 2015. http://www.theses.fr/2015PA114814.
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Le mépliement et l'agrégation des protéines sont à l'origine de nombreuses maladies neurodégénératives, dont la maladie de Huntington (HD) et la maladie de Parkinson (PD). Même si l’agrégation de différentes protéines liées à des maladies est bien documentée, on en sait peu sur l'interaction entre les protéines mal repliées et les cellules neuronales, qui leur permettent de se propager et affecter différentes régions du cerveau. L'objectif de ma thèse était de générer des modèles cellulaires rapporteurs de la huntingtine et l’α-synucléine, protéines dont le mauvais repliement et l'agrégation sont à l'origine de HD et PD respectivement, et utiliser ces modèles cellulaires pour étudier les interactions entre les agrégats et des lignées cellulaires de mammifères. Notre but c’était de documenter les propriétés de liaison et d’absorption de ces agrégats par les cellules rapporteuses, et les conséquences de leur internalisation pour les cellules. Deux modèles cellulaires de neuroblastome (SH-SY5Y et Neuro2A) et un modèle de cellules d’ostéoblastome (U2OS) exprimant la protéine fluorescente ChFP ont été générés pour HD. Pour simuler ce qui se passe au sein de neurones réels, des cellules de neuroblastome ont été induites à se différencier. Des différences de fixation, internalisation, nucléation de la protéine endogène et localisation finale des agrégats de polyglutamine internalisés ont été observées entre les cellules différenciées et non différenciées. Des cellules rapporteuses U2OS ont été utilisées pour déterminer les différences d’infectiosité entre des fibres de HttExon1 assemblés en présence ou en l’absence de la protéine de choc thermique constitutivement exprimée chez l'Homme Hsc70. Hsc70 a un effet protecteur car il rend les fibres moins infectieuses pour les cellules de mammifères en culture. Enfin, un modèle cellulaire de neuroblastome (Neuro2A) rapporteur pour PD exprimant l’α-synucléine fusionnée à la protéine ChFP a été utilisé pour déterminer des différences de liaison, pénétration, absorption, nucléation de la protéine endogène et persistance entre deux polymorphismes d’α-synucléine générés par notre équipe. L'hétérogénéité observée dans différents patients souffrant de synucléinopaties pourrait s'expliquer par différents polymorphes d’assemblages protéiques d’α-synucléine présents dans les cerveaux des malades, ce qui doit être pris en compte pour les développements thérapeutiques futurs.Ces modèles cellulaires rapporteurs pour différentes maladies sont un système valable pour l'étude de différents processus cellulaires liés à l'interaction entre les protéines agrégées exogènes et des cellules de mammifères en culture. Nos résultats indiquent un mécanisme commun par lequel les différentes protéines agrégées peuvent interagir avec des cellules en culture: les protéines mal repliées exogènes sont capables de se lier à des membranes cellulaires, les pénétrer, entrer dans l'espace intracellulaire et recruter des protéines endogènes solubles. Même si cela semble être un mécanisme générique pour des protéines infectieuses telles que la α-synucléine ou la huntingtine, des lignées cellulaires avec différents phénotypes montrent différences de vulnérabilité à la présence de protéines agrégées. Ceci suggère la présence de récepteurs spécifiques à la surface de la cellule capables de reconnaître des structures de type amyloïde. D'autres études sont nécessaires pour déterminer la nature de ces récepteurs et si sa modulation pourrait être utile pour contrôler la propagation des ces maladies dans le cerveauProtein misfolding and aggregation are at the origin of many neurodegenerative diseases, including Huntington’s disease (HD) and Parkinson’s disease (PD). Even if the aggregation of different disease-related proteins is well documented, little is known about the interaction between those misfolded proteins and neuronal cells that allow them to spread and affect several regions of the brain. The objective of my thesis was to generate reporter cellular models of huntingtin and α-synuclein, proteins whose misfolding and aggregation are at the origin of HD and PD respectively, and use these cell models for studying the interactions between misfolded protein aggregates and mammalian cell lines. We aimed to document the binding and uptake properties of those aggregates by reporter cells and the consequences of their internalization for the cells. Two neuroblastoma cell models (SH-SY5Y and Neuro2A) and an osteoblastoma cell model (U2OS) expressing the fluorescent protein ChFP were generated as mammalian reporter cell lines for HD. To mimic what happens in real neurons, neuroblastoma reporter cells were induced to differentiate. Differences in binding, internalization, nucleation of the endogenous protein and final localization of the internalized polyglutamine aggregates were observed between differentiated and undifferentiated cells. U2OS reporter cells were used for determining differences in the infectivity of HttExon1 fibrils assembled in the presence or in the absence of the constitutively expressed heat shock protein Hsc70, suggesting a protective effect of Hsc70, since it renders the fibrils less