Interpretando a crítica cultural

Oligomeric aggregates are widely suspected as toxic agents in diseases caused by protein aggregation, yet they remain poorly characterized, partly because they are challenging to isolate from a heterogeneous mixture of species. We developed an assay for characterizing structure, stability, and kinetics of individual oligomers at high resolution and sensitivity using single-molecule force spectroscopy, and applied it to observe the formation of transient structured aggregates within single oligomers of α-synuclein, an intrinsically-disordered protein linked to Parkinson's disease. Measurements of the molecular extension as the proteins unfolded under tension in optical tweezers revealed that even small oligomers could form numerous metastable structures, with a surprisingly broad range of sizes. Comparing the structures formed in monomers, dimers and tetramers, we found that the average mechanical stability increased with oligomer size. Most structures formed within a minute, with size-dependent rates. These results provide a new window onto the complex α-synuclein aggregation landscape, characterizing the microscopic structural heterogeneity and kinetics of different pathways

Energy landscape analysis of native folding of the prion protein yields the diffusion constant, transition path time, and rates

Protein folding is described conceptually in terms of diffusion over a configurational free-energy landscape, typically reduced to a one-dimensional profile along a reaction coordinate. In principle, kinetic properties can be predicted directly from the landscape profile using Kramers theory for diffusive barrier crossing, including the folding rates and the transition time for crossing the barrier. Landscape theory has been widely applied to interpret the time scales for protein conformational dynamics, but protein folding rates and transition times have not been calculated directly from experimentally measured free-energy profiles. We characterized the energy landscape for native folding of the prion protein using force spectroscopy, measuring the change in extension of a single protein molecule at high resolution as it unfolded/refolded under tension. Key parameters describing the landscape profile were first recovered from the distributions of unfolding and refolding forces, allowing the diffusion constant for barrier crossing and the transition path time across the barrier to be calculated. The full landscape profile was then reconstructed from force-extension curves, revealing a double-well potential with an extended, partially unfolded transition state. The barrier height and position were consistent with the previous results. Finally, Kramers theory was used to predict the folding rates from the landscape profile, recovering the values observed experimentally both under tension and at zero force in ensemble experiments. These results demonstrate how advances in single-molecule theory and experiment are harnessing the power of landscape formalisms to describe quantitatively the mechanics of folding.Peer reviewed: YesNRC publication: Ye

FECs of α-synuclein dimers.

<p>(A, B) Representative FECs of a dimer show unfolding of stable structures with a wide range of sizes and unfolding forces. WLC fits to determine contour length changes are displayed as dashed lines (grey: folded states, red: unfolded state). Inset: the dimer contains two monomers connected by short, flexible peptides linkers. (C) Histogram of Δ<i>L</i>_c for all identifiable transitions in dimer FECs.</p

Clinical and biochemical phenotypes, genotypes, and long-term outcomes of individuals with galactosemia type I from a single metabolic genetics center in Alberta

