Molecular characterization of an aggregation-prone variant of alpha-synuclein used to model synucleinopathies

The misfolding and aggregation of alpha-synuclein (aSyn) are thought to be central events in synucleinopathies. The physiological function of aSyn has been related to vesicle binding and trafficking, but the precise molecular mechanisms leading to aSyn pathogenicity are still obscure. In cell models, aSyn does not readily aggregate, even upon overexpression. Therefore, cellular models that enable the study of aSyn aggregation are essential tools for our understanding of the molecular mechanisms that govern such processes. Here, we investigated the structural features of SynT, an artificial variant of aSyn that has been widely used as a model of aggregation in mammalian cell systems, since it is more prone to aggregation than aSyn. Using Nuclear Magnetic Resonance (NMR) spectroscopy we performed a detailed structural characterization of SynT through a systematic comparison with normal, unmodified aSyn. Interestingly, we found that the conformations adopted by SynT resemble those described for the unmodified protein, demonstrating the usefulness of SynT as a model for aSyn aggregation. However, subtle differences were observed at the N-terminal region involving transient intra and/or intermolecular interactions that are known to regulate aSyn aggregation. Importantly, our results indicate that disturbances in the N-terminal region of SynT, and the consequent decrease in membrane binding of the modified protein, might contribute to the observed aggregation behavior of aSyn, and validate the use of SynT, one of the few models of aSyn aggregation in cultured cells

Probing the interstitial calcium compartment

Calcium in interstitial fluids is a crucial ion pool for entry into cells through a plethora of calcium-permeable channels. It is also sensed actively by dedicated receptors. While the mechanisms of global calcium homeostasis and regulation in body fluids appear well understood, more efforts and new technology are needed to elucidate local calcium handling in the small and relatively isolated interstitial spaces between cells. Here we review current methodology for monitoring interstitial calcium and highlight the potential of new approaches for its study. In particular, new generations of high-performance low-affinity genetically encoded calcium indicators could allow imaging of calcium in relatively inaccessible intercellular structures in live tissues and organisms

Binding modes of phthalocyanines of amyloid β peptide and their effects on amyloid fibril formation

The inherent tendency of proteins to convert from their native states into amyloid aggregates is associated with a range of human disorders, including Alzheimer's and Parkinson's diseases. In that sense, the use of small molecules as probes for the structural and toxic mechanism related to amyloid aggregation has become an active area of research. Compared with other compounds, the structural and molecular basis behind the inhibitory interaction of phthalocyanine tetrasulfonate (PcTS) with proteins such as αS and tau has been well established, contributing to a better understanding of the amyloid aggregation process in these proteins. We present here the structural characterization of the binding of PcTS and its Cu(II) and Zn(II)-loaded forms to the amyloid β-peptide (Aβ) and the impact of these interactions on the peptide amyloid fibril assembly. Elucidation of the PcTS binding modes to Aβ40 revealed the involvement of specific aromatic and hydrophobic interactions in the formation of the Aβ40-PcTS complex, ascribed to a binding mode in which the planarity and hydrophobicity of the aromatic ring system in the phthalocyanine act as main structural determinants for the interaction. Our results demonstrated that formation of the Aβ40-PcTS complex does not interfere with the progression of the peptide toward the formation of amyloid fibrils. On the other hand, conjugation of Zn(II) but not Cu(II) at the center of the PcTS macrocyclic ring modified substantially the binding profile of this phthalocyanine to Aβ40 and became crucial to reverse the effects of metal-free PcTS on the fibril assembly of the peptide. Overall, our results provide a firm basis to understand the structural rules directing phthalocyanine-protein interactions and their implications on the amyloid fibril assembly of the target proteins; in particular, our results contradict the hypothesis that PcTS might have similar mechanisms of action in slowing the formation of a variety of pathological aggregates

Structural basis behind the interaction of Zn2+ with the protein α-synuclein and the Aβ peptide: A comparative analysis.

α-Synuclein (AS) aggregation is associated to neurodegeneration in Parkinson's disease (PD). At the same time, alterations in metal ion homeostasis may play a pivotal role in the progression of AS amyloid assembly and the onset of PD. Elucidation of the structural basis directing AS–metal interactions and their effect on AS aggregation constitutes a key step towards understanding the role of metal ions in AS amyloid formation and neurodegeneration. Despite of the reported evidences that link Zn2+ with the pathophysiology of PD and the fact that this metal ion was shown to promote AS fibrillation in vitro, neither the structural characterization of the binding sites nor the identification of the amino acids involved in the interaction of Zn2+ with the protein AS has been carried out. By using NMR spectroscopy, we have addressed here unknown structural details related to the binding of Zn2+ to the protein AS through the design of site-directed and domain truncated mutants of AS. The binding of zinc to the Aβ peptide was also studied and discussed comparatively. Although the results of this study contribute to the understanding of the structural and molecular basis behind the acceleration of AS fibrillation mediated by Zn2+, the low affinity that characterizes the interaction of Zn2+ with AS contrasts strongly with the high-affinity features reported for the binding of this metal ion to other target proteins linked to human amylodosis such as Aβ peptide and the Islet Amyloid Polypeptide (IAPP), challenging the biological relevance of zinc interactions in the pathogenesis of PD

Phthalocyanines as Molecular Scaffolds to Block Disease-Associated Protein Aggregation

ConspectusThe aggregation of proteins into toxic conformations plays a critical role in the development of different neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Creutzfled-Jakob's disease (CJD). These disorders share a common pathological mechanism that involves the formation of aggregated protein species including toxic oligomers and amyloid fibrils. The aggregation of alpha-synuclein (αS) in PD and the amyloid beta peptide (Aβ) and tau protein in AD results in neuronal death and disease onset. In the case of CJD, the misfolding of the physiological prion protein (PrP) induces a chain reaction that results in accumulation of particles that elicit brain damage. Currently, there is no preventive therapy for these diseases and the available therapeutic approaches are based on the treatment of the symptoms rather than the underlying causes of the disease. Accordingly, the aggregation pathway of these proteins represents a useful target for therapeutic intervention. Therefore, understanding the mechanism of amyloid formation and its inhibition is of high clinical importance. The design of small molecules that efficiently inhibit the aggregation process and/or neutralize its associated toxicity constitutes a promising tool for the development of therapeutic strategies against these disorders. In this accounts, we discuss current knowledge on the anti-amyloid activity of phthalocyanines and their potential use as drug candidates in neurodegeneration. These tetrapyrrolic compounds modulate the amyloid assembly of αS, tau, Aβ, and the PrP in vitro, and protect cells from the toxic effects of amyloid aggregates. In addition, in scrapie-infected mice, these compounds showed important prophylactic antiscrapie properties. The structural basis for the inhibitory effect of phthalocyanines on amyloid filament assembly relies on specific π-π interactions between the aromatic ring system of these molecules and aromatic residues in the amyloidogenic proteins. Analysis of the structure-activity relationship in phthalocyanines revealed that their anti-amyloid activity is highly dependent on the type of metal ion coordinated to the tetrapyrrolic system but is not sensitive to the number of peripheral charged substituents. The tendency of phthalocyanines to oligomerize (self-association) via aromatic-aromatic stacking interactions correlates precisely with their binding capabilities to target proteins and, more importantly, determines their efficiency as anti-amyloid agents. The ability to block different types of disease-associated protein aggregation raises the possibility that these cyclic tetrapyrrole compounds have a common mechanism of action to impair the formation of a variety of pathological aggregates. Because the structural and molecular basis for the anti-amyloid effects of these molecules is starting to emerge, combined efforts from the fields of structural, cellular, and animal biology will result critical for the rational design and discovery of new drugs for the treatment of amyloid related neurological disorders.Fil: Valiente Gabioud, Ariel Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: Miotto, Marco César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: Chesta, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: Lombardo, Veronica Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: Binolfi, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: Fernandez, Claudio Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; Argentin

Effect of repetitiveness on the immunogenicity and antigenicity of Trypanosoma cruzi FRA protein

Repetitive proteins (RP) of Trypanosoma cruzi are highly present in the parasite and are strongly recognized by sera from Chagas' disease patients. Flagelar Repetitive Antigen (FRA), which is expressed in all steps of the parasite life cycle, is the RP that displays the greatest number of aminoacids per repeat and has been indicated as one of the most suitable candidate for diagnostic test because of its high performance in immunoassays. Here we analyzed the influence of the number of repeats on the immunogenic and antigenic properties of the antigen. Recombinant proteins containing one, two, and four tandem repeats of FRA (FRA1, FRA2, and FRA4, respectively) were obtained and the immune response induced by an equal amount of repeats was evaluated in a mouse model. The reactivity of specific antibodies present in sera from patients naturally infected with T. cruzi was also assessed against FRA1, FRA2, and FRA4 proteins, and the relative avidity was analyzed. We determined that the number of repeats did not increase the humoral response against the antigen and this result was reproduced when the repeated motifs were alone or fused to a non-repetitive protein. By contrast, the binding affinity of specific human antibodies increases with the number of repeated motifs in FRA antigen. We then concluded that the high ability of FRA to be recognized by specific antibodies from infected individuals is mainly due to a favorable polyvalent interaction between the antigen and the antibodies. In accordance with experimental results, a 3D model was proposed and B epitope in FRA1, FRA2, and FRA4 were predicted