Nasrollah Rezaei-Ghaleh

Pre-fibrillar α-synuclein variants with impaired β-structure increase neurotoxicity in Parkinson's disease models

The relation of α‐synuclein (αS) aggregation to Parkinsons disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated αS species have in neurotoxicity in vivo, we generated αS variants by a structure‐based rational design. Biophysical analysis revealed that the αS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre‐fibrillar αS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between αS aggregates with impaired β‐structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric αS species and their in vivo function.

Extracellular phosphorylation of the amyloid β‐peptide promotes formation of toxic aggregates during the pathogenesis of Alzheimer's disease

Alzheimers disease (AD) is the most common form of dementia and associated with progressive deposition of amyloid β‐peptides (Aβ) in the brain. Aβ derives by sequential proteolytic processing of the amyloid precursor protein by β‐ and γ‐secretases. Rare mutations that lead to amino‐acid substitutions within or close to the Aβ domain promote the formation of neurotoxic Aβ assemblies and can cause early‐onset AD. However, mechanisms that increase the aggregation of wild‐type Aβ and cause the much more common sporadic forms of AD are largely unknown. Here, we show that extracellular Aβ undergoes phosphorylation by protein kinases at the cell surface and in cerebrospinal fluid of the human brain. Phosphorylation of serine residue 8 promotes formation of oligomeric Aβ assemblies that represent nuclei for fibrillization. Phosphorylated Aβ was detected in the brains of transgenic mice and human AD brains and showed increased toxicity in Drosophila models as compared with non‐phosphorylated Aβ. Phosphorylation of Aβ could represent an important molecular mechanism in the pathogenesis of the most common sporadic form of AD.

Inhibition of amyloid fibrillation of lysozyme by indole derivatives ) possible mechanism of action

Amyloid aggregation of polypeptides is related to a growing number of pathologic states known as amyloid disorders. There is a great deal of interest in developing small molecule inhibitors of the amyloidogenic processes. In the present article, the inhibitory effects of some indole derivatives on amyloid fibrillation of hen egg white lysozyme (HEWL) are reported. Acidic pH and high temperatures were used to drive HEWL towards amyloid formation. A variety of techniques, ranging from thioflavin T fluorescence and Congo red absorbance assays to far‐UV CD and transmission electron microscopy, were employed to characterize the HEWL fibrillation process. Among the indole derivatives tested, indole 3‐acetic acid, indole 3‐carbinol and tryptophol had the most inhibitory effects on amyloid formation, indole and indