Effect of spermidine on misfolding and interactions of alpha-synuclein.

Alpha-synuclein (α-Syn) is a 140 aa presynaptic protein which belongs to a group of natively unfolded proteins that are unstructured in aqueous solutions. The aggregation rate of α-Syn is accelerated in the presence of physiological levels of cellular polyamines. Here we applied single molecule AFM force spectroscopy to characterize the effect of spermidine on the very first stages of α-Syn aggregation--misfolding and assembly into dimers. Two α-Syn variants, the wild-type (WT) protein and A30P, were studied. The two protein molecules were covalently immobilized at the C-terminus, one at the AFM tip and the other on the substrate, and intermolecular interactions between the two molecules were measured by multiple approach-retraction cycles. At conditions close to physiological ones at which α-Syn misfolding is a rare event, the addition of spermidine leads to a dramatic increase in the propensity of the WT and mutant proteins to misfold. Importantly, misfolding is characterized by a set of conformations, and A30P changes the misfolding pattern as well as the strength of the intermolecular interactions. Together with the fact that spermidine facilitates late stages of α-Syn aggregation, our data demonstrate that spermidine promotes the very early stages of protein aggregation including α-Syn misfolding and dimerization. This finding suggests that increased levels of spermidine and potentially other polyamines can initiate the disease-related process of α-Syn

Metabolic Signatures of Lung Cancer in Biofluids: NMR-Based Metabonomics of Blood Plasma

In this work, the variations in the metabolic profile of blood plasma from lung cancer patients and healthy controls were investigated through NMR-based metabonomics, to assess the potential of this approach for lung cancer screening and diagnosis. PLS-DA modeling of CPMG spectra from plasma, subjected to Monte Carlo Cross Validation, allowed cancer patients to be discriminated from controls with sensitivity and specificity levels of about 90%. Relatively lower HDL and higher VLDL + LDL in the patients' plasma, together with increased lactate and pyruvate and decreased levels of glucose, citrate, formate, acetate, several amino acids (alanine, glutamine, histidine, tyrosine, valine), and methanol, could be detected. These changes were found to be present at initial disease stages and could be related to known cancer biochemical hallmarks, such as enhanced glycolysis, glutaminolysis, and gluconeogenesis, together with suppressed Krebs cycle and reduced lipid catabolism, thus supporting the hypothesis of a systemic metabolic signature for lung cancer. Despite the possible confounding influence of age, smoking habits, and other uncontrolled factors, these results indicate that NMR-based metabonomics of blood plasma can be useful as a screening tool to identify suspicious cases for subsequent, more specific radiological tests, thus contributing to improved disease management.ERDF - Competitive Factors Thematic Operational ProgrammeFCT/PTDC/ QUI/68017/2006FCOMP-01-0124-FEDER-007439SFRH/BD/ 63430/2009National UNESCO Committee - L'Oréal Medals of Honor for Women in Science 200Portuguese National NMR Network - RNRM

Effect of Spermidine on Misfolding and Interactions of Alpha-Synuclein

Alpha-synuclein (α-Syn) is a 140 aa presynaptic protein which belongs to a group of natively unfolded proteins that are unstructured in aqueous solutions. The aggregation rate of α-Syn is accelerated in the presence of physiological levels of cellular polyamines. Here we applied single molecule AFM force spectroscopy to characterize the effect of spermidine on the very first stages of α-Syn aggregation – misfolding and assembly into dimers. Two α-Syn variants, the wild-type (WT) protein and A30P, were studied. The two protein molecules were covalently immobilized at the C-terminus, one at the AFM tip and the other on the substrate, and intermolecular interactions between the two molecules were measured by multiple approach-retraction cycles. At conditions close to physiological ones at which α-Syn misfolding is a rare event, the addition of spermidine leads to a dramatic increase in the propensity of the WT and mutant proteins to misfold. Importantly, misfolding is characterized by a set of conformations, and A30P changes the misfolding pattern as well as the strength of the intermolecular interactions. Together with the fact that spermidine facilitates late stages of α-Syn aggregation, our data demonstrate that spermidine promotes the very early stages of protein aggregation including α-Syn misfolding and dimerization. This finding suggests that increased levels of spermidine and potentially other polyamines can initiate the disease-related process of α-Syn

