Single-molecule approaches to prion protein misfolding

The structural conversion of the prion protein PrP into a transmissible, misfolded form is the central element of prion disease, yet there is little consensus as to how it occurs. Key aspects of conversion into the diseased state remain unsettled, from details about the earliest stages of misfolding such as the involvement of partially- or fully-unfolded intermediates to the structure of the infectious state. Part of the difficulty in understanding the structural conversion arises from the complexity of the underlying energy landscapes. Single molecule methods provide a powerful tool for probing complex folding pathways as in prion misfolding, because they allow rare and transient events to be observed directly. We discuss recent work applying single-molecule probes to study misfolding in prion proteins, and what it has revealed about the folding dynamics of PrP that may underlie its unique behavior. We also discuss single-molecule studies probing the interactions that stabilize non-native structures within aggregates, pointing the way to future work that may help identify the microscopic events triggering pathogenic conversion. Although single-molecule approaches to misfolding are relatively young, they have a promising future in prion science.     

A new perspective on Hsp104-mediated propagation and curing of the yeast prion [PSI+]

Most prions in yeast form amyloid fibrils that must be severed by the protein disaggregase Hsp104 to be propagated and transmitted efficiently to newly formed buds. Only one yeast prion, [PSI⁺], is cured by Hsp104 overexpression. We investigated the interaction between Hsp104 and Sup35, the priongenic protein in yeast that forms the [PSI⁺] prion.1 Helsen CW, Glover JR. Insight into molecular basis of curing of [PSI+] prion by overexpression of 104-kDa heat shock protein (Hsp104). J Biol Chem 2012; 287:542 - 56; http://dx.doi.org/10.1074/jbc.M111.302869; PMID: 22081611 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] We found that a 20-amino acid segment within the highly-charged, unstructured middle domain of Sup35 contributes to the physical interaction between the middle domain and Hsp104. When this segment was deleted from Sup35, the efficiency of [PSI⁺] severing was substantially reduced, resulting in larger Sup35 particles and weakening of the [PSI⁺] phenotype. Furthermore, [PSI⁺] in these cells was completely resistant to Hsp104 curing. The affinity of Hsp104 was considerably weaker than that of model Hsp104-binding proteins and peptides, implying that Sup35 prions are not ideal substrates for Hsp104-mediated remodeling. In light of this finding, we present a modified model of Hsp104-mediated [PSI⁺] propagation and curing that requires only partial remodeling of Sup35 assembled into amyloid fibrils.     

Engineering a therapeutic IgG molecule to address cysteinylation,aggregation and enhance thermal stability and expression

Antibodies can undergo a variety of covalent and non-covalent degradation reactions that have adverse effects on efficacy, safety, manufacture and storage. We had identified an antibody to Angiopoietin 2 (Ang2 mAb) that neutralizes Ang2 binding to its receptor in vitro and inhibits tumor growth in vivo. Despite favorable pharmacological activity, the Ang2 mAb preparations were heterogeneous, aggregated rapidly and were poorly expressed. Here, we report the engineering of the antibody variable and constant domains to generate an antibody with reduced propensity to aggregate, enhanced homogeneity, 11°C elevated T_m, 26-fold improved level of expression and retained activity. The engineered molecule, MEDI-3617, is now compatible with the large scale material supply required for clinical trials and is currently being evaluated in Phase 1 in cancer patients. This is the first report to describe the stability engineering of a therapeutic antibody addressing non canonical cysteine residues and the design strategy reported here is generally applicable to other therapeutic antibodies and proteins.     

DISC1 and the aggresome

Disrupted in Schizophrenia 1 (DISC1) is a key susceptibility gene for major psychiatric disorders. DISC1 plays a role in key neuronal processes such as neuronal proliferation, migration, integration and function via DISC1's roles at the centrosome and synapse, and in the regulation of intracellular signaling pathways. Recently, the idea of protein aggregation as a disease mechanism for DISC1 has been suggested. In our recent paper we explore these DISC1 protein aggregates in cell lines and neurons and find they are recruited to the aggresome and cause disruption of DISC1 function in intracellular transport.     