infectious to mammalian cells. Finally, a neuroblastoma reporter cell model (Neuro2A) of PD expressing α-synuclein fused to the fluorescent and reporter protein ChFP was used to determine the different binding, penetration, uptake, nucleation of the endogenous protein and persistence properties of two α-synuclein polymorphs generated by our team. The heterogeneity observed in different patients suffering from synucleinopathies could be explained due to different α-synuclein assemblies present in diseased brains, what needs to be taken into account for future therapeutic developments. These reporter cellular models for different diseases are a valid system for the study of different cellular processes related with the interaction between exogenous aggregated proteins and mammalian cells in culture. Our results indicate a common mechanism by which different aggregated proteins can interact with cells in culture: exogenous misfolded proteins are able to bind cell membranes, penetrate them, enter the intracellular space and recruit endogenous soluble proteins. Even if this seems to be a generic mechanism for infectious proteins such as α-synuclein or huntingtin, different cell lines or cell phenotypes show distinct vulnerability to the presence of aggregated proteins. This strongly suggests the presence of specific receptors at the surface of the cell able to recognize amyloid-like structures. Further investigations are needed to determine the nature of these receptors and whether their modulation might be helpful for controlling the spread of these diseases within the brain
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Cardeal, Isabel Cristina Mendonça de Azevedo. "Uso terapêutico de chaperones em doenças conformacionais." Master's thesis, [s.n.], 2013. http://hdl.handle.net/10284/4093.
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Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências FarmacêuticasOs chaperones são proteínas que têm por função principal assistir e promover o enrolamento adequado de cadeias polipeptídicas, quer as cadeias recém-sintetizadas nos ribossomas do retículo endoplasmático quer pós-traducionalmente durante o seu processo de translocação através das membranas intracelulares. No ambiente celular existem várias classes de chaperones não relacionadas estruturalmente que se organizam formando redes cooperativas de vigilância e manutenção da conformação nativa de proteínas ou de indução da destruição de proteínas misfolded através da formação de corpos de inclusão e posterior degradação pelas proteases do sistema lisosomal ou proteossomal. As doenças conformacionais, como por exemplo as doenças amiloides, são caracterizadas pela redução do nível de proteína nativa e pela acumulação da respetiva proteína misfolded, resultando na sua aglomeração e deposição em tecidos específicos que está associada a um aumento de morbilidade e mortalidade. A investigação ao nível terapêutico sugere que o tratamento com chaperones farmacológicos pode ser preventivo, ao reduzir o stress oxidativo que é um agente causador comum a estas doenças, ou curativo, seja pela aplicação/administração de chaperones farmacológicos ou pelo meio de indução de produção destes chaperones pelo próprio organismo. No entanto, ainda existe um longo caminho para percorrer até que seja identificado um fármaco que consiga devolver a estes doentes a qualidade de vida que eles merecem, facto que torna fundamental a continuidade da investigação sobre chaperones, desde a elucidação do seu funcionamento à sua aplicação farmacológica. Chaperones are proteins whose function is to assist and promote the correct folding of proteins, either newly proteins synthesized at ribosomes of the endoplasmic reticulum or post-translationally during the process of translocation across intracellular membranes. In the cellular environment, there are several classes of structurally unrelated chaperones. These molecules are organized in cooperative networks involved in surveillance and maintenance of the native conformation of proteins, or in the destruction of misfolded proteins through the formation of inclusion bodies that are subsequently degraded by lysosomal or proteosomal systems. Protein conformational diseases, such as amyloid disorders, are characterized by a reduction in the level of native protein and, simultaneously, by the accumulation of misfolded proteins. These alterations result in the agglomeration of misfolded proteins and their accumulation at toxic levels in a specific tissue is associated with disorders with an increased morbidity and mortality. Data from investigation of therapeutic options suggest that pharmacological chaperons may act preventively, by reducing oxidative stress which is a common causative agent of these diseases or correctively by either the application/administration of these molecules or the induction of its production by the body itself. However, there is still a long way until the identification of a drug that can return to these patients the quality of life they deserve, thus underline the importance of future research on chaperones, not only to better elucidate its molecular mechanism in the cell but also to identify more effective drugs for the treatment of conformational diseases.
