Influence of Gold Nanoparticles on the Kinetics of alpha-Synuclein Aggregation
2013; 13 (12): 6156-6163
Highly Solvatochromic 7-Aryl-3-hydroxychromones
JOURNAL OF PHYSICAL CHEMISTRY LETTERS
2012; 3 (8): 1011-1016
Supramolecular Non-Amyloid Intermediates in the Early Stages of alpha-Synuclein Aggregation
2012; 102 (5): 1127-1136
α-synuclein (AS) is a small (140 amino acids), abundant presynaptic protein, which lacks a unique secondary structure in aqueous solution. Amyloid aggregates of AS in dopaminergic neurons of the midbrain are the hallmark of Parkinson's disease (PD). The process of aggregation involves a series of complex structural transitions from innocuous monomeric AS to oligomeric, presumably neurotoxic, forms and finally to fibril formation. Despite its potential importance for understanding PD pathobiology and devising rational, targeted therapeutic strategies, the details of the aggregation process remain largely unknown. Methodologies and reagents capable of controlling the aggregation kinetics are essential tools for the investigation of the molecular mechanisms of amyloid diseases. In this work, we investigated the influence of citrate-capped gold nanoparticles on the aggregation kinetics of AS using a fluorescent probe (MFC) sensitive to the polarity of the molecular microenvironment via excited state intramolecular proton transfer (ESIPT). The particular effects on the half time, nucleation time, and growth rate were ascertained. Gold nanoparticles produced a strong acceleration of protein aggregation with an influence on both the nucleation and growth phases of the overall mechanism. The effects were dependent on the size and concentration of the nanoparticles, being strongest for nanoparticles 10 nm in diameter, which produced a 3-fold increase in the overall aggregation rate at concentrations as low as 20 nM.
View details for DOI 10.1021/nl403490e
View details for Web of Science ID 000328439200061
View details for PubMedID 24219503
Fluorescent Ratiometric MFC Probe Sensitive to Early Stages of alpha-Synuclein Aggregation
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2010; 132 (23): 7860-?
The aggregation of α-synuclein is associated with progression of Parkinson's disease. We have identified submicrometer supramolecular structures that mediate the early stages of the overall mechanism. The sequence of structural transformations between metastable intermediates were captured and characterized by atomic force microscopy guided by a fluorescent probe sensitive to preamyloid species. A novel ~0.3-0.6 μm molecular assembly, denoted the acuna, nucleates, expands, and liberates fibers with distinctive segmentation and a filamentous fuzzy fringe. These fuzzy fibers serve as precursors of mature amyloid fibrils. Cryo-electron tomography resolved the acuna inner structure as a scaffold of highly condensed colloidal masses interlinked by thin beaded threads, which were perceived as fuzziness by atomic force microscopy. On the basis of the combined data, we propose a sequential mechanism comprising molecular, colloidal, and fibrillar stages linked by reactions with disparate temperature dependencies and distinct supramolecular forms. We anticipate novel diagnostic and therapeutic approaches to Parkinson's and related neurodegenerative diseases based on these new insights into the aggregation mechanism of α-synuclein and intermediates, some of which may act to cause and/or reinforce neurotoxicity.
View details for DOI 10.1016/j.bpj.2012.01.051
View details for Web of Science ID 000301280900019
View details for PubMedID 22404935
Biophysical properties and cellular toxicity of covalent crosslinked oligomers of alpha-synuclein formed by photoinduced side-chain tyrosyl radicals
FREE RADICAL BIOLOGY AND MEDICINE
2012; 53 (4): 1004-1015
We introduce a sensor molecule, AS140-MFC, consisting of a covalent adduct of an Ala-to-Cys mutant of alpha-synuclein with the 3-hydroxychromone dual emission dye MFC. We show that the AS140-MFC construct is a multiparametric fluorescent probe suitable for the continuous monitoring of protein aggregation and is sensitive to the early and intermediate stages of alpha-synuclein aggregation, a process associated with Parkinson's disease.
View details for DOI 10.1021/ja102838n
View details for Web of Science ID 000278717700019
View details for PubMedID 20491471
Voltamperometric discrimination of urea and melamine adulterated skimmed milk powder.
2012; 12 (9): 12220-12234
Alpha-synuclein (αS), a 140 amino acid presynaptic protein, is the major component of the fibrillar aggregates (Lewy bodies) observed in dopaminergic neurons of patients affected by Parkinson's disease. It is currently believed that noncovalent oligomeric forms of αS, arising as intermediates in its aggregation, may constitute the major neurotoxic species. However, attempts to isolate and characterize such oligomers in vitro, and even more so in living cells, have been hampered by their transient nature, low concentration, polymorphism, and inherent instability. In this work, we describe the preparation and characterization of low molecular weight covalently bound oligomeric species of αS obtained by crosslinking via tyrosyl radicals generated by blue-light photosensitization of the metal coordination complex ruthenium (II) tris-bipyridine in the presence of ammonium persulfate. Numerous analytical techniques were used to characterize the αS oligomers: biochemical (anion-exchange chromatography, SDS-PAGE, and Western blotting); spectroscopic (optical: UV/Vis absorption, steady state, dynamic fluorescence, and dynamic light scattering); mass spectrometry; and electrochemical. Light-controlled protein oligomerization was mediated by formation of Tyr-Tyr (dityrosine) dimers through -C-C- bonds acting as covalent bridges, with a predominant involvement of residue Y39. The diverse oligomeric species exhibited a direct effect on the in vitro aggregation behavior of wild-type monomeric αS, decreasing the total yield of amyloid fibrils in aggregation assays monitored by thioflavin T (ThioT) fluorescence and light scattering, and by atomic force microscopy (AFM). Compared to the unmodified monomer, the photoinduced covalent oligomeric species demonstrated increased toxic effects on differentiated neuronal-like SH-SY5Y cells. The results highlight the importance of protein modification induced by oxidative stress in the initial molecular events leading to Parkinson's disease.
View details for DOI 10.1016/j.freeradbiomed.2012.06.035
View details for Web of Science ID 000307920100035
View details for PubMedID 22771470
Nitrogen compounds like urea and melamine are known to be commonly used for milk adulteration resulting in undesired intoxication; a well-known example is the Chinese episode occurred in 2008. The development of a rapid, reliable and economic test is of relevance in order to improve adulterated milk identification. Cyclic voltammetry studies using an Au working electrode were performed on adulterated and non-adulterated milk samples from different independent manufacturers. Voltammetric data and their first derivative were subjected to functional principal component analysis (f-PCA) and correctly classified by the KNN classifier. The adulterated and non-adulterated milk samples showed significant differences. Best results of prediction were obtained with first derivative data. Detection limits in milk samples adulterated with 1% of its total nitrogen derived from melamine or urea were as low as 85.0 mg · L(-1) and 121.4 mg · L(-1), respectively. We present this method as a fast and robust screening method for milk adulteration analysis and prevention of food intoxication.
View details for DOI 10.3390/s120912220
View details for PubMedID 23112709