Stimulation of autophagy is neuroprotective in a mouse model of human tauopathy

The most common neurodegenerative diseases are characterized by the accumulation of misfolded proteins. Tauopathies, which include Alzheimer disease, progressive supranuclear palsy, corticobasal degeneration, Pick disease and cases of frontotemporal dementia and parkinsonism linked to chromosome 17, are characterized by the accumulation of hyperphosphorylated and filamentous MAPT/tau protein. The pathological mechanisms involved in MAPT protein accumulation are not well understood, but a possible impairment of protein degradation pathways has been suggested. We investigated the effects of autophagy stimulation on MAPT pathology in a model tauopathy, the human mutant P301S MAPT transgenic mouse line. In the brain of the trehalose-treated mutant mice, autophagy is activated and a reduced number of neurons containing MAPT inclusions, as well as a decreased amount of insoluble MAPT, are observed. The improvement of MAPT pathology is associated with increased nerve cell survival. Moreover, MAPT inclusions colocalize with SQSTM1/p62- and LC3-positive puncta, suggesting the colocalization of MAPT aggregates with autophagic vacuoles. Autophagy is not activated in the spinal cord of the human P301S MAPT transgenic mice and neuronal survival, as well as MAPT pathology, is unaffected. This study supports a role for autophagy stimulation in the degradation of MAPT aggregates and opens new perspectives for the investigation of autophagy as a pathological mechanism involved in neurodegenerative diseases.     

Investigating food limitations in wild fisheries: the attendance and growth responses of fish at an anthropogenic feeding station within a temperate estuary

To investigate whether wild fish populations are food limited, this study explored whether the provision of supplementary food had a positive effect on the abundance, condition and growth characteristics of estuarine fish assemblages in New Zealand. Feed (7690 kg) was delivered from an anthropogenic feeding station over a 60-week period to a naturally occurring assemblage of wild fish. Yellow-eyed mullet (Aldrichetta forsteri) of juvenile, sub-adult and newly matured size classes were the dominant species actively foraging at the feeding station throughout its operation, whereas larger piscivorous species visited and foraged from the feed station over the summer period only. Other species were present in the wider area of Nelson Haven, but not at the feed station. No obvious changes in the condition factor of yellow-eyed mullet were observed across the period of monitoring, and no changes in their catchability were detected – although marked seasonal variation in catch rates was observed. Results from tag-recapture data identify that the length-based growth rates of yellow-eyed mullet recaptured near the feed station were higher than those of tagged individuals recaptured at a nearby comparison site, and were higher than the growth rates observed in natural populations of yellow-eyed mullet in the wider region. Shifts in the median size of fish, as observed by acoustic observations, agreed with the tag-recapture data. Although some of the results identified were not amenable to statistical analyses, the attendance of yellow-eyed mullet at the feeding station, in combination with the improved estimates of growth by most of the techniques employed, indicates that yellow-eyed mullet are food limited in their natural environment and that the growth performance of these fish can be positively affected by the increased availability of food.     

A study of the abundance and patchiness of cicada nymphs (Homoptera: Tibicinidae) in a New Zealand subalpine shrub-grassland