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CORDA, ERICA. "TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSES): EXPERIMENTAL APPROACHES TO PATHOGENESIS, THERAPY AND PREVENTION IN ANIMAL MODELS." Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/169556.
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Prion diseases are perhaps the most mysterious and peculiar diseases in nature. These diseases do not rely on the general dogmas of modern biology, seen in other infectious diseases caused by conventional pathogens, such as viruses and bacteria. On the contrary, their infectious agent is an unconventional proteinaceous pathogen, termed prion, that lacks functional nucleic acids. Prion diseases are also known as Transmissible Spongiform Encephalopathies (TSEs), since the diseases are transmissible from one host to another and manifest a spongiform appearance as result of the destruction of brain tissue during a long incubation period. Prion diseases include Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE, “mad cow disease”) in ruminants, scrapie in sheep and goats and chronic wasting disease in deer and elks. As demonstrated in the BSE outbreak and its transmission to humans, the onset of diseases is not limited to a certain species but can be transmissible from one host species to another. Such a striking nature of prions has generated huge concerns in public health and attracted serious attention in the scientific communities.
To date, the potential transmission of prions to human has not been alleviated and TSEs still have no reliable preclinical screening tests and effective treatments.
This doctoral thesis deals widely with the prion diseases, from epidemiology to pathogenesis, from diagnosis to therapy and prevention. Moreover it describes in detail three experimental projects aimed to clarify different aspects of TSEs. In all of them wild-type mouse bioassays are used, as they are the gold standard for assessing the biological properties of prions.
The goal of the first study was to assess the therapeutic and/or preventive activity on TSEs of the chronic administration of a new γ-secretase modulator. The second research investigated the ability to identify BSE in presence of scrapie. The third project was aimed to study the effects induced by chronic administration of lipid enriched/depleted specific diets on the pathogenesis of prion diseases.
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Wörner, Andreas Christian [Verfasser], and Franz-Ulrich [Akademischer Betreuer] Hartl. "Cytoplasmic protein aggregates interfere with nucleo-cytoplasmic transport of protein and RNA : designed β-sheet proteins and their structural properties reveal novel toxicity mechanisms in a gain-of-function model of protein misfolding diseases / Andreas Christian Wörner. Betreuer: Franz-Ulrich Hartl." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/1108130100/34.
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Boopathy, Sivakumar. "Investigating Structural and Functional Defects in ALS-causing Profilin 1 Variants." eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/923.
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Mutations in profilin 1 (PFN1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that targets motor neurons. PFN1 is a 15 kDa protein that is best known for its role in actin dynamics. However, little is known about the pathological mechanisms of PFN1 in ALS. In this dissertation, it is demonstrated that certain familial ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in neuronal cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported functional defects in cell-based assays. The source of this destabilization is illuminated by the crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of one ALS variant and predicting a non-surface exposed cavity in another. Functional biochemical experiments point to abnormalities in actin filament nucleation and elongation caused by PFN1 mutants. In HeLa cells, PFN1 is essential for the generation of actin-rich filopodia and expression of mutant PFN1 alters filopodia density further supporting a pathogenesis mechanism involving actin cytoskeleton. Taken together, this dissertation infers that the pathogenesis of ALS due to mutations in PFN1 can be mediated at least by two possibly related mechanisms, a destabilization of the native PFN1 structure and an impact on the actin assembly processes.
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Boopathy, Sivakumar. "Investigating Structural and Functional Defects in ALS-causing Profilin 1 Variants." eScholarship@UMMS, 2017. https://escholarship.umassmed.edu/gsbs_diss/923.