Bioinformatics tools for cancer metabolomics

It is well known that significant metabolic change take place as cells are transformed from normal to malignant. This review focuses on the use of different bioinformatics tools in cancer metabolomics studies. The article begins by describing different metabolomics technologies and data generation techniques. Overview of the data pre-processing techniques is provided and multivariate data analysis techniques are discussed and illustrated with case studies, including principal component analysis, clustering techniques, self-organizing maps, partial least squares, and discriminant function analysis. Also included is a discussion of available software packages

Schematic representation of the experimental approach.

<p>A) The unstructured form of alpha-synuclein immobilized on the tip and a substrate reveals no interaction. B) Misfolding of alpha-synuclein results in the formation of aggregation-prone conformation with elevated propensity of intermolecular interactions. C) A representative force-distance curve measured in the absence of spermidine. D) A representative force –distance curve with a rupture event in the presence of spermidine: (1) an adhesion peak due to short-range non-specific interactions between the tip and the surface, (2) gradual increase in force characteristic of polymer stretching, (3) complete rupture at 110 pN and (4) region where tip comes free from the surface. The inset shows worm-like chain fitting yielding contour length of 31 nm.</p

Interaction model of alpha-synuclein molecules.

<p>The model describes three major peaks in the contour length histograms as identical sites in each monomer interacting with each other with the formation of a dimer. A) Position of the interacting site further from the C-terminus (point of attachment) results in longer contour length value. B) Positions at the beginning of the detected interaction sites. Colored arrows correspond to three detected interaction sites schematically shown in A), and the black arrow shows the position of the A30P mutation in alpha-synuclein; C), D) and E) superposition of representative force-distance curves for the detected rupture events corresponding to L_C1, L_C2 and L_C3, red, green and blue lines on the graphs are WLC curves calculated with L_C values from Fig. 4.</p

α‑Synuclein Misfolding Assessed with Single Molecule AFM Force Spectroscopy: Effect of Pathogenic Mutations

Misfolding and subsequent aggregation of alpha-synuclein (α-Syn) protein are critically involved in the development of several neurodegenerative diseases, including Parkinson’s disease (PD). Three familial single point mutations, A30P, E46K, and A53T, correlate with early onset PD; however, the molecular mechanism of the effects of these mutations on the structural properties of α-Syn and its propensity to misfold remains unclear. Here, we address this issue utilizing a single molecule AFM force spectroscopy approach in which structural details of dimers formed by all four variants of α-Syn are characterized. Analysis of the force spectroscopy data reflecting contour length distribution for α-Syn dimer dissociation suggests that multiple segments are involved in the assembly of the dimer. The interactions are not limited to the central nonamyloid-beta component (NAC) of the protein but rather expand beyond this segment. All three mutations alter the protein’s folding and interaction patterns affecting interactions far beyond their immediate locations. Implementation of these findings to our understanding of α-Syn aggregation pathways is discussed

Complex force-distance curves with multiple rupture peaks detected with WT alpha-synuclein (A, B) and A30P mutant (C, D).

<p>Red, green and blue lines on the graphs are calculated WLC curves using maxima of Gaussians determined from contour length distributions corresponding to 26, 36 and 47 nm (WT) and 22, 34 and 49 nm (A30P). E) Pie charts showing relative contribution of multiple rupture events to total rupture events: 31% (WT) and 15% (A30P).</p

Effect of spermidine on the appearance of force-distance curves with A30P alpha-synuclein.

<p>Superposition of representative force-distance curves measured upon probing of interactions between A30P alpha-synuclein molecules: A) in the absence of spermidine (40 curves), B) with addition of 5 mM spermidine (58 curves).</p