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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 monom

Introduction: The history of levothyroxine has been linked to advances in the treatment of thyroid disease and to date it is the standard therapy for the treatment of hypothyroidism. Bioequivalence studies are the most widely used method to demonstrate interchangeability, although controversy persists regarding the best design for this molecule declared as a narrow therapeutic index product in many countries. This study aimed to evaluate the pharmacokinetic profile of two formulations of levothyroxine to determine bioequivalence between them., Methods: This two-period, randomized, crossover, blind study was conducted in 80 healthy volunteers, of both sexes, using a single levothyroxine dose of 600 μg with a washout period of 42 days. Blood sampling was performed at - 30 min, - 15 min, and 0 h pre-dose and 30 min, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 16, 24, and 48 h post-dose., Results: A total of 78 subjects successfully completed both periods. There were no serious adverse events during the study and both formulations were well tolerated. Baseline correction of serum levothyroxine concentrations was performed before statistical analysis. The mean maximum plasma concentration of the test product (Levotiroxina MK ® ) was 57.49 ng/mL while for the reference product it reached 59.32 ng/mL. Importantly, both test and reference formulations reached maximum concentrations in plasma at about the same time. The areas under the pharmacokinetic curves with the test product showed AUC 0-t of 1407.1 ng h/mL and the reference product 1394.3 ng h/mL. The bioequivalence statistical analysis showed that the 90% confidence interval (CI 90% ) of the ratio of test over reference formulation was within the bioequivalence margins of 90-111%. For C max , the test/reference ratio was 96.2% with CI 90% of 91.6-100.9%, and for AUC 0-t the test/reference ratio was 99.9 with CI 90% of 93.3-107.0%., Conclusions: Both formulations have the same pharmacokinetic profile and are bioequivalent in the narrow therapeutic index required by some health authorities., (© 2022. The Author(s).)

The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson's disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes. Using a surface plasmon resonance high-throughput screen, in which monomeric αSyn is incubated with microchips arrayed with tethered compounds, we identified novel αSyn interacting drug-like compounds. Because these small molecules could impact a variety of αSyn forms present in the ensemble, we tested representative hits for impact on multiple αSyn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits αSyn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by αSyn overexpression, and a compound blocking cellular transmission of αSyn. Our studies demonstrate that drug-like small molecules that interact with native αSyn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of αSyn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinson's disease.

The androgen receptor is a transcription factor that plays a key role in the development of prostate cancer, and its interactions with general transcription regulators are therefore of potential therapeutic interest. The mechanistic basis of these interactions is poorly understood due to the intrinsically disordered nature of the transactivation domain of the androgen receptor and the generally transient nature of the protein-protein interactions that trigger transcription. Here, we identify a motif of the transactivation domain that contributes to transcriptional activity by recruiting the C-terminal domain of subunit 1 of the general transcription regulator TFIIF. These findings provide molecular insights into the regulation of androgen receptor function and suggest strategies for treating castration-resistant prostate cancer., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Castration-resistant prostate cancer is the lethal condition suffered by prostate cancer patients that become refractory to androgen deprivation therapy. EPI-001 is a recently identified compound active against this condition that modulates the activity of the androgen receptor, a nuclear receptor that is essential for disease progression. The mechanism by which this compound exerts its inhibitory activity is however not yet fully understood. Here we show, by using high resolution solution nuclear magnetic resonance spectroscopy, that EPI-001 selectively interacts with a partially folded region of the transactivation domain of the androgen receptor, known as transactivation unit 5, that is key for the ability of prostate cells to proliferate in the absence of androgens, a distinctive feature of castration-resistant prostate cancer. Our results can contribute to the development of more potent and less toxic novel androgen receptor antagonists for treating this disease.

Correlated inter-domain motions in proteins can mediate fundamental biochemical processes such as signal transduction and allostery. Here we characterize at structural level the inter-domain coupling in a multidomain enzyme, Adenylate Kinase (AK), using computational methods that exploit the shape information encoded in residual dipolar couplings (RDCs) measured under steric alignment by nuclear magnetic resonance (NMR). We find experimental evidence for a multi-state equilibrium distribution along the opening/closing pathway of Adenylate Kinase, previously proposed from computational work, in which inter-domain interactions disfavour states where only the AMP binding domain is closed. In summary, we provide a robust experimental technique for study of allosteric regulation in AK and other enzymes.