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Mutations in profilin 1 (PFN1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that targets motor neurons. PFN1 is a 15 kDa protein that is best known for its role in actin dynamics. However, little is known about the pathological mechanisms of PFN1 in ALS. In this dissertation, it is demonstrated that certain familial ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in neuronal cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported functional defects in cell-based assays. The source of this destabilization is illuminated by the crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of one ALS variant and predicting a non-surface exposed cavity in another. Functional biochemical experiments point to abnormalities in actin filament nucleation and elongation caused by PFN1 mutants. In HeLa cells, PFN1 is essential for the generation of actin-rich filopodia and expression of mutant PFN1 alters filopodia density further supporting a pathogenesis mechanism involving actin cytoskeleton. Taken together, this dissertation infers that the pathogenesis of ALS due to mutations in PFN1 can be mediated at least by two possibly related mechanisms, a destabilization of the native PFN1 structure and an impact on the actin assembly processes.
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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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Mulvihill, Cory Michael. "Structural Basis for Misfolding at Disease Phenotypic Positions in CFTR." Thesis, 2012. http://hdl.handle.net/1807/34817.
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Misfolding of membrane proteins as a result of mutations that disrupt their functions in substrate transport across the membrane or signal transduction is the cause of many significant human diseases. Yet, we still have a limited understanding of the direct consequences of these mutations on folding and function - a necessary step toward the rational design of corrective therapeutics. This thesis addresses the gap in understanding the residue-specific implications for folding through a series of experiments that utilize the cystic fibrosis transmembrane conductance regulator (CFTR) as a model in various contexts. We first examined the thermodynamic implications of mutations in the soluble nucleotide binding domain 1 (NBD1) of CFTR. We found that mutations can have a significant effect on thermodynamic stability that is masked in non-physiological conditions. Our studies were then focussed on a membrane-embedded hairpin CFTR fragment comprised of transmembrane segments 3 (TM3) and 4 (TM4) to evaluate the direct effects of mutations on folding in a systematic manner. It was found that the translocon-mediated membrane insertion of helices closely parallels a basic hydrophobic-aqueous partitioning event. This study was then extended to determine residue-specific effects on helix-helix association. We found that this process is not solely dependent on hydropathy, but there is a context dependence of these results with regard to residue position within the helix. Overall, these findings constitute a key step in relating mutation-derived effects on membrane protein folding to the underlying basis of human disease such as cystic fibrosis.
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Khan, Muhammad Qasim. "Misfolding of Particular PrP and Susceptibility to Prion Infection." Thesis, 2010. http://hdl.handle.net/1807/24589.
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Pathogenesis of prion diseases in animals is associated with the misfolding of the cellular prion protein PrPC to the infectious form, PrPSc. We hypothesized that an animal’s susceptibility to prions is correlated with the propensity of an animal’s PrPC to adopt a β-sheet, PrPSc-like, conformation. We have developed a method which uses circular dichroism (CD) to directly calculate the relative population of PrP molecules that adopt a β-sheet conformation or the ‘β-state’, as a function of denaturant concentration and pH.
We find that the PrP from animals that are more susceptible to prion diseases, like
hamsters and mice, adopt the β-state more readily than the PrP from rabbits. The X-ray
crystal structure of rabbit PrP reveals a helix-capping motif that may lower the propensity to form the β-state. PrP in the β-state contains both monomeric and octameric β-structured species, and possesses cytotoxic properties.
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Sun, Chia-Sui, and 孫嘉穗. "Explore the protein misfolding and aggregation process in Huntington’s disease and amyotrophic lateral sclerosis." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/3e6n5r.
