Protein misfolding and amyloid formation

Most proteins need to adopt a three-dimensional structure in order to function properly. Misfolding, or inability of proteins to fold, is associated with a number of diseases. In a subset of these disorders, the misfolded protein or peptide selfassembles into stable, β-sheet rich structures known as amyloid fibrils. Alzheimer's disease is associated with the aggregation of the amyloid β-peptide (Aβ) into oligomers and amyloid fibrils. Aβ has a discordant, i.e β-sheet preferring, helix prone to misfold and it has been proposed that stabilization of this helix could prevent aggregation. We have designed small molecules that bind to this region and stabilize Aβ in a helical conformation. This interaction reduced fibril formation and cell toxicity of the peptide and also restored a memory-linked electrophysiological function in mouse hippocampal slices treated with Aβ. Moreover, when administered orally, these compounds had a rescuing effect in a Drosophila melanogaster model of Aβ aggregation. Another protein capable of forming amyloid-like fibrils in association with disease, is the human lung surfactant protein C, SP-C, which has a discordant transmembrane helix. SP-C is expressed as a pro-protein with a C-terminal, CTC, which has a Brichos domain with unknown function. Here, we show that CTC is important for the stability and folding of the pro-protein in the endoplasmic reticulum (ER). It is able to prevent the mature SP-C from aggregating in vitro, and is shown to bind specifically to non-helical segments and to amino acids that have been reported to promote membrane insertion in the ER. Together these data suggest a chaperone function for CTC, targeting transmembrane regions that have not attained an α-helical conformation. CTC interacts with and reduces amyloidlike fibril formation of Aβ as well as an additional amyloidogenic peptide – medin. In conclusion this thesis explores two new strategies for preventing protein misfolding and amyloid fibril formation. The first approach utilizes designed ligands to trap the Alzheimer's disease associated Aβ in its native helical structure. The second employs a novel, natural chaperone that bridges folding of transmembrane regions and anti-amyloid properties

Prediction of peptide and protein propensity for amyloid formation

Understanding which peptides and proteins have the potential to undergo amyloid formation and what driving forces are responsible for amyloid-like fiber formation and stabilization remains limited. This is mainly because proteins that can undergo structural changes, which lead to amyloid formation, are quite diverse and share no obvious sequence or structural homology, despite the structural similarity found in the fibrils. To address these issues, a novel approach based on recursive feature selection and feed-forward neural networks was undertaken to identify key features highly correlated with the self-assembly problem. This approach allowed the identification of seven physicochemical and biochemical properties of the amino acids highly associated with the self-assembly of peptides and proteins into amyloid-like fibrils (normalized frequency of β-sheet, normalized frequency of β-sheet from LG, weights for β-sheet at the window position of 1, isoelectric point, atom-based hydrophobic moment, helix termination parameter at position j+1 and ΔGº values for peptides extrapolated in 0 M urea). Moreover, these features enabled the development of a new predictor (available at http://cran.r-project.org/web/packages/appnn/index.html) capable of accurately and reliably predicting the amyloidogenic propensity from the polypeptide sequence alone with a prediction accuracy of 84.9 % against an external validation dataset of sequences with experimental in vitro, evidence of amyloid formation

4-Phenylbutyric acid treatment rescues trafficking and processing of a mutant surfactant protein C

