Amyloid

Although most proteins can assemble into amyloid-like fibrils in vitro under extreme conditions, how proteins form amyloid fibrils in vivo remains unresolved. Identifying rare aggregation-prone species under physiologically relevant conditions and defining their structural properties is therefore an important challenge. By solving the folding mechanism of the naturally amyloidogenic protein beta-2-microglobulin at pH 7.0 and 37 degrees C and correlating the concentrations of different species with the rate of fibril elongation, we identify a specific folding intermediate, containing a non-native trans-proline isomer, as the direct precursor of fibril elongation. Structural analysis using NMR shows that this species is highly native-like but contains perturbation of the edge strands that normally protect beta-sandwich proteins from self-association. The results demonstrate that aggregation pathways can involve self-assembly of highly native-like folding intermediates, and have implications for the prevention of this, and other, amyloid disorders.

Many relevant questions in biology and medicine require both topography and chemical information with high spatial resolution. Several biological events that occur at the nanometer scale level need to be investigated in physiological conditions. In this regard Atomic Force Microscopy (AFM) is one of the most powerful tools for label-free nanoscale characterization of biological samples in liquid environment. Recently, the coupling of Raman spectroscopy to scanning probe microscopies has opened new perspectives on this subject; however, the coupling of quality AFM spectroscopy with Raman spectroscopy in the same probe is not trivial. In this work we report about the AFM capabilities of an advanced high-resolution probe that has been previously nanofabricated by our group for coupling with Raman spectroscopy applications. We investigate its use for liquid AFM measurements on biological model samples like lipid bilayers, amyloid fibrils, and titin proteins. We demonstrate topography resolution down to nanometer level, force measurement and stable imaging capability. We also discuss about its potential as nanoscale chemical probe in liquid phase. Microsc. Res. Tech. 00:000-000, 2012. V V C 2012 Wiley Periodicals, Inc.

Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes). They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF. Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded β-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The transmembrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric ...

Purpose Despite good to excellent inter-reader agreement in
the evaluation of amyloid load on PET scans in subjects with
Alzheimer's disease, some equivocal findings have been reported
in the literature.We aimed to describe the clinical characteristics
of subjects with equivocal PET images.
Methods Nondemented subjects aged 70 years or more were
enrolled from the MAPT trial. Cognitive and functional assessments
were conducted at baseline, at 6months, and annually for
3 years. During the follow-up period, 271 subjects had 18FAV45
PET scans. Images were visually assessed by three observers
and classified as positive, negative or equivocal (if one
observer disagreed). After debate, equivocal images were
reclassified as positive (EP+) or negative (EP−). Scans were
also classified by semiautomated quantitative analysis using
mean amyloid uptake of cortical regions. We evaluated agreement
among the observers, and between visual and quantitative
assessments using kappa coefficients, and compared the clinical
characteristics of the subjects according to their PET results.
Results In 158 subjects (58.30 %) the PET scan was negative
for amyloid, in 77 (28.41 %) the scan was positive and in 36
(13.28 %) the scan was equivocal. Agreement among the three
observers was excellent (kappa 0.80). Subjects with equivocal
images were more frequently men (58 % vs. 37 %) and exhibited
intermediate scores on cognitive and functional scales between
those of subjects with positive and negative scans. Amyloid
load differed between the EP− and negative groups and
between the EP+ and positive groups after reclassification.
Conclusion Equivocal amyloid PET images could represent a
neuroimaging entity with intermediate amyloid load but without a specific neuropsychological pattern. Clinical
follow-up to assess cognitive evolution in subjects with equivocal scans is needed.

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Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer's disease, Parkinson's disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin prevents amyloid fibril formation of kappa-casein, amyloid beta peptide and a-synuclein by blocking b-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and cs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases.

Amyloid is associated with debilitating human ailments including Alzheimer's and prion diseases. Biochemical, biophysical, and imaging analyses revealed that fibers produced by Escherichia coli called curli were amyloid. The CsgA curlin subunit, purified in the absence of the CsgB nucleator, adopted a soluble, unstructured form that upon prolonged incubation assembled into fibers that were indistinguishable from curli. In vivo, curli biogenesis was dependent on the nucleation-precipitation machinery requiring the CsgE and CsgF chaperone-like and nucleator proteins, respectively. Unlike eukaryotic amyloid formation, curli biogenesis is a productive pathway requiring a specific assembly machinery.

Amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease and similar neurodegenerative disorders take their toll on patients, caregivers and society. A common denominator for these disorders is the accumulation of aggregated proteins in nerve cells, yet the triggers for these aggregation processes are currently unknown. In ALS, protein aggregation has been described
for the SOD1, C9orf72, FUS and TDP-43 proteins. The latter is a nuclear protein normally binding to both DNA and RNA, contributing to gene expression and mRNA life cycle regulation. TDP-43 seems to have a specific role in ALS pathogenesis, and ubiquitinated and hyperphosphorylated cytoplasmic inclusions of aggregated TDP-43 are present in nerve cells in almost all sporadic ALS cases. ALS pathology appears to include metal imbalances, and environmental metal exposure is a known risk factor in ALS. However, studies on metal-to-TDP-43 interactions are scarce, even though this protein
seems to have the capacity to bind to metals. This review discusses the possible role of metals in TDP-43 aggregation, with respect to ALS pathology.