Full textAbstract:
博士國立陽明大學
生化暨分子生物研究所
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Protein misfolding and aggregation plays an important role in many neurodegenerative diseases such as Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS). In this study, we show two cases of structural transformation in turning the amyloid-prone pathological proteins/peptides from filamentous to amorphous aggregates by either introducing isomerase in the protein system (Chapter 1), or substitution with de novo-designed proline mutations in peptide model (Chapter 2). Moreover, we have also disclosed that these conformational conversions reduced the proteinopathy of amyloidogenic proteins/peptides, which further elicit neuroprotective effect. The abundant accumulation of inclusion bodies containing polyglutamine-expanded mutant huntingtin (mHTT) aggregates is considered as the key pathological event in HD. In Chapter 1, we have disclosed that FK506 binding protein 12 (FKBP12), an peptidyl-prolyl cis-trans isomerase that exhibits reduced expression in HD, shows profound neuroprotection and motility improvement against polyglutamine (polyQ)-mediated neurotoxicity in Neuro2a cell and Caenorhabditis elegans. Instead of decreasing the aggregation level, FKBP12 structurally transforms mHTT and other disease-related (i.e. polyQ disease and ALS) peptides into benign amorphous aggregates as examined by multiple cell-biological, biochemical, and novel biophysical approaches. Besides, the oligomerization state of mHTT has also been remodeled by FKBP12. Our results suggest a novel function of FKBP12 in ameliorating the proteotoxicity of mHTT and its possible role in other neurodegenerative diseases. Besides HD, we have also focused our attention on TAR DNA-binding protein (TDP-43), the major ubiquitinated deposits in ALS patients in Chapter 2. Numerous ALS-related mutations have been identified at the C-terminal region of TDP-43, which implies the possible role of TDP-43 mutations during pathogenesis. Here, we synthesize various peptides harboring pathogenic or de novo-designed mutations and discover that peptides with pathological mutations are able to form twisted amyloid fibrils, cause liposome leakage, and mediate cellular toxicity. We also show that by replacing glycines with prolines, known to obstruct the -sheet formation, at the different positions in these peptides may influence their amyloidogenesis process and toxicity to the neuronal cell. Particularly, GGG308PPP mutant peptide could neither form beta-amyloid, cause the leakage of liposome, nor jeopardized cell survival, which implies the importance of the glycines residues at position 308-310 during amyloidogenesis. Collectively, our study provide insights into the field of protein folding/misfolding and shed light on the linkage between structural aberration and pathophysiological mechanism, which may benefit for future therapeutic development. By delineating the correlation between proteinopathy and neurotoxicity, we truly hope the derived results may benefit future therapeutic development against protein misfolding diseases.
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Yin, Guowei. "Mechanistic insights into alpha-Synuclein neuronal toxicity: misfolding, serine phosphorylation and interactions with Rab GTPases." Doctoral thesis, 2013. http://hdl.handle.net/11858/00-1735-0000-0001-BC56-C.
Full text
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GHADAMI, SEYYED ABOLGHASEM. "Study of the conformational changes occurring in human transthyretin that are necessary for amyloid fibril formation in disease and for its role as a detoxifier." Doctoral thesis, 2017. http://hdl.handle.net/2158/1076898.
Full textAbstract:
Transthyretin (TTR) is an extracellular protein able to deposit into well-defined protein aggregates called amyloid, in pathological conditions known as senile systemic amyloidosis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and leptomeningeal amyloidosis. At least three distinct partially folded states have been described for TTR, including the widely studied amyloidogenic state at mildly acidic pH. In this study, I have used fluorescence resonance energy transfer (FRET) experiments in a monomeric variant of TTR (M-TTR) and in its W41F and W79F mutants, taking advantage of the presence of a unique, solvent-exposed, cysteine residue at position 10, that I have labelled with a coumarin derivative (DACM, acceptor), and of the two natural tryptophan residues at positions 41 and 79 (donors). Trp41 is located in an ideal position as it is one of the residues of -strand C, whose degree of unfolding is debated. I found that the amyloidogenic state at low pH has the same FRET efficiency as the folded state at neutral pH in both M-TTR and W79F-M-TTR, indicating an unmodified Cys10-Trp41 distance. The partially folded state populated at low denaturant concentrations also has a similar FRET efficiency, but other spectroscopic probes indicate that it is distinct from the amyloidogenic state at acidic pH. By contrast, the off-pathway state accumulating transiently during refolding has a higher FRET efficiency, indicating nonnative interactions that reduce the Cys10-Trp41 spatial distance, revealing a third distinct conformational state. Overall, these results clarify a negligible degree of unfolding of -strand C in the formation of the amyloidogenic state and establish the concept that TTR is a highly plastic protein able to populate at least three distinct conformational states.