Insight into how amyloid β (Aβ) aggregation occurs in vivo is vital for understanding the molecular pathways that underlie Alzheimer's disease and requires new techniques that provide detailed kinetic and mechanistic information. Using noninvasive fluorescence lifetime recordings, we imaged the formation of Aβ(1-40) and Aβ(1-42) aggregates in live cells. For both peptides, the cellular uptake via endocytosis is rapid and spontaneous. They are then retained in lysosomes, where their accumulation leads to aggregation. The kinetics of Aβ(1-42) aggregation are considerably faster than those of Aβ(1-40) and, unlike those of the latter peptide, show no detectable lag phase. We used superresolution fluorescence imaging to examine the resulting aggregates and could observe compact amyloid structures, likely because of spatial confinement within cellular compartments. Taken together, these findings provide clues as to how Aβ aggregation may occur within neurons., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 monomeric state of such intrinsically disordered proteins by small molecules has, however, been a major challenge because of their heterogeneous conformational properties. We show here that a combination of computational and experimental techniques has led to the identification of a drug-like phenyl-sulfonamide compound (ELN484228), that targets α-synuclein, a key protein in Parkinson's disease. We found that this compound has substantial biological activity in cellular models of α-synuclein-mediated dysfunction, including rescue of α-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of α-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting α-synuclein by small molecules represents a promising approach to the development of therapeutic treatments of Parkinson's disease and related conditions.

Structural conversion of the presynaptic, intrinsically disordered protein α-synuclein into amyloid fibrils underlies neurotoxicity in Parkinson's disease. The detailed mechanism by which this conversion occurs is largely unknown. Here, we identify a discrete pattern of transient tertiary interactions in monomeric α-synuclein involving amino acid residues that are, in the fibrillar state, part of β-strands. Importantly, this pattern of pairwise interactions does not correspond to that found in the amyloid state. A redistribution of this network of fibril-like contacts must precede aggregation into the amyloid structure., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In the last decade it has become evident that disordered states of proteins play important physiological and pathological roles and that the transient tertiary interactions often present in these systems can play a role in their biological activity. The structural characterization of such states has so far largely relied on ensemble representations, which in principle account for both their local and global structural features. However, these approaches are inherently of low resolution due to the large number of degrees of freedom of conformational ensembles and to the sparse nature of the experimental data used to determine them. Here, we overcome these limitations by showing that tertiary interactions in disordered states can be mapped at high resolution by fitting paramagnetic relaxation enhancement data to a small number of conformations, which can be as low as one. This result opens up the possibility of determining the topology of cooperatively collapsed and hidden folded states when these are present in the vast conformational landscape accessible to disordered states of proteins. As a first application, we study the long-range tertiary interactions of acid-unfolded apomyoglobin from experimentally measured paramagnetic relaxation enhancement data., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We report observations of an intrinsic fluorescence in the visible range, which develops during the aggregation of a range of polypeptides, including the disease-related human peptides amyloid-β(1-40) and (1-42), lysozyme and tau. Characteristic fluorescence properties such as the emission lifetime and spectra were determined experimentally. This intrinsic fluorescence is independent of the presence of aromatic side-chain residues within the polypeptide structure. Rather, it appears to result from electronic levels that become available when the polypeptide chain folds into a cross-β sheet scaffold similar to what has been reported to take place in crystals. We use these findings to quantify protein aggregation in vitro by fluorescence imaging in a label-free manner.

Inefficient gene transfer and low virion concentrations are common limitations of retroviral transduction. We and others have previously shown that peptides derived from human semen form amyloid fibrils that boost retroviral gene delivery by promoting virion attachment to the target cells. However, application of these natural fibril-forming peptides is limited by moderate efficiencies, the high costs of peptide synthesis, and variability in fibril size and formation kinetics. Here, we report the development of nanofibrils that self-assemble in aqueous solution from a 12-residue peptide, termed enhancing factor C (EF-C). These artificial nanofibrils enhance retroviral gene transfer substantially more efficiently than semen-derived fibrils or other transduction enhancers. Moreover, EF-C nanofibrils allow the concentration of retroviral vectors by conventional low-speed centrifugation, and are safe and effective, as assessed in an ex vivo gene transfer study. Our results show that EF-C fibrils comprise a highly versatile, convenient and broadly applicable nanomaterial that holds the potential to significantly facilitate retroviral gene transfer in basic research and clinical applications.

