In-cell NMR characterization of the secondary structure populations of a disordered conformation of α-Synuclein within E. coli cells

α-Synuclein is a small protein strongly implicated in the pathogenesis of Parkinson’s disease and related neurodegenerative disorders. We report here the use of in-cell NMR spectroscopy to observe directly the structure and dynamics of this protein within E. coli cells. To improve the accuracy in the measurement of backbone chemical shifts within crowded in-cell NMR spectra, we have developed a deconvolution method to reduce inhomogeneous line broadening within cellular samples. The resulting chemical shift values were then used to evaluate the distribution of secondary structure populations which, in the absence of stable tertiary contacts, are a most effective way to describe the conformational fluctuations of disordered proteins. The results indicate that, at least within the bacterial cytosol, α-synuclein populates a highly dynamic state that, despite the highly crowded environment, has the same characteristics as the disordered monomeric form observed in aqueous solution

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

Atomic force microscopy based nanoassay: A new method to study \u3b1-Synuclein-dopamine bioaffinity interactions

Intrinsically Disordered Proteins (IDPs) are characterized by the lack of well-defined 3-D structure and show high conformational plasticity. For this reason, they are a strong challenge for the traditional characterization of structure, supramolecular assembly and biorecognition phenomena. We show here how the fine tuning of protein orientation on a surface turns useful in the reliable testing of biorecognition interactions of IDPs, in particular \u3b1-Synuclein. We exploited atomic force microscopy (AFM) for the selective, nanoscale confinement of \u3b1-Synuclein on gold to study the early stages of \u3b1-Synuclein aggregation and the effect of small molecules, like dopamine, on the aggregation process. Capitalizing on the high sensitivity of AFM topographic height measurements we determined, for the first time in the literature, the dissociation constant of dopamine-\u3b1-Synuclein adducts

Inhibition of alpha-synuclein fibrillization by dopamine is mediated by interactions with five C-terminal residues and with E83 in the NAC region

The interplay between dopamine and alpha-synuclein (AS) plays a central role in Parkinson's disease (PD). PD results primarily from a severe and selective devastation of dopaminergic neurons in substantia nigra pars compacta. The neuropathological hallmark of the disease is the presence of intraneuronal proteinaceous inclusions known as Lewy bodies within the surviving neurons, enriched in filamentous AS. In vitro, dopamine inhibits AS fibril formation, but the molecular determinants of this inhibition remain obscure. Here we use molecular dynamic (MD) simulations to investigate the binding of dopamine and several of its derivatives onto conformers representative of an NMR ensemble of AS structures in aqueous solution. Within the limitations inherent to MD simulations of unstructured proteins, our calculations suggest that the ligands bind to the (125)YEMPS(129) region, consistent with experimental findings. The ligands are further stabilized by long-range electrostatic interactions with glutamate 83 (E83) in the NAC region. These results suggest that by forming these interactions with AS, dopamine may affect AS aggregation and fibrillization properties. To test this hypothesis, we investigated in vitro the effects of dopamine on the aggregation of mutants designed to alter or abolish these interactions. We found that point mutations in the (125)YEMPS(129) region do not affect AS aggregation, which is consistent with the fact that dopamine interacts non-specifically with this region. In contrast, and consistent with our modeling studies, the replacement of glutamate by alanine at position 83 (E83A) abolishes the ability of dopamine to inhibit AS fibrillization

Glial Innate Immunity Generated by Non-Aggregated Alpha-Synuclein in Mouse: Differences between Wild-type and Parkinson's Disease-Linked Mutants

Background: Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized pathologically by the presence in the brain of intracellular protein inclusions highly enriched in aggregated alpha-synuclein (alpha-Syn). Although it has been established that progression of the disease is accompanied by sustained activation of microglia, the underlying molecules and factors involved in these immune-triggered mechanisms remain largely unexplored. Lately, accumulating evidence has shown the presence of extracellular alpha-Syn both in its aggregated and monomeric forms in cerebrospinal fluid and blood plasma. However, the effect of extracellular alpha-Syn on cellular activation and immune mediators, as well as the impact of familial PD-linked alpha-Syn mutants on this stimulation, are still largely unknown.Methods and Findings: In this work, we have compared the activation profiles of non-aggregated, extracellular wild-type and PD-linked mutant alpha-Syn variants on primary glial and microglial cell cultures. After stimulation of cells with alpha-Syn, we measured the release of Th1- and Th2-type cytokines as well as IP-10/CXCL10, RANTES/CCL5, MCP-1/CCL2 and MIP-1 alpha/CCL3 chemokines. Contrary to what had been observed using cell lines or for the case of aggregated alpha-Syn, we found strong differences in the immune response generated by wild-type alpha-Syn and the familial PD mutants (A30P, E46K and A53T).Conclusions: These findings might contribute to explain the differences in the onset and progression of this highly debilitating disease, which could be of value in the development of rational approaches towards effective control of immune responses that are associated with PD