TTR is also a protein able to inhibit amyloid sibril formation of A, the peptide associate with alzheimer diseases, and to suppres the toxcicity of performed oligomers of Aβ. Pervious analyses of the in vitro interaction between human TTR and Aβ, have shown that TTR binds to all forms of Aβ: monomers, oligomers and fibrils. The binding occurs with higher affinity for A oligomers, aggregates and fibrils with respect to A monomers. Previous data do not offer any insight into the mechanism by which TTR inhibits A amyloid fibril formation, oligomer toxicity and on the TTR form responsible for such an effect. In this thesis, the interaction between different variants of TTR (WT-TTR, M-TTR and W79F M-TTR) and the A peptide in both the process of aggregation, as well as on pre-formed toxic oligomers called amyloid-derived diffusible ligands (ADDLs), was studied by FRET technique. The time course of amyloid fibril formation was studied under conditions close to physiological and starting from a monomeric state of the A40 peptide, in the presence of WT-TTR, M-TTR and W97F M-TTR at different concentrations. This study showed inhibition of aggregation of Aβ40 by transthyretin molecules. I also studied the conformational change of all three TTR variants following the interaction with Aβ40 during aggregation at a molar ratio of 1:3 (TTR:Aβ40). In particular, the FRET E of TTRs during aggregation of Aβ40 was monitored and the results showed a change in FRET E of all TTR molecules as Aβ40 aggregation proceeds, indicating a conformational conversion upon Aβ40/TTR interaction. The far-UV CD spectra of WT-TTR, M-TTR and W79F M-TTR in the absence and presence of Aβ40 undergoing aggregation were compared, showing that there are no changes in the secondary structure of TTRs after binding to Aβ40 while it converts from monomers to oligomers. The fluorescence spectra recorded for both M-TTR and DACM-M-TTR in the presence of A42 ADDLs decreased in intensity with time, indicating a progressive interaction with the ADDLs. The FRET E value of M-TTR measured during incubation with A42 ADDLs was found to decrease progressively, indicating that a conformational change occurs for M-TTR following the interaction with A42 ADDLs. This analysis was repeated for the W79F mutant of M-TTR leading to very similar results. This confirms that a conformational change occurs also for W79F M-TTR following the interaction with A42 ADDLs and indicates an increased spatial distance between the DACM moiety attached to Cys10 and the two tryptophan residues, particularly Trp41.
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Bigi, Alessandra. "Study of the relationship between structure and toxicity of different α-synuclein aggregates and related cellular dysfunctions." Doctoral thesis, 2020. http://hdl.handle.net/2158/1191838.
Full textAbstract:
α-Synucleinopathies are a vast group of neurodegenerative disorders characterized by the abnormal accumulation of insoluble aggregates, both in neurons and in oligodendrocytes, whose major component is the protein α-synuclein (αS). Among them, Parkinson’s disease (PD) is the most widespread; it is defined by the progressive loss of dopaminergic neurons in the substantia nigra, responsible for several motor disturbances, such as
bradykinesia, muscular rigidity and resting tremor.
This work is focused on αS, whose abnormal self-assembly gives rise to insoluble inclusions called Lewy bodies and neurites, the most relevant neuropathological hallmarks of PD. The aggregation process of αS is extremely complex and leads to the formation of a wide range of assemblies such as oligomers, protofibrils and fibrils. To define the nature of the species responsible for neuronal damage and their mechanism of action, in the first part of this work we have evaluated the evolution in time of different readouts of cellular dysfunction in neuronal cells. We found that, at early incubation times, small oligomeric species with a rudimentary cross-β structure and high solvent exposed hydrophobicity are by far the most toxic to cells, whereas unstructured monomers and hydrophilic and disordered oligomers are unable to cause any cellular dysfunction. We also found that αS fibrils induce the same cascade of events as toxic oligomers, but more slowly and at a rate dependent on their length, despite their inability to be internalized by the cells. Thus, we associated the toxic capacity of αS fibrillar assemblies with their ability to release small hydrophobic oligomers with a cross-β architecture, particularly effective in crossing neuronal membranes and in inducing neurotoxicity. Our results indicate that oligomers are the most toxic among the analyzed αS species, but fibrillar assemblies can generate neurotoxicity through the release of small oligomeric aggregates, that can in turn contribute to the toxicity associated with their well-characterized ability to transfer from neuron-to-neuron, causing the spreading of
Lewy body pathology.