Mutations in the SFTPC gene, encoding surfactant protein–C (SP-C), are associated with interstitial lung disease (ILD). Knowledge of the intracellular fate of mutant SP-C is essential in the design of therapies to correct trafficking/processing of the proprotein, and to prevent the formation of cytotoxic aggregates. We assessed the potential of a chemical chaperone to correct the trafficking and processing of three disease-associated mutant SP-C proteins. HEK293 cells were stably transfected with wild-type (SP-C(WT)) or mutant (SP-C(L188Q), SP-C(Δexon4), or SP-C(I73T)) SP-C, and cell lines with a similar expression of SP-C mRNA were identified. The effects of the chemical chaperone 4-phenylbutyric acid (PBA) and lysosomotropic drugs on intracellular trafficking to the endolysosomal pathway and the subsequent conversion of SP-C proprotein to mature peptide were assessed. Despite comparable SP-C mRNA expression, proprotein concentrations varied greatly: SP-C(I73T) was more abundant than SP-C(WT) and was localized to the cell surface, whereas SP-C(Δexon4) was barely detectable. In contrast, SP-C(L188Q) and SP-C(WT) proprotein concentrations were comparable, and a small amount of SP-C(L188Q) was localized to the endolysosomal pathway. PBA treatment restored the trafficking and processing of SP-C(L188Q) to SP-C(WT) concentrations, but did not correct the mistrafficking of SP-C(I73T) or rescue SP-C(Δexon4). PBA treatment also promoted the aggregation of SP-C proproteins, including SP-C(L188Q). This study provides proof of the principle that a chemical chaperone can correct the mistrafficking and processing of a disease-associated mutant SP-C proprotein

Curcumin Promotes A-beta Fibrillation and Reduces Neurotoxicity in Transgenic Drosophila

The pathology of Alzheimer's disease (AD) is characterized by the presence of extracellular deposits of misfolded and aggregated amyloid-β (Aβ) peptide and intraneuronal accumulation of tangles comprised of hyperphosphorylated Tau protein. For several years, the natural compound curcumin has been proposed to be a candidate for enhanced clearance of toxic Aβ amyloid. In this study we have studied the potency of feeding curcumin as a drug candidate to alleviate Aβ toxicity in transgenic Drosophila. The longevity as well as the locomotor activity of five different AD model genotypes, measured relative to a control line, showed up to 75% improved lifespan and activity for curcumin fed flies. In contrast to the majority of studies of curcumin effects on amyloid we did not observe any decrease in the amount of Aβ deposition following curcumin treatment. Conformation-dependent spectra from p-FTAA, a luminescent conjugated oligothiophene bound to Aβ deposits in different Drosophila genotypes over time, indicated accelerated pre-fibrillar to fibril conversion of Aβ1–42 in curcumin treated flies. This finding was supported by in vitro fibrillation assays of recombinant Aβ1–42. Our study shows that curcumin promotes amyloid fibril conversion by reducing the pre-fibrillar/oligomeric species of Aβ, resulting in a reduced neurotoxicity in Drosophila

The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation

<p>Abstract</p> <p>Background</p> <p>Surfactant protein C (SP-C) is important for the function of pulmonary surfactant. Heterozygous mutations in <it>SFTPC</it>, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD) in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We also evaluated the ability of drugs currently used in ILD therapy to counteract these effects.</p> <p>Methods</p> <p>SP-C^A116Dwas expressed in MLE-12 alveolar epithelial cells. We assessed in vitro the consequences for cellular homeostasis, immune response and effects of azathioprine, hydroxychloroquine, methylprednisolone and cyclophosphamide.</p> <p>Results</p> <p>Stable expression of SP-C^A116Din MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-C^A116Dexpression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC) and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-C^A116Dcells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4⁺lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-C^A116Don neighboring cells in the alveolar space.</p> <p>Conclusions</p> <p>We show that the A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy. Our findings shed new light on the pathomechanisms underlying SP-C deficiency associated ILD and provide insight into the mechanisms by which drugs currently used in ILD therapy act.</p

Unfolding of the Amyloid β-Peptide Central Helix: Mechanistic Insights from Molecular Dynamics Simulations