Non-native hydrophobic interactions detected in unfolded apoflavodoxin by paramagnetic relaxation enhancement

Transient structures in unfolded proteins are important in elucidating the molecular details of initiation of protein folding. Recently, native and non-native secondary structure have been discovered in unfolded A. vinelandii flavodoxin. These structured elements transiently interact and subsequently form the ordered core of an off-pathway folding intermediate, which is extensively formed during folding of this α–β parallel protein. Here, site-directed spin-labelling and paramagnetic relaxation enhancement are used to investigate long-range interactions in unfolded apoflavodoxin. For this purpose, glutamine-48, which resides in a non-native α-helix of unfolded apoflavodoxin, is replaced by cysteine. This replacement enables covalent attachment of nitroxide spin-labels MTSL and CMTSL. Substitution of Gln-48 by Cys-48 destabilises native apoflavodoxin and reduces flexibility of the ordered regions in unfolded apoflavodoxin in 3.4 M GuHCl, because of increased hydrophobic interactions in the unfolded protein. Here, we report that in the study of the conformational and dynamic properties of unfolded proteins interpretation of spin-label data can be complicated. The covalently attached spin-label to Cys-48 (or Cys-69 of wild-type apoflavodoxin) perturbs the unfolded protein, because hydrophobic interactions occur between the label and hydrophobic patches of unfolded apoflavodoxin. Concomitant hydrophobic free energy changes of the unfolded protein (and possibly of the off-pathway intermediate) reduce the stability of native spin-labelled protein against unfolding. In addition, attachment of MTSL or CMTSL to Cys-48 induces the presence of distinct states in unfolded apoflavodoxin. Despite these difficulties, the spin-label data obtained here show that non-native contacts exist between transiently ordered structured elements in unfolded apoflavodoxin

Inhibiting α-Synuclein Oligomerization by Stable Cell-Penetrating β-Synuclein Fragments Recovers Phenotype of Parkinson's Disease Model Flies

The intracellular oligomerization of α-synuclein is associated with Parkinson's disease and appears to be an important target for disease-modifying treatment. Yet, to date, there is no specific inhibitor for this aggregation process. Using unbiased systematic peptide array analysis, we indentified molecular interaction domains within the β-synuclein polypeptide that specifically binds α-synuclein. Adding such peptide fragments to α-synuclein significantly reduced both amyloid fibrils and soluble oligomer formation in vitro. A retro-inverso analogue of the best peptide inhibitor was designed to develop the identified molecular recognition module into a drug candidate. While this peptide shows indistinguishable activity as compared to the native peptide, it is stable in mouse serum and penetrates α-synuclein over-expressing cells. The interaction interface between the D-amino acid peptide and α-synuclein was mapped by Nuclear Magnetic Resonance spectroscopy. Finally, administering the retro-inverso peptide to a Drosophila model expressing mutant A53T α-synuclein in the nervous system, resulted in a significant recovery of the behavioral abnormalities of the treated flies and in a significant reduction in α-synuclein accumulation in the brains of the flies. The engineered retro-inverso peptide can serve as a lead for developing a novel class of therapeutic agents to treat Parkinson's disease

Probing the urea dependence of residual structure in denatured human α-lactalbumin

Backbone 15N relaxation parameters and 15N–1HN residual dipolar couplings (RDCs) have been measured for a variant of human α-lactalbumin (α-LA) in 4, 6, 8 and 10 M urea. In the α-LA variant, the eight cysteine residues in the protein have been replaced by alanines (all-Ala α-LA). This protein is a partially folded molten globule at pH 2 and has been shown previously to unfold in a stepwise non-cooperative manner on the addition of urea. 15N R2 values in some regions of all-Ala α-LA show significant exchange broadening which is reduced as the urea concentration is increased. Experimental RDC data are compared with RDCs predicted from a statistical coil model and with bulkiness, average area buried upon folding and hydrophobicity profiles in order to identify regions of non-random structure. Residues in the regions corresponding to the B, D and C-terminal 310 helices in native α-LA show R2 values and RDC data consistent with some non-random structural propensities even at high urea concentrations. Indeed, for residues 101–106 the residual structure persists in 10 M urea and the RDC data suggest that this might include the formation of a turn-like structure. The data presented here allow a detailed characterization of the non-cooperative unfolding of all-Ala α-LA at higher concentrations of denaturant and complement previous studies which focused on structural features of the molten globule which is populated at lower concentrations of denaturant

Targeting the intrinsically disordered structural ensemble of α-synuclein by small molecules as a potential therapeutic strategy for Parkinson's disease.

The misfolding of intrinsically disordered proteins such as α-synuclein, tau and the Aβ peptide has been associated with many highly debilitating neurodegenerative syndromes including Parkinson's and Alzheimer's diseases. Therapeutic targeting of the mono