Conformational equilibria in monomeric alpha-synuclein at the single molecule level

Natively unstructured proteins defy the classical "one sequence-one structure" paradigm of protein science. Monomers of these proteins in pathological conditions can aggregate in the cell, a process that underlies socially relevant neurodegenerative diseases such as Alzheimer and Parkinson. A full comprehension of the formation and structure of the so-called misfolded intermediates from which the aggregated states ensue is still lacking. We characterized the folding and the conformational diversity of alpha-synuclein (aSyn), a natively unstructured protein involved in Parkinson disease, by mechanically stretching single molecules of this protein and recording their mechanical properties. These experiments permitted us to directly observe directly and quantify three main classes of conformations that, under in vitro physiological conditions, exist simultaneously in the aSyn sample, including disordered and "beta-like" structures. We found that this class of "beta-like" structures is directly related to aSyn aggregation. In fact, their relative abundance increases drastically in three different conditions known to promote the formation of aSyn fibrils: the presence of Cu2+, the occurrence of the pathogenic A30P mutation, and high ionic strength. We expect that a critical concentration of aSyn with a "beta-like" structure must be reached to trigger fibril formation. This critical concentration is therefore controlled by a chemical equilibrium. Novel pharmacological strategies can now be tailored to act upstream, before the aggregation process ensues, by targeting this equilibrium. To this end, Single Molecule Force Spectroscopy can be an effective tool to tailor and test new pharmacological agents.Comment: 37 pages, 9 figures (including supplementary material

Toward a better definition of EPCAM deletions in Lynch Syndrome: Report of new variants in Italy and the associated molecular phenotype

BackgroundInherited epimutations of Mismatch Repair (MMR) genes are responsible for Lynch Syndrome (LS) in a small, but well defined, subset of patients. Methylation of the MSH2 promoter consequent to the deletion of the upstream EPCAM gene is found in about 1%-3% of the LS patients and represents a classical secondary, constitutional and tissue-specific epimutation. Several different EPCAM deletions have been reported worldwide, for the most part representing private variants caused by an Alu-mediated recombination.Methods712 patients with suspected LS were tested for MMR mutation in our Institute. EPCAM deletions were detected by multiplex ligation-dependent probe amplification (MLPA) and then defined by Long-Range polymerase chain reaction (PCR)/Sanger sequencing. A comprehensive molecular characterization of colorectal cancer (CRC) tissues was carried out by immunohistochemistry of MMR proteins, Microsatellite Instability (MSI) assay, methylation specific MLPA and transcript analyses. In addition, somatic deletions and/or variants were investigated by MLPA and next generation sequencing (NGS).ResultsAn EPCAM deletion was found in five unrelated probands in Italy: variants c.556-490_*8438del and c.858+1193_*5826del are novel; c.859-1430_*2033del and c.859-670_*530del were previously reported. All probands were affected by CRC at young age; tumors showed MSI and abnormal MSH2/MSH6 proteins expression. MSH2 promoter methylation, as well as aberrant in-frame or out-of-frame EPCAM/MSH2 fusion transcripts, were detected in CRCs and normal mucosae.ConclusionAn EPCAM deletion was the causative variant in about 2% of our institutional series of 224 LS patients, consistent with previously estimated frequencies. Early age and multiple CRCs was the main clinical feature of this subset of patients

Phenotype and genotype of 87 patients with Mowat-Wilson syndrome and recommendations for care

Mowat-Wilson syndrome (MWS) is a rare intellectual disability/multiple congenital anomalies syndrome caused by heterozygous mutation of the ZEB2 gene. It is generally underestimated because its rarity and phenotypic variability sometimes make it difficult to recognize. Here, we aimed to better delineate the phenotype, natural history, and genotype-phenotype correlations of MWS.MethodsIn a collaborative study, we analyzed clinical data for 87 patients with molecularly confirmed diagnosis. We described the prevalence of all clinical aspects, including attainment of neurodevelopmental milestones, and compared the data with the various types of underlying ZEB2 pathogenic variations.ResultsAll anthropometric, somatic, and behavioral features reported here outline a variable but highly consistent phenotype. By presenting the most comprehensive evaluation of MWS to date, we define its clinical evolution occurring with age and derive suggestions for patient management. Furthermore, we observe that its severity correlates with the kind of ZEB2 variation involved, ranging from ZEB2 locus deletions, associated with severe phenotypes, to rare nonmissense intragenic mutations predicted to preserve some ZEB2 protein functionality, accompanying milder clinical presentations.ConclusionKnowledge of the phenotypic spectrum of MWS and its correlation with the genotype will improve its detection rate and the prediction of its features, thus improving patient care.GENETICS in MEDICINE advance online publication, 4 January 2018; doi:10.1038/gim.2017.221