Abstract

Cicada emergence skins in a subalpine shrub grassland have been sampled during 1969–75 to determine the abundance and spatial distributions of nymphs feeding on plant roots. A guild of six cicada species is primarily associated with two forms of vegetation: shrubs (Dracophyllum and Cassinia) and tall tussock (Chionochloa). Skin locations were mapped relative to dominant vegetation species, litter zones, and soil and rock pavements over a range of aspects, altitudes, and vegetation types, and sampling methods were scaled at four levels: the locality, plot, quadrat, and individual plant. There were significant differences in skin counts over four years, and different measures of mean skin densities are given for the four sampling scales. The two primary vegetation types had cumulative 1969–72 mean densities of 5.2 ± 4.0 and 12.9 ± 10.0 skins/quadrat (2.3 m²) , and the 1969–72 mean productivities of the upper 25% of quadrats (adjusted for percent ground cover) were, respectively, 5.5 and 35.5 skins/m². These productivities are believed to be conservative estimates of the maturing nymph numbers per individual host plant over the span of one cicada generation. Over a 17-year span, such productivities lie within the upper range of mean densities recorded for 17-year periodical cicadas in the United States. As the dominant subalpine vegetation species are very slow-growing,it is suggested that high densities of nymphs feeding on root sap may affect plant vitality, although 1971/1987 comparisons of vitality in 52 Chionochloa tussocks could not positively demonstrate a correlation across all data. Skin dispersion analyses indicated significant levels of patchiness, in agreement with other nymphal studies and with known cicada oviposition behaviour. No single dispersion model fitted the data comprehensively, and it is suggested that a gradual shifting of the centres of cicada aggregation may occur over a cumulative period of several generations.     

Regulation of β-catenin stabilization in human platelets

The Wnt/β-catenin pathway controls developmental processes and homeostasis; however, abnormal activation of this pathway has been linked to several human diseases. Recent reports have demonstrated regulation of platelet function by canonical and non-canonical Wnt signalling. Platelet aggregation plays a crucial role in haemostasis and thrombosis. Here we report for the first time that, induction of sustained aggregation of platelets by a strong agonist in the presence of calcium was associated with nearly complete proteolysis of β-catenin, which was abrogated upon depletion of calcium from platelet suspension. β-catenin cleavage was disallowed in absence of aggregation, thus implicating integrin α_IIbβ₃ engagement in β-catenin proteolysis. Degradation of β-catenin was blocked partially by inhibitors of either proteasome or calpain and completely when cells were exposed to both the inhibitors. Protein kinase C inhibition, too, abolished β-catenin degradation. Thus activities of proteasome, calpain and protein kinase C regulate stabilization of β-catenin in aggregated human platelets.     

Aerosol generation using nanometer liposome suspensions for pulmonary drug delivery applications

Abstract

Pulmonary lung targeting finds applications in drug delivery to the lung itself and to other body organs, via blood circulation following transfer across alveolar membranes. Understanding pulmonary drug delivery systems towards improving their efficacy needs identification of particle sizes of relevance and elucidation of links between suspension properties, techniques of atomisation and properties of the generated aerosols. This review article is focussed on understanding the elements of pulmonary drug delivery, specifically related to suspensions of small liposomes. Specific objectives of this review include (a) understanding aerosol particle deposition and absorption on pulmonary surface, (b) links between properties of aerosol generation and colloidal drug carriers used for drug encapsulation, and (c) investigation on the controlled properties of liposome aerosols generated using different atomisation techniques for efficacious aerosol therapy.     

Tryptophan to Glycine mutation in the position 116 leads to protein aggregation and decreases the stability of the LITAF protein

Mutations in the gene-encoding vesicle lipopolysaccharide-induced tumor necrosis factor (LITAF) protein cause Charcot–Marie–Tooth type 1C (CMT1C) disease, a neurological disorder. The LITAF gene is mapped to chromosome number 16 and can be found at cytogenetic location 16p13 of the chromosome. CMT1C-linked small integral membrane protein of lysosome/late endosome mutants are loss-of-function mutants that act in a dominant negative manner to impair endosomal trafficking, leading to prolonged extracellular signal-regulated kinases 1/2 signaling downstream of ErbB activation. Mutation W116G in the LITAF decreases the stability of the protein and also interrupts the functioning of gene. We have analyzed the single nucleotide polymorphism (SNP) results of 28 nsSNPs obtained from dbSNP. We also carried out multiple molecular dynamics simulations of 200 ns and obtained results of root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent-accessible surface area, H-bond, and principal component analysis to check and prove the stability of both the wild type and the mutant. The protein was then checked for its aggregation and the results showed loss of helix. The loss of helix leads to the instability of the protein.