Background: Galactosemia type I is an autosomal recessive disorder of galactose metabolism due to galactose-1-phosphate uridyltransferase deficiency, encoded by GALT. To investigate the phenotypes, genotypes and long-term outcomes of galactosemia, we performed a retrospective cohort study in our center. Methods: All individuals with galactosemia type I were included. We divided individuals into two groups to compare the outcomes of those treated symptomatically (SymX) and asymptomatically (AsymX). We reviewed electronic patient charts for clinical features, biochemical investigations, molecular genetic investigations, treatments, and outcomes. Results: There were 25 individuals including classic (n = 17), clinical variant (n = 4), and biochemical variant (Duarte) galactosemia (n = 4). Twelve individuals were diagnosed symptomatically (SymX), and 9 individuals were diagnosed asymptomatically (AsymX). We did not include individuals with biochemical variant (Duarte) galactosemia into any of these groups. At the time of the diagnosis, conjugated hyperbilirubinemia was present in 83.3% of SymX group, whereas only 22% of AsymX group. SymX group had hepatomegaly (25%), failure to thrive (33.3%), cataract (16.7%) and sepsis (25%), whereas none of the individuals in the AsymX group had these clinical features. Fourteen variants in GALT were identified including pathogenic/likely pathogenic (n = 12), and likely benign/benign (n = 2) variants. The vast majority of individuals with classic and clinical variant galactosemia were treated with a galactose-lactose-free diet for life (n = 20/21). Intellectual disability was present in 54.5% of the SymX group, and in 37.5% of the AsymX group as a long-term outcome. Tremors were present 50% of the SymX group, and in 22% of the AsymX group as a long-term outcome. Although, intellectual disability and tremors seem to be less common in the AsymX group, there was no statistically significant difference between both groups. Primary ovarian insufficiency was present 50% of the SymX group, whereas in 20% of the AsymX group in post-pubertal females. We report a novel hypomorphic GALT variant (p.Ala303Ser) in one individual with clinical variant galactosemia. We also report an individual with clinical variant galactosemia with normal urine galactitol levels on a normal diet. Conclusion: It seems that newborn screening and early administration of a galactose-lactose-free diet decreases the long-term galactosemia-associated complications but does not prevent them completely. It may be that not all individuals with clinical variant galactosemia may need a galactose-lactose-free diet. It is timely to find new therapeutic strategies that can reduce the frequency of late-onset complications in galactosemia

Dynamic force spectroscopy.

<p>Loading rate dependence of the average unfolding force for the two most frequent transitions: Δ<i>L</i>_c = 11–13 nm for the dimer (blue) and Δ<i>L</i>_c = 16–18 nm for the tetramer (red). Fits to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086495#pone.0086495.e002" target="_blank">Equation 2</a> yield the unfolding rates at zero force, ∼0.1 s⁻¹, and the distance to the barrier for unfolding, ∼1 nm.</p

Contour length and unfolding force distributions.

<p>(A) Histogram of Δ<i>L</i>_c for all identifiable transitions in FECs of the tetramer (red), dimer (blue), and monomer (black). (B) Scatterplot of <i>F</i>_u vs Δ<i>L</i>_c for tetramer (red), dimer (blue), and monomer (black). Arrows indicate Δ<i>L</i>_c values consistent with a β-sandwich structure, asterisks indicate Δ<i>L</i>_c values expected from a helical multimer structure (blue: dimer and tetramer, red: tetramer only). Dashed lines indicate the contour lengths of the entire monomer (black), dimer (blue), or tetramer (red). (C) Histograms of <i>F</i>_u for the tetramer (red), dimer (blue), and monomer (black) show an increase in <i>F</i>_u with increasing oligomer size.</p

FECs of α-synuclein tetramers.

<p>(A, B) Representative FECs of a tetramer reveal many structures with different sizes and unfolding forces. WLC fits are shown as dashed lines (grey: folded states, red: unfolded state). Inset: the tetramer contains four α-synuclein domains connected by short, flexible peptide linkers. (C) Histogram of Δ<i>L</i>_c for all identifiable transitions in FECs of the tetramer.</p

Direct observation of multiple misfolding pathways in a single prion protein molecule

Protein misfolding is a ubiquitous phenomenon associated with a wide range of diseases. Single-molecule approaches offer a powerful tool for deciphering the mechanisms of misfolding by measuring the conformational fluctuations of a protein with high sensitivity. We applied single-molecule force spectroscopy to observe directly the misfolding of the prion protein PrP, a protein notable for having an infectious misfolded state that is able to propagate by recruiting natively folded PrP. By measuring folding trajectories of single PrP molecules held under tension in a high-resolution optical trap, we found that the native folding pathway involves only two states, without evidence for partially folded intermediates that have been proposed to mediate misfolding. Instead, frequent but fleeting transitions were observed into off-pathway intermediates. Three different misfolding pathways were detected, all starting from the unfolded state. Remarkably, the misfolding rate was even higher than the rate for native folding. A mutant PrP with higher aggregation propensity showed increased occupancy of some of the misfolded states, suggesting these states may act as intermediates during aggregation. These measurements of individual misfolding trajectories demonstrate the power of single-molecule approaches for characterizing misfolding directly by mapping out nonnative folding pathways.Peer reviewed: YesNRC publication: Ye