Here, we use single-molecule techniques to study the aggregation of α-synuclein, the protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The fibrillation of amyloidogenic proteins is a critical step in the etiology of neurodegenerative disorders such as Alzheimer and Parkinson diseases. There is major interest in the therapeutic intervention on such aberrant aggregation phenomena, and the utilization of polyaromatic scaffolds has lately received considerable attention. In this regard, the molecular and structural basis of the anti-amyloidogenicity of polyaromatic compounds, required to evolve this molecular scaffold toward therapeutic drugs, is not known in detail. We present here biophysical and biochemical studies that have enabled us to characterize the interaction of metal-substituted, tetrasulfonated phthalocyanines (PcTS) with α-synuclein (AS), the major protein component of amyloid-like deposits in Parkinson disease. The inhibitory activity of the assayed compounds on AS amyloid fibril formation decreases in the order PcTS[Ni(II)] ~ PcTS > PcTS[Zn(II)] >> PcTS[Al(III)] ≈ 0. Using NMR and electronic absorption spectroscopies we demonstrated conclusively that the differences in binding capacity and anti-amyloid activity of phthalocyanines on AS are attributed to their relative ability to self-stack through π-π interactions, modulated by the nature of the metal ion bound at the molecule. Low order stacked aggregates of phthalocyanines were identified as the active amyloid inhibitory species, whose effects are mediated by residue specific interactions. Such sequence-specific anti-amyloid behavior of self-stacked phthalocyanines contrasts strongly with promiscuous amyloid inhibitors with self-association capabilities that act via nonspecific sequestration of AS molecules. The new findings reported here constitute an important contribution for future drug discovery efforts targeting amyloid formation.

Misfolding and aggregation of peptides and proteins is a characteristic of many neurodegenerative disorders, including Alzheimer's disease (AD). In AD the β-amyloid peptide (Aβ) aggregates to form characteristic fibrillar structures, which are the deposits found as plaques in the brains of patients. We have used direct stochastic optical reconstruction microscopy, dSTORM, to probe the process of in situ Aβ aggregation and the morphology of the ensuing aggregates with a resolution better than 20 nm. We are able to distinguish different types of structures, including oligomeric assemblies and mature fibrils, and observe a number of morphological differences between the species formed in vitro and in vivo, which may be significant in the context of disease. Our data support the recent view that intracellular Aβ could be associated with Aβ pathogenicity in AD, although the major deposits are extracellular, and suggest that this approach will be widely applicable to studies of the molecular mechanisms of protein deposition diseases.

The misfolding and aggregation of amyloidogenic polypeptides are characteristics of many neurodegenerative syndromes including Alzheimer's and Parkinson's disease. There is a major interest in the availability of amyloid-specific probes that exhibit fluorescence properties, for its use as reporters of protein aggregation in spectroscopy and microscopy methodologies. In this review, we intend to provide an overview of novel fluorescence-based probes and procedures applied for addressing fundamental aspects of amyloid self-assembly in vitro and in vivo. We highlight the utilization in vitro of several small-molecule fluorescent probes as extrinsic and site-specific reporters of amyloid formation, including single-molecule determinations. Detection of amyloid self-assembly employing compounds such as JC-1, DCVJ, ANS derivatives and luminescent conjugated polymers, as well as site-specific probes such as pyrene and ESIPT is discussed. We further review novel fluorescent probes developed for the non-invasive optical imaging of protein aggregates in vivo, including BTA-1, Methoxy-X04, NIAD-4 and CRANAD-2. Availability of increasingly versatile amyloid-specific fluorescent probes is having a very positive impact in the drug discovery and diagnostics fields.

Misfolding and aggregation of amyloidogenic polypeptides lie at the root of many neurodegenerative diseases. Whilst protein aggregation can be readily studied in vitro by established biophysical techniques, direct observation of the nature and kinetics of aggregation processes taking place in vivo is much more challenging. We describe here, however, a Förster resonance energy transfer sensor that permits the aggregation kinetics of amyloidogenic proteins to be quantified in living systems by exploiting our observation that amyloid assemblies can act as energy acceptors for variants of fluorescent proteins. The observed lifetime reduction can be attributed to fluorescence energy transfer to intrinsic energy states associated with the growing amyloid species. Indeed, for a-synuclein, a protein whose aggregation is linked to Parkinson's disease, we have used this sensor to follow the kinetics of the self-association reactions taking place in vitro and in vivo and to reveal the nature of the ensuing aggregated species. Experiments were conducted in vitro, in cells in culture and in living Caenorhabditis elegans. For the latter the readout correlates directly with the appearance of a toxic phenotype. The ability to measure the appearance and development of pathogenic amyloid species in a living animal and the ability to relate such data to similar processes observed in vitro provides a powerful new tool in the study of the pathology of the family of misfolding disorders. Our study confirms the importance of the molecular environment in which aggregation reactions take place, highlighting similarities as well as differences between the processes occurring in vitro and in vivo, and their significance for defining the molecular physiology of the diseases with which they are associated., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intrinsically disordered proteins (IDPs) lack well-defined structure but are widely represented in eukaryotic proteomes. Although the functions of most IDPs are not understood, some have been shown to have molecular recognition and/or regulatory roles where their disordered nature might be advantageous. Anhydrin is an uncharacterized IDP induced by dehydration in an anhydrobiotic nematode, Aphelenchus avenae. We show here that anhydrin is a moonlighting protein with two novel, independent functions relating to desiccation tolerance. First, it has a chaperone-like activity that can reduce desiccation-induced enzyme aggregation and inactivation in vitro. When expressed in a human cell line, anhydrin localizes to the nucleus and reduces the propensity of a polyalanine expansion protein associated with oculopharyngeal muscular dystrophy to form aggregates. This in vivo activity is distinguished by a loose association of anhydrin with its client protein, consistent with a role as a molecular shield. In addition, anhydrin exhibits a second function as an endonuclease whose substrates include supercoiled, linear, and chromatin linker DNA. This nuclease activity could be involved in either repair of desiccation-induced DNA damage incurred during anhydrobiosis or in apoptotic or necrotic processes, for example, but it is particularly unexpected for anhydrin because IDP functions defined to date anticorrelate with enzyme activity. Enzymes usually require precise three-dimensional positioning of residues at the active site, but our results suggest this need not be the case. Anhydrin therefore extends the range of IDP functional categories to include catalysis and highlights the potential for the discovery of new functions in disordered proteomes.

Misfolding and aggregation of proteins are characteristics of a range of increasingly prevalent neurodegenerative disorders including Alzheimer's and Parkinson's diseases. In Parkinson's disease and several closely related syndromes, the protein alpha-synuclein (AS) aggregates and forms amyloid-like deposits in specific regions of the brain. Fluorescence microscopy using fluorescent proteins, for instance the yellow fluorescent protein (YFP), is the method of choice to image molecular events such as protein aggregation in living organisms. The presence of a bulky fluorescent protein tag, however, may potentially affect significantly the properties of the protein of interest; for AS in particular, its relative small size and, as an intrinsically unfolded protein, its lack of defined secondary structure could challenge the usefulness of fluorescent-protein-based derivatives. Here, we subject a YFP fusion of AS to exhaustive studies in vitro designed to determine its potential as a means of probing amyloid formation in vivo. By employing a combination of biophysical and biochemical studies, we demonstrate that the conjugation of YFP does not significantly perturb the structure of AS in solution and find that the AS-YFP protein forms amyloid deposits in vitro that are essentially identical with those observed for wild-type AS, except that they are fluorescent. Of the several fluorescent properties of the YFP chimera that were assayed, we find that fluorescence anisotropy is a particularly useful parameter to follow the aggregation of AS-YFP, because of energy migration Förster resonance energy transfer (emFRET or homoFRET) between closely positioned YFP moieties occurring as a result of the high density of the fluorophore within the amyloid species. Fluorescence anisotropy imaging microscopy further demonstrates the ability of homoFRET to distinguish between soluble, pre-fibrillar aggregates and amyloid fibrils of AS-YFP. Our results validate the use of fluorescent protein chimeras of AS as representative models for studying protein aggregation and offer new opportunities for the investigation of amyloid aggregation in vivo using YFP-tagged proteins., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The identification of aggregation inhibitors and the investigation of their mechanism of action are fundamental in the quest to mitigate the pathological consequences of amyloid formation. Here, characterization of the structural and mechanistic basis for the antiamyloidogenic effect of phthalocyanine tetrasulfonate (PcTS) on alpha-synuclein (AS) allowed us to demonstrate that specific aromatic interactions are central for ligand-mediated inhibition of amyloid formation. We provide evidence indicating that the mechanism behind the antiamyloidogenic effect of PcTS is correlated with the trapping of prefibrillar AS species during the early stages of the assembly process. By using NMR spectroscopy, we have located the primary binding region for PcTS to a specific site in the N terminus of AS, involving the amino acid Tyr-39 as the anchoring residue. Moreover, the residue-specific structural characterization of the AS-PcTS complex provided the basis for the rational design of nonamyloidogenic species of AS, highlighting the role of aromatic interactions in driving AS amyloid assembly. A comparative analysis with other proteins involved in neurodegenerative disorders reveals that aromatic recognition interfaces might constitute a key structural element to target their aggregation pathways. These findings emphasize the use of aggregation inhibitors as molecular probes to assess structural and toxic mechanisms related to amyloid formation and the potential of small molecules as therapeutics for amyloid-related pathologies.

The ATP-dependent protein chaperone heat-shock protein 70 (Hsp70) displays broad anti-aggregation functions and has a critical function in preventing protein misfolding pathologies. According to in vitro and in vivo models of Parkinson's disease (PD), loss of Hsp70 activity is associated with neurodegeneration and the formation of amyloid deposits of alpha-synuclein (alphaSyn), which constitute the intraneuronal inclusions in PD patients known as Lewy bodies. Here, we show that Hsp70 depletion can be a direct result of the presence of aggregation-prone polypeptides. We show a nucleotide-dependent interaction between Hsp70 and alphaSyn, which leads to the aggregation of Hsp70, in the presence of ADP along with alphaSyn. Such a co-aggregation phenomenon can be prevented in vitro by the co-chaperone Hip (ST13), and the hypothesis that it might do so also in vivo is supported by studies of a Caenorhabditis elegans model of alphaSyn aggregation. Our findings indicate that a decreased expression of Hip could facilitate depletion of Hsp70 by amyloidogenic polypeptides, impairing chaperone proteostasis and stimulating neurodegeneration.

Increasing evidence links the misfolding and aberrant self-assembly of proteins with the molecular events that underlie a range of neurodegenerative diseases, yet the mechanistical details of these processes are still poorly understood. The fact that many of these proteins are intrinsically unstructured makes it particularly challenging to develop strategies for discovering small molecule inhibitors of their aggregation. We present here a broad biophysical approach that enables us to characterize the mechanisms of interaction between alpha-synuclein, a protein whose aggregation is closely connected with Parkinson's disease, and two small molecules, Congo red and Lacmoid, which inhibit its fibrillization. Both compounds are found to interact with the N-terminal and central regions of the monomeric protein although with different binding mechanisms and affinities. The differences can be attributed to the chemical nature of the compounds as well as their abilities to self-associate. We further show that alpha-synuclein binding and aggregation inhibition are mediated by small oligomeric species of the compounds that interact with distinct regions of the monomeric protein. These findings provide potential explanations of the nonspecific antiamyloid effect observed for these compounds as well as important mechanistical information for future drug discovery efforts targeting the misfolding and aggregation of intrinsically unstructured proteins.

We report here the use of protonless NMR spectroscopy to extract structural information under biologically relevant conditions when conventional proton-detection NMR spectroscopy fails due to the loss of labile proton resonances. By direct (13)C detection, correlations between nonlabile nuclei of a given biomolecule can be determined with high resolution, which becomes particularly useful when the system of interests is sensitive to solvent exchange at elevated temperatures, such as intrinsically disordered proteins. Human alpha-synuclein, which is associated with Parkinson's disease, is used as a model system to illustrate the usefulness of protonless NMR spectroscopy in recovering hitherto missing spectral information.

The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. Protein-metal interactions play a critical role in AS aggregation and might represent the link between the pathological processes of protein aggregation and oxidative damage. Our previous studies established a hierarchy in AS-metal ion interactions, where Cu(II) binds specifically to the protein and triggers its aggregation under conditions that might be relevant for the development of PD. In this work, we have addressed unresolved structural details related to the binding specificity of Cu(II) through the design of site-directed and domain-truncated mutants of AS and by the characterization of the metal-binding features of its natural homologue beta-synuclein (BS). The structural properties of the Cu(II) complexes were determined by the combined application of nuclear magnetic resonance, electron paramagnetic resonance, UV-vis, circular dichroism spectroscopy, and matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). Two independent, noninteracting copper-binding sites with significantly different affinities for the metal ion were detected in the N-terminal regions of AS and BS. MALDI MS provided unique evidence for the direct involvement of Met1 as the primary anchoring residue for Cu(II) in both proteins. Comparative spectroscopic analysis of the two proteins allowed us to deconvolute the Cu(II) binding modes and unequivocally assign the higher-affinity site to the N-terminal amino group of Met1 and the lower-affinity site to the imidazol ring of the sole His residue. Through the use of competitive chelators, the affinity of the first equivalent of bound Cu(II) was accurately determined to be in the submicromolar range for both AS and BS. Our results prove that Cu(II) binding in the C-terminal region of synucleins represents a nonspecific, very low affinity process. These new insights into the bioinorganic chemistry of PD are central to an understanding of the role of Cu(II) in the fibrillization process of AS and have implications for the molecular mechanism by which BS might inhibit AS amyloid assembly.