In the second part of this study we focused on the ability of αS species to interact with neuronal membranes and we analyzed the involvement of the different membrane components, in particular the exposed proteins, on this interaction. Our study revealed that αS oligomers accumulate on the plasma membrane in close proximity to the cellular prion protein, subsequently inducing an increase of intracellular calcium influx in cells by both channel-independent and channel-dependent mechanisms, with the N-methyl-D-aspartate receptor-channels (NMDARs) triggering a prompt and transient calcium influx, followed by a massive calcium dysregulation due to the disruption of the plasma membrane integrity. Accordingly, the pharmacological inhibition of NMDARs, as well as the blockade of the cellular prion protein and the removal of the proteins exposed on the cell membrane transiently delayed the early calcium influx, but not the sustained late one caused by αS oligomers. Furthermore, αS fibrils caused calcium dyshomeostasis with slower kinetics with respect to the oligomers, and the observed ionic alterations were not rescued by the blockade of NMDARs or by the removal of the proteins exposed in neuronal membranes. Thus, the experimental evidences accumulated in the second part of this work shed light into the interplay between αS aggregates and the plasma membrane of neuronal cells, thus expanding the range of molecular targets for the therapeutic intervention in PD.
Overall, the data presented in this work provide a robust body of evidence on the prominent role of oligomeric species with high solvent exposed hydrophobicity and cross-β structure, formed either during the aggregation process of αS, or released from mature fibrils, in the neurotoxicity of αS, giving a detailed description of the toxic effects they evoke. The experimental evidences accumulated in this study also emphasize the importance of the membrane binding properties of such species for their pathological features, proposing possible strategies with therapeutic value in PD.
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Mulligan, Vikram. "Probing the Molecular Mechanisms Underlying Familial Amyotrophic Lateral Sclerosis: New Insight into Unfolding and Misfolding Mechanisms of the Cu, Zn Superoxide Dismutase." Thesis, 2012. http://hdl.handle.net/1807/34815.
Full textAbstract:
While great strides have been made in treating many classes of human disease, the late-onset neurodegenerative diseases continue to elude modern medicine. These diseases, which include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), the transmissible spongiform encephalopathies (TSEs), and amyotrophic lateral sclerosis (ALS), involve accumulation of insoluble aggregates of one or more causative proteins, leading to progressive loss of central nervous system neurons, progressively worsening neurological symptoms, and eventual patient death. All of these diseases are currently incurable and fatal.
In the case of ALS, progressive death of upper and lower motor neurons leads to full-body paralysis, respiratory difficulty, and patient death. Of the subset of ALS cases showing familial inheritance, approximately 20% are caused by mutations in the SOD1 gene, encoding the Cu, Zn superoxide dismutase (SOD1). These mutations do not have the common property of impairing SOD1's normal function as a free radical scavenger. Instead, they are thought to increase the protein's likelihood of misfolding and aggregating via a poorly-understood aggregation cascade. It is believed that species populated along the misfolding and aggregation pathway may prove to be good targets for therapies designed to block accumulation of downstream toxic species, or to prevent aberrant protein-protein interactions responsible for neurotoxicity.
In this thesis, several new techniques are developed to enable detailed elucidation of the SOD1 unfolding and misfolding pathways. Time-resolved measurements collected during SOD1 unfolding or misfolding of release of bound Cu and Zn, of changes in intrinsic fluorescence, of exposure of hydrophobic surface area, and of alterations in the chemical environment of histidine residues, are presented. A new mathematical analysis technique named the Analytical Laplace Inversion Algorithm is developed for rapid extraction of mechanistic information from these time-resolved signals. These tools are applied to the construction of the most detailed models to date of the unfolding and misfolding mechanisms of WT and ALS-causing mutant SOD1. The models presented identify several well-populated unfolding and misfolding intermediates that could serve as good targets for therapies designed to address the fundamental molecular mechanisms underlying SOD1-associated ALS, and to treat what is currently a devastating and incurable disease.