Alzheimer's disease (AD) pathogenesis is associated with formation of amyloid fibrils caused by polymerization of the amyloid β-peptide (Aβ), which is a process that requires unfolding of the native helical structure of Aβ. According to recent experimental studies, stabilization of the Aβ central helix is effective in preventing Aβ polymerization into toxic assemblies. To uncover the fundamental mechanism of unfolding of the Aβ central helix, we performed molecular dynamics simulations for wild-type (WT), V18A/F19A/F20A mutant (MA), and V18L/F19L/F20L mutant (ML) models of the Aβ central helix. It was quantitatively demonstrated that the stability of the α-helical conformation of both MA and ML is higher than that of WT, indicating that the α-helical propensity of the three nonpolar residues (18, 19, and 20) is the main factor for the stability of the whole Aβ central helix and that their hydrophobicity plays a secondary role. WT was found to completely unfold by a three-step mechanism: 1) loss of α-helical backbone hydrogen bonds, 2) strong interactions between nonpolar sidechains, and 3) strong interactions between polar sidechains. WT did not completely unfold in cases when any of the three steps was omitted. MA and ML did not completely unfold mainly due to the lack of the first step. This suggests that disturbances in any of the three steps would be effective in inhibiting the unfolding of the Aβ central helix. Our findings would pave the way for design of new drugs to prevent or retard AD

LÄRARES ATTITYDER OCH INTERVENTIONSSTRATEGIER MOT OLIKA FORMER AV MOBBNING

Denna kvantitativa vinjettstudie undersökte lärares skillnader i attityder och interventionsstrategier mot fysisk-, verbal-, social- och nätmobbning. Åtta vinjetter beskrev samtliga mobbningsformer i en enkät som besvarades av 114 grundskolelärare från olika städer i Sverige. Resultatet indikerade att lärare är mer benägna att ingripa mot situationer av fysisk mobbning jämfört med situationer av nät-, verbal- och social mobbning, att lärare värderar social- och verbal mobbning som mindre allvarligt än fysisk- och nätmobbning samt att lärare upplever bristande kunskaper i att hantera situationer av social- och nätmobbning i jämförelse med situationer av fysisk- och verbal mobbning. Studiens resultat diskuteras utifrån tidigare forskning och understryker vikten av läraren i det förebyggande arbetet mot alla former av mobbning i skolan.The present study examines teachers’ attitudes and intervention strategies toward physical-, verbal-, social- and cyber bullying. Eight vignettes described these various types of bullying in one survey that 114 teachers completed from different cities in Sweden. The results indicated that teachers were more likely to intervene towards physical bullying rather than cyber-, verbal- and social bullying.  Teachers perceived social-, and verbal bullying less serious than physical- and cyber bullying.  Teachers also felt that they lacked the knowledge on how to handle situations in forms of social- and cyber bullying rather than situations of physical- and verbal bullying. These findings are discussed based on previous research and emphasize the importance of teachers’ role in intervening and preventing all types of bullying

Ungskogsröjning i Älvdalens socken

Abstract Syftet med denna studie var att undersöka: Om delägarna i Älvdalens Besparingsskog röjer sin ungskog i större omfattning än andra skogsägare i Sverige. Hur delägarna i Älvdalens Besparingsskog utnyttjar sina skogsbruksplaner. Hur entreprenörerna får sina uppdrag. Studien genomfördes som dokument-och litteraturstudier, enkät samt personliga intervjuer. Röjningsfrekvensen i Sverige år 2012 var 18,4 procent av röjningsbehovet, enligt Riksskogstaxeringen. Skogsvårdsaktiviteten i Älvdalens socken är sannolikt högre än i landet i övrigt. Enkäten visar att endast cirka 38 procent av delägarna i Älvdalens besparingsskog konsulterar sin skogsbruksplan när beslut skall tas angående röjning. Skogsbruksplanerna används i liten omfattning. Entreprenörerna får sina uppdrag till cirka 80 till 90 procent genom att skogägarna kontaktar entreprenören. Cirka 80 till 85 procent av röjningsarbetet görs av entreprenörer med en ökande trend. Ungskogsröjning, Älvdalens Besparingsskog, skogsbruksplan, entreprenöre