Parkinson's disease (PD) is associated with the formation and deposition of amyloid fibrils of the protein alpha-synuclein (AS). It has been proposed that oligomeric intermediates on the pathway to fibrilization rather than the fibrils themselves are the pathogenic agents of PD, but efficient methods for their detection are lacking. We have studied the interfacial properties of wild-type AS and the course of its aggregation in vitro using electrochemical analysis and dynamic light scattering. The oxidation signals of tyrosine residues of AS at carbon electrodes and the ability of fibrils to adsorb and catalyze hydrogen evolution at hanging mercury drop electrodes (HMDEs) decreased during incubation. HMDEs were particularly sensitive to pre-aggregation changes in AS. Already after 1 h of a standard aggregation assay in vitro (stirring at 37 degrees C), the electrocatalytic peak H increased greatly and shifted to less negative potentials. Between 3 and 9 h of incubation, an interval during which dynamic light scattering indicated AS oligomerization, peak H diminished and shifted to more negative potentials, and AS adsorbability decreased. We tentatively attribute the very early changes in the interfacial behavior of the protein after the first few hours of incubation to protein destabilization with disruption of long-range interactions. The subsequent changes can be related to the onset of oligomerization. Our results demonstrate the utility of electrochemical methods as new and simple tools for the investigation of amyloid formation.

The synuclein family of intrinsically unfolded proteins is composed of three highly homologous members, alpha-synuclein (alphaS), beta-synuclein (betaS) and gamma-synuclein (gammaS), which are linked to neurodegenerative disorders and cancer. alphaS has been studied intensively after its identification as the major protein component of amyloid-like deposits in Parkinson's disease and dementia with Lewy bodies. betaS, on the other hand, was found to act as a potent inhibitor of alphaS amyloid formation, and it is proposed as a natural regulator of its neurotoxicity. It is then of particular interest to elucidate the structural and dynamic features of the soluble state of betaS as a first step to understand the molecular basis of its anti-amyloidogenic effect on alphaS. We present here the characterization of natively unstructured betaS by high resolution heteronuclear NMR techniques. A combination of pulse-field gradient, three-dimensional heteronuclear correlation, residual dipolar couplings, paramagnetic relaxation enhancement and backbone relaxation experiments were employed to characterize the ensemble of conformations populated by the protein. The results indicate that betaS adopts extended conformations in its native state, characterized by the lack of the long-range contacts as previously reported for alphaS. Despite the lack of defined secondary structure, we found evidence for transient polyproline II conformations clustered at the C-terminal region. The structuring of the backbone at the C terminus is locally encoded, stabilized by the presence of eight proline residues embedded in a polypeptide stretch rich in hydrophilic and negatively charged amino acids. The structural and functional implications of these findings are analyzed via a thorough comparison with its neurotoxic homolog alphaS.

Alpha-synuclein is a major component of intraneuronal protein aggregates constituting a distinctive feature of Parkinson disease. To date, fluorescence imaging of dynamic processes leading to such amyloid deposits in living cells has not been feasible. To address this need, we generated a recombinant alpha-synuclein (alpha-synuclein-C4) bearing a tetracysteine target for fluorogenic biarsenical compounds. The biophysical, biochemical and aggregation properties of alpha-synuclein-C4 matched those of the wild-type protein in vitro and in living cells. We observed aggregation of alpha-synuclein-C4 transfected or microinjected into cells, particularly under oxidative stress conditions. Fluorescence resonance energy transfer (FRET) between FlAsH and ReAsH confirmed the close association of fibrillized alpha-synuclein-C4 molecules. Alpha-synuclein-C4 offers the means for directly probing amyloid formation and interactions of alpha-synuclein with other proteins in living cells, the response to cellular stress and screening drugs for Parkinson disease.

The aggregation of alpha-synuclein (AS) is characteristic of Parkinson's disease and other neurodegenerative synucleinopathies. Interactions with metal ions affect dramatically the kinetics of fibrillation of AS in vitro and are proposed to play a potential role in vivo. We recently showed that Cu(II) binds at the N-terminus of AS with high affinity (K(d) approximately 0.1 microM) and accelerates its fibrillation. In this work we investigated the binding features of the divalent metal ions Fe(II), Mn(II), Co(II), and Ni(II), and their effects on AS aggregation. By exploiting the different paramagnetic properties of these metal ions, NMR spectroscopy provides detailed information about the protein-metal interactions at the atomic level. The divalent metal ions bind preferentially and with low affinity (millimolar) to the C-terminus of AS, the primary binding site being the (119)DPDNEA(124) motif, in which Asp121 acts as the main anchoring residue. Combined with backbone residual dipolar coupling measurements, these results suggest that metal binding is not driven exclusively by electrostatic interactions but is mostly determined by the residual structure of the C-terminus of AS. A comparative analysis with Cu(II) revealed a hierarchal effect of AS-metal(II) interactions on AS aggregation kinetics, dictated by structural factors corresponding to different protein domains. These findings reveal a strong link between the specificity of AS-metal(II) interactions and the enhancement of aggregation of AS in vitro. The elucidation of the structural basis of AS metal binding specificity is then required to elucidate the mechanism and clarify the role of metal-protein interactions in the etiology of Parkinson's disease.

Intrinsically unstructured proteins play key biochemical roles in a vast range of normal and pathological processes. To study these systems, it is necessary to invoke an ensemble of rapidly interconverting conformations. Residual dipolar couplings (RDCs) are particularly powerful probes of the behavior of unfolded proteins, reporting on time and ensemble-averaged conformations up to and beyond the millisecond time scale. In this study, we present a novel interpretation of RDCs in unfolded systems that simultaneously defines long-range structural order and local conformational sampling. This approach is used to describe the structure and dynamics of alpha-Synuclein (alphaS), a protein that is strongly implicated in the development of Parkinson's disease (PD), allowing unambiguous detection of strongly populated conformers containing long-range contacts between the N- and C-terminal domains. The structural model combines two features required for the description of alphaS in solution: local conformational fluctuation based on random sampling of residue-specific phi/psi distributions, and long-range contacts induced by the presence of nonbonding interactions between domains that are distant in primary sequence. Both aspects are found to be necessary for the reproduction of the nonaveraged RDCs from alphaS. Although RDCs have previously been shown to report on local conformational preferences in unstructured proteins, this study shows the additional sensitivity of these measurements to the presence of long-range order in highly flexible systems.

A30P and A53T mutations of the presynaptic protein alpha-synuclein are associated with familial forms of Parkinson disease. NMR spectroscopy demonstrates that Parkinsonism-linked mutations greatly perturb specific tertiary interactions essential for the native state of alpha-synuclein. However, alpha-synuclein is not completely unfolded but exhibits structural fluctuations on the time scale of secondary structure formation and loses its native conformation gradually when protein stability decreases. The redistribution of the ensemble of alpha-synuclein conformers may underlie toxic gain-of-function by fostering self-association and altered binding affinity to ligands and receptors.

The aggregation of alpha-synuclein (AS) is characteristic of Parkinson's disease and other neurodegenerative synucleinopathies. We demonstrate here that Cu(II) ions are effective in accelerating AS aggregation at physiologically relevant concentrations without altering the resultant fibrillar structures. By using numerous spectroscopic techniques (absorption, CD, EPR, and NMR), we have located the primary binding for Cu(II) to a specific site in the N terminus, involving His-50 as the anchoring residue and other nitrogen/oxygen donor atoms in a square planar or distorted tetragonal geometry. The carboxylate-rich C terminus, originally thought to drive copper binding, is able to coordinate a second Cu(II) equivalent, albeit with a 300-fold reduced affinity. The NMR analysis of AS-Cu(II) complexes reveals the existence of conformational restrictions in the native state of the protein. The metallobiology of Cu(II) in Parkinson's disease is discussed by a comparative analysis with other Cu(II)-binding proteins involved in neurodegenerative disorders.

In idiopathic Parkinson's disease, intracytoplasmic neuronal inclusions (Lewy bodies) containing aggregates of the protein alpha-synuclein (alphaS) are deposited in the pigmented nuclei of the brainstem. The mechanisms underlying the structural transition of innocuous, presumably natively unfolded, alphaS to neurotoxic forms are largely unknown. Using paramagnetic relaxation enhancement and NMR dipolar couplings, we show that monomeric alphaS assumes conformations that are stabilized by long-range interactions and act to inhibit oligomerization and aggregation. The autoinhibitory conformations fluctuate in the range of nanoseconds to micro-seconds corresponding to the time scale of secondary structure formation during folding. Polyamine binding and/or temperature increase, conditions that induce aggregation in vitro, release this inherent tertiary structure, leading to a completely unfolded conformation that associates readily. Stabilization of the native, autoinhibitory structure of alphaS constitutes a potential strategy for reducing or inhibiting oligomerization and aggregation in Parkinson's disease.

Several families of plant transcription factors contain a conserved DNA binding motif known as the homeodomain. In two of these families, named Hd-Zip and glabra2, the homeodomain is associated with a leucine zipper-like dimerization motif. A group of Hd-Zip proteins, namely Hd-ZipII, contain a set of conserved cysteines within the dimerization motif and adjacent to it. Incubation of one of these proteins, Hahb-10, in the presence of thiol-reducing agents such as dithiothreitol or reduced glutathione produced a significant increase in DNA binding. Under such conditions, the protein migrated as a monomer in non-reducing SDS-polyacrylamide gels. Under oxidizing conditions, a significant proportion of the protein migrated as dimers, suggesting the formation of intermolecular disulfide bonds. A similar behavior was observed for the glabra2 protein HAHR1, which also contains two conserved cysteines within its dimerization domain. Site-directed mutagenesis of the cysteines to serines indicated that each of them has different roles in the activation of the proteins. Purified thioredoxin was able to direct the NADPH-dependent activation of Hahb-10 and HAHR1 in the presence of thioredoxin reductase. The results suggest that redox conditions may operate to regulate the activity of these groups of plant transcription factors within plant cells.

Four groups of plant homeodomain proteins contain a dimerization motif closely linked to the homeodomain. We here show that two sunflower homeodomain proteins, Hahb-4 and HAHR1, which belong to the Hd-Zip I and GL2/Hd-Zip IV groups, respectively, show different binding preferences at a defined position of a pseudopalindromic DNA-binding site used as a target. HAHR1 shows a preference for the sequence 5'-CATT(A/T)AATG-3', rather than 5'-CAAT(A/T)ATTG-3', recognized by Hahb-4. To analyze the molecular basis of this behavior, we have constructed a set of mutants with exchanged residues (Phe-->Ile and Ile-->Phe) at position 47 of the homeodomain, together with chimeric proteins between HAHR1 and Hahb-4. The results obtained indicate that Phe47, but not Ile47, allows binding to 5'-CATT(A/T)AATG-3'. However, the preference for this sequence is determined, in addition, by amino acids located C-terminal to residue 53 of the HAHR1 homeodomain. A double mutant of Hahb-4 (Ile47-->Phe/Ala54-->Thr) shows the same binding behavior as HAHR1, suggesting that combinatorial interactions of amino acid residues at positions 47 and 54 of the homeodomain are involved in establishing the affinity and selectivity of plant dimeric homeodomain proteins with different DNA target sequences.

Plant homeodomain-leucine zipper proteins, unlike most animal homeodomains, bind DNA efficiently only as dimers. In the present work, we report that the deletion of the homeodomain N-terminal arm (first nine residues) of the homeodomain-leucine zipper protein Hahb-4 dramatically affects its DNA-binding affinity, causing a 70-fold increase in dissociation constant. The addition of the N-terminal arm of Drosophila Antennapedia to the truncated form restores the DNA-binding affinity of dimers to values similar to those of the native form. However, the Antennapedia N-terminal arm is not able to confer increased binding affinity to monomers of Hahb-4 lacking the leucine zipper motif, indicating that the inefficient binding of monomers must be due to structural differences in other parts of the molecule. The construction of proteins with modifications at residues 5 to 7 of the homeodomain suggests strongly that positively charged amino acids at these positions play essential roles in determining the DNA-binding affinity. However, the effect of mutations at positions 6 and 7 can be counteracted by introducing a stretch of positively charged residues at positions 1 to 3 of the homeodomain. Sequence comparisons indicate that all homeodomain-leucine zipper proteins might use contacts of the N-terminal arm with DNA for efficient binding. The occurrence of a homeodomain with a DNA-interacting N-terminal arm must then be an ancient acquisition in evolution, earlier than the separation of lines leading to metazoa, fungi and plants., (Copyright 2001 Academic Press.)