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Lucas, Tânia. "Protein misfolding and cellular responses in metabolic disorders." Doctoral thesis, 2018. http://hdl.handle.net/10451/42536.
Full textAbstract:
This dissertation concerns studies on the electron transfer flavoprotein:ubiquininone oxidoreductase (ETF:QO), a structurally complex membrane protein containing three cofactors organized into three different structural domains. ETF:QO and its partner electron transfer flavoprotein (ETF), constitute an important hub in mitochondrial metabolism mediating electron transfer from several dehydrogenases to the respiratory chain for ATP production. Defects in any of these two proteins result in multiple acyl-CoA dehydrogenase deficiency (MADD), a rare inborn metabolic disorder presenting a broad range of clinical phenotypes, some responding to riboflavin supplementation. Although several MADD cases have been identified, the molecular studies to clarify disease mechanism are scarce, and clinicians have difficulty predicting disease progression and response to riboflavin therapy. Therefore, conformational and structural characterization of ETF:QO is an important step to identify how different mutations impact on clinical phenotypes.
In order to elucidate the molecular aspects behind MADD and riboflavin supplementation, we revisited a riboflavin responsive MADD clinically relevant mutation in ETF:QO (ETF:QO-p.Pro456Leu) and investigated how it affects the enzyme structure and fold. As model, we took advantage of the higher expression levels of the Rhodobacter sphaeroides ETF:QO in Escherichia coli and its 67 % sequence similarity to the mature human protein.
Nevertheless, not all MADD mutations are conserved between these species, and using the recombinant human ETF:QO would have been a better choice. However, human ETF:QO heterologous expression in E. coli has not been a straight forward process. Here, we describe a tour de force approach to better understand the critical steps underlying the folding of human ETF:QO. We investigated possible origins for these difficulties and analysed the possible codon-usage effects, a factor known to influence and regulate protein folding. We also analysed and hypothesized that the codon usage frequency variation might be a possible cause for some MADD related phenotypes. In the last chapter the relationship between fatty acid oxidation and ETF:QO in cancer is discussed.FEBS – Federation of European Biochemical Societies
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Fani, Giulia. "IDENTIFICATION OF MEMBRANE CA2+ CHANNELS ACTIVATED BY PROTEIN MISFOLDED OLIGOMERS AND THEIR ACTIVATION MECHANISM." Doctoral thesis, 2021. http://hdl.handle.net/2158/1260286.
Full textAbstract:
Alzheimer’s disease is the most common form of dementia, characterized by the aggregation of the amyloid β peptide (Aβ) in the brain and by an impairment of calcium homeostasis caused by excessive activation of glutamatergic receptors, named excitotoxicity. Here, we studied the effects on calcium homeostasis caused by the formation of Aβ oligomeric assemblies, formed with Aβ40 and Aβ42 peptide, and by the model protein HypF-N. We found that these oligomers cause a rapid influx of calcium ions (Ca2+) across the cell membrane by rapidly activating extrasynaptic N-methyl-D-aspartate receptors (NMDAr) and, to a lower extent, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAr), and also by causing a perforation of lipid bilayers. Instead, none of the Ca2+ channels, including those found in previous interactome studies to physically interact with the oligomers, were found to participate to the observed Ca2+ influx. We also observed, however, that misfolded oligomers do not interact directly with NMDAr and AMPAr. Further experiments with lysophosphatidylcholine and arachidonic acid, which cause membrane compression and stretch, respectively, indicated that these receptors are activated through a change in membrane tension induced by the oligomers and transmitted mechanically to the receptors via the lipid bilayer. Indeed, lysophosphatidylcholine is able to neutralise the oligomer-induced activation of the NMDAr, whereas arachidonic acid activates the receptors similarly to the oligomers with no additive effects. An increased rotational freedom observed for a fluorescent probe embedded within the membrane in the presence of the oligomers also indicates a membrane stretch. These results reveal a further mechanism of toxicity of Aβ oligomers in Alzheimer’s disease through the perturbation of the mechanical properties of lipid membranes sensed by NMDAr and AMPAr, in addition to others based on the direct binding to membrane receptors and on permeabilization of lipid membranes.