Phenotype and genotype of 87 patients with Mowat–Wilson syndrome and recommendations for care

Purpose: Mowat–Wilson syndrome (MWS) is a rare intellectual disability/multiple congenital anomalies syndrome caused by heterozygous mutation of the ZEB2 gene. It is generally underestimated because its rarity and phenotypic variability sometimes make it difficult to recognize. Here, we aimed to better delineate the phenotype, natural history, and genotype–phenotype correlations of MWS. Methods: In a collaborative study, we analyzed clinical data for 87 patients with molecularly confirmed diagnosis. We described the prevalence of all clinical aspects, including attainment of neurodevelopmental milestones, and compared the data with the various types of underlying ZEB2 pathogenic variations. Results: All anthropometric, somatic, and behavioral features reported here outline a variable but highly consistent phenotype. By presenting the most comprehensive evaluati

Interaction Between alpha-Synuclein and Metal Ions, Still Looking for a Role in the Pathogenesis of Parkinson's Disease

The most recent literature on the interaction between \u3b1-synuclein in its several aggregation states and metal ions is discussed. This analysis shows two major types of interactions. Binding sites are present in the C-terminal region, and similar, low affinity (in the millimolar range) is exhibited toward many different metal ions, including copper and iron. A more complex scenario emerges for these latter metal ions, which are also able to coordinate with high affinity (in the micromolar range) to the N-terminal region of \u3b1-synuclein. Moreover, these redox-active metal ions may induce chemical modifications on the protein in vitro and in the reducing intracellular environment, and these modifications might be relevant for the aggregation properties of \u3b1-synuclein. Finally, an attempt is made to contextualize the interaction between \u3b1-synuclein and these metal ions in the framework of the elusive and multifactorial pathogenesis of Parkinson\u2019s disease

Cloning, expression, purification, and spectroscopic analysis of the fragment 57-102 of human alpha-synuclein

The protein alpha-synuclein plays an important role in many neurodegenerative disorders.. referred to as alpha-synucleinopathies, that include, among others, Parkinson's and Alzheimer's diseases. The central region of the wild type protein, known as the non-Abeta component of amyloid plaques (NAC, amino acids 61-95), seems to be responsible for its aggregation process. To structurally characterize this fragment by nuclear magnetic resonance, we produced it by DNA recombinant technology. This technique, unlike chemical synthesis, allows the production of labeled samples (C-13, N-15) required for NMR studies. Because the NAC region is very sparingly soluble in aqueous buffer, we cloned a slightly larger portion of alpha-synuclein, alphasyn57-102, with the presence of several charged residues in both extremities of the NAC region. The conformational preferences of purified alphasyn57-102, in solution and bound to SDS micelles, was studied. Our results indicate that the protein is largely unfolded in solution but exhibits a helical conformation in the lipid-associated state. The methodology that we have used in this work for the cloning, expression, and purification of alphasyn57-102 can be easily applied to most small proteins, thus representing a powerful tool for structural NMR analysis of labeled peptides

Toward a better definition of EPCAM deletions in Lynch Syndrome: Report of new variants in Italy and the associated molecular phenotype

BackgroundInherited epimutations of Mismatch Repair (MMR) genes are responsible for Lynch Syndrome (LS) in a small, but well defined, subset of patients. Methylation of the MSH2 promoter consequent to the deletion of the upstream EPCAM gene is found in about 1%-3% of the LS patients and represents a classical secondary, constitutional and tissue-specific epimutation. Several different EPCAM deletions have been reported worldwide, for the most part representing private variants caused by an Alu-mediated recombination.Methods712 patients with suspected LS were tested for MMR mutation in our Institute. EPCAM deletions were detected by multiplex ligation-dependent probe amplification (MLPA) and then defined by Long-Range polymerase chain reaction (PCR)/Sanger sequencing. A comprehensive molecular characterization of colorectal cancer (CRC) tissues was carried out by immunohistochemistry of MMR proteins, Microsatellite Instability (MSI) assay, methylation specific MLPA and transcript analyses. In addition, somatic deletions and/or variants were investigated by MLPA and next generation sequencing (NGS).ResultsAn EPCAM deletion was found in five unrelated probands in Italy: variants c.556-490_*8438del and c.858+1193_*5826del are novel; c.859-1430_*2033del and c.859-670_*530del were previously reported. All probands were affected by CRC at young age; tumors showed MSI and abnormal MSH2/MSH6 proteins expression. MSH2 promoter methylation, as well as aberrant in-frame or out-of-frame EPCAM/MSH2 fusion transcripts, were detected in CRCs and normal mucosae.ConclusionAn EPCAM deletion was the causative variant in about 2% of our institutional series of 224 LS patients, consistent with previously estimated frequencies. Early age and multiple CRCs was the main clinical feature of this subset of patients