Structural biology of bacterial functional amyloid formation

Amyloids are proteinaceous aggregates best known for their role in degenerative diseases involving protein misfolding. Research into amyloid has intensified in recent times due to its prominence in many debilitating human diseases and limited understanding of the causes. The discovery of functional amyloids in a broad range of species has enhanced our understanding of amyloid, of these the curli system of E. coli has been extensively studied, in this system CsgC was identified as a potent inhibitor of amyloid. An additional protein was discovered in some curli operons in other species termed CsgH and warrants further study. A morphologically similar but genetically distinct bacterial functional amyloid system was identified in Pseudomonas encoded by the fapABCDEF operon and termed amyloid-like fibres (Alf). The study of functional amyloid has the potential to provide insights into how amyloid can be controlled. The aims of this thesis were to investigate the novel functional amyloid system of Pseudomonas with a view to structural and functional characterisation of the individual components. The structure and function of the CsgH protein were also studied by nuclear magnetic resonance (NMR) and the ThioflavinT (ThT) amyloid fibrillation assay. Constructs were produced for all the Alf proteins and the more structured components, FapD and FapF, were optimised to produce constructs for structural study. The structure of CsgH was solved successfully using NMR and showed that the protein shared a similar tertiary structure to CsgC. The function of the CsgH was shown to be similar to CsgC inhibiting amyloid formation by CsgA at substoichiometric concentrations. Mutagenesis, ThT assay and NMR were used to show that CsgH and CsgA interact and that several charged residues have an important role in function. It was also interesting to note that CsgH was capable of inhibiting amyloid formation by the FapC amyloid protein of Pseudomonas.Open Acces

Mechanisms of Brain Region-Specific Amyloid-beta Deposition

Alzheimer\u27s disease: AD) is the most common cause of dementia. A fundamental feature of AD is brain region-specific deposition of extracellular amyloid plaques principally comprised of the amyloid-β: Aβ) peptide. Using mouse models of cerebral Aβ deposition, we examined molecular, cellular and systems-level mechanisms that regulate brain region-specific Aβ accumulation and aggregation. Parallel studies using in vivo multiphoton microscopy and in vivo microdialysis revealed that modest pharmacological reduction of soluble interstitial fluid: ISF) Aβ levels was associated with a dramatic reduction in amyloid plaque formation and growth. We found that ISF Aβ concentrations in several brain regions of APP transgenic mice prior to the onset of plaque deposition were proportional to the degree of subsequent plaque deposition and with the concentration of lactate, a marker of neuronal activity. Moreover, we found that physiological modulation of endogenous neuronal activity by vibrissal manipulation was sufficient to modulate ISF Aβ levels and amyloid plaque growth dynamics. Using a novel optical intrinsic signal imaging approach, we found that bilateral functional connectivity magnitude in APP/PS1 mice prior to plaque deposition was proportional to the amount of regional plaque deposition in aged APP/PS1 mice. Furthermore, we found that bilateral functional connectivity was reduced in normal aging and was markedly exacerbated by Aβ deposition. Together, these data suggest that endogenous neuronal activity and functional connectivity may regulate region-specific Aβ plaque deposition. These data advance our understanding of the mechanisms by which the intrinsic metabolic and functional organization of the brain may contribute to AD pathogenesis

Factors that Contribute to de novo Protein Misfolding and Prion Formation in Saccharomyces cerevisiae

Protein misfolding is a common phenomenon that can have severe consequences on cellular and organismal health. Despite this, the causes of protein misfolding remain poorly understood. Prions are a class of proteins that, when misfolded, can convert other molecules into a heritable, non-native conformation. The yeast Saccharomyces cerevisiae naturally harbors several diverse prion-forming proteins; thus, it is an ideal model with which to investigate the factors that influence misfolding and aggregation.This thesis utilizes the yeast prions [PSI+] and [RNQ+] to investigate two distinct steps of the protein misfolding pathway: interactions with chaperones and their cofactors, and heterologous templating by other misfolded proteins. Chaperones are proteins that help other proteins fold correctly, yet we have found that chaperones can have non-intuitive effects upon cells that harbor the prion [PSI+]. An overabundance of the Hsp70 chaperone Ssa1 relative to the Hsp70 Ssb1 exacerbates [PSI+]-related toxicity. This toxicity can be rescued by overexpressing a Hsp70 nucleotide exchange factor, Sse1, that may improve Ssb1 functionality in the presence of excess available Ssa1. Our results imply that the balance of molecular chaperones is finely tuned and is crucial to maintaining protein homeostasis.Interestingly, the [PSI+] prion cannot form without the presence of an inducing factor, which is most commonly the [RNQ+] prion. The nature of the interaction between [PSI+] and [RNQ+] was previously unknown. Here, we have demonstrated that the two proteins undergo cross-seeding reaction, wherein the prion-forming proteins bind to one another to template the formation of [PSI+]. Blocking or restoring a binding site can have significant impacts upon the frequency of prion formation. As cross-seeding has been implicated in several human pathologies, these results may inform key principles that can be utilized to research disease prevention

The role of amyloid-beta assembly state in monocyte maturation and smooth muscle cell degeneration

Alzheimer\u27s Disease (AD) is a progressive, neurodegenerative disorder which is ranked as a leading cause of death among Americans. AD is characterized by the presence of intracellular neurofibrillary tangles and extracellular plaques made of amyloid ß (Aß). Together these pathologies lead to severe memory impairment in patients, but research has implicated the presence of the Aß deposits as likely causes for AD progression. Aß is produced through the proteolytic cleavage of the integral membrane amyloid precursor protein, which occurs through the action of beta- and gamma-secretases and produces 39-43 amino acid Aß peptides. In AD, the Aß plaques are comprised of mostly 40 or 42 amino acid Aß (Aß(1-40) and Aß(1-42) respectively). Evidence suggests that in response to the presence of Aß in the brain, monocytic cells circulating in the blood are recruited across the blood brain barrier and transformed into brain macrophages, known as microglia. Here we investigate the ability of Aß to transform cultured THP-1 monocytes into macrophage-like cells as a model of the in vivo process. Our results indicate that an early-formed Aß oligomer, which is formed when Aß(1-42) is aggregated in water, has the ability to potently transform the non-adherent monocytes into adherent cells with many properties consistent with macrophages. Our data also shows that Aß(1-40) cannot form a species with a similar activity. We have determined that the transforming activity of Aß(1-42) occurs through formyl peptide receptor-like 1, but not through an NF-kappaB dependent mechanism. We also study the involvement of cAMP in a model system of cerebral amyloid angiopathy (CAA), a condition in which Aß deposits within the walls of cerebral vessels leading to hemorrhages. CAA occurs in many cases of AD, but especially in early onset AD cases. We studied the ability of cAMP to rescue human aortic vascular smooth muscle cells from Aß induced toxicity. We found that in our experiments treatment with some cAMP elevating compounds can subtly protect the cells from Aß. Overall we show that Aß is a peptide which has a wide variety of activities that are dependent upon the peptide assembly state

Étude numérique des premières étapes d'agrégation du peptide amyloïde GNNQQNY, impliqué dans une maladie à prion

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

Identifying, Targeting, and Exploiting a Common Misfolded, Toxic Conformation of SOD1 in ALS: A Dissertation

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a loss of voluntary movement over time, leading to paralysis and death. While 10% of ALS cases are inherited or familial (FALS), the majority of cases (90%) are sporadic (SALS) with unknown etiology. Approximately 20% of FALS cases are genetically linked to a mutation in the anti-oxidizing enzyme, superoxide dismutase (SOD1). SALS and FALS are clinically indistinguishable, suggesting a common pathogenic mechanism exists for both types. Since such a large number of genetic mutations in SOD1 result in FALS (\u3e170), it is reasonable to suspect that non-genetic modifications to SOD1 induce structural perturbations that result in ALS pathology as well. In fact, misfolded SOD1 lacking any genetic mutation was identified in end stage spinal cord tissues of SALS patients using misfolded SOD1-specific antibodies. In addition, this misfolded WT SOD1 found in SALS tissue inhibits axonal transport in vitro, supporting the notion that misfolded WT SOD1 exhibits toxic properties like that of FALS-linked SOD1. Indeed, aberrant post-translational modifications, such as oxidation, cause WT SOD1 to mimic the toxic properties of FALS-linked mutant SOD1. Based on these data, I hypothesize that modified, misfolded forms of WT SOD1 contribute to SALS disease progression in a manner similar to FALS linked mutant SOD1 in FALS. The work presented in this dissertation supports this hypothesis. Specifically, one common misfolded form of SOD1 is defined and exposure of this toxic region is shown to enhance SOD1 toxicity. Preventing exposure, or perhaps stabilization, of this “toxic” region is a potential therapeutic target for a subset of both familial and sporadic ALS patients. Further, the possibility of exploiting this misfolded SOD1 species as a biomarker is explored. For example, an over-oxidized SOD1 species was identified in peripheral blood mononuclear cells (PBMCs) from SALS patients that is reduced in controls. Moreover, 2-dimensional gel electrophoresis revealed a more negatively charged species of SOD1 in PBMCs of healthy controls greatly reduced in SALS patients. This species is hypothesized to be involved in the degradation of SOD1, further implicating both misfolded SOD1 and altered protein homeostasis in ALS pathogenesis

Investigating the role of proteostasis pathways in regulating the intracellular inclusion formation of firefly luciferase: a model system to study protein aggregation in cells

The maintenance of cellular protein homeostasis, or proteostasis, is dependent upon a complex network of molecular chaperones, degradation machinery and other regulatory factors, which together act to keep the proteome soluble and functional. Disturbances to proteostasis can lead to protein aggregation and inclusion formation, processes associated with a variety of neurodegenerative disorders. The heat shock proteins (Hsps) are a superfamily of molecular chaperones that are dramatically upregulated in response to cellular stress. The Hsps can bind aggregation-prone proteins and either refold or traffic them for degradation. One class of Hsps, the DNAJBs, act as co-factors of the Hsp70 machine and have been previously identified as potent suppressors of disease-related protein aggregation. This has raised the potential of targeting DNAJB chaperone action in the context of protein aggregation associated with disease. In the work described in this thesis, a destabilised isoform of the protein firefly luciferase (R188Q/R261Q Fluc; FlucDM) was overexpressed in cells to assess how components of the proteostasis machinery engage with aggregation-prone proteins to prevent them from forming intracellular inclusions

The first step towards the development of an electrophoretic prion detector

In nanopore analysis, peptides and proteins can be detected by the change in current when single molecules interact with an α-hemolysin pore embedded in a lipid membrane. Studies into the effects of fluorenylmethoxycarbonyl (Fmoc), acetylation or proline modification to negatively charged α-helical peptides showed that Fmoc peptides give more translocations than acetylated peptides. The addition of a proline in the middle of an acetylated peptide further reduces the number of translocations compared to Fmoc. The effect of peptide conformation on translocation or intercalation was studied with small α-helical and β-sheet hairpins. The capped β-hairpin increased translocations compared to the uncapped. The Fmoc-α-helical hairpin, containing a disulfide link, displayed both bumping and translocations whereas in the unlinked peptide the proportion of translocations was greater. Prion diseases arise from the misfolding and aggregation of the normal cellular prion protein. Nanopore analysis of prion peptides with α-helical and β-strand sequences show changes to the event parameters that help distinguish them. The interaction of bovine prion protein (bPrP), with α-hemolysin showed both bumping (type-I) and intercalation/translocation (type-II) events. There are several lines of evidence that indicate these type-II events with a blockade current of -65 pA for bPrP, represent translocations. Nanopore analysis showed that about 37% events were translocations. The interaction of metal ions with bPrP showed that Cu(II) or Zn(II) reduced translocations. Surprisingly, Mn(II) caused an increase in translocation events to about 64%. Complex formation between antibodies and prion peptides and proteins can be detected by nanopore analysis. The PrP/antibody complex is too large to translocate whereas the event parameters for unbound molecules are unchanged. In principle, a nanopore can detect a single molecule; thus, this work represents the first step towards the development of a prion detector. The nanopore will provide the sensitivity and the antibodies will provide the specificity to distinguish between PrPC and PrPSc. Also, the prion N- and C-terminal signal peptides interact with bPrP changing the event parameters, relating to a new mechanism. Finally, the folding intermediates of bPrP at 0.86 M Gdn-HCl suggests that the protein unfolds and then refolds into a different conformation with event parameters similar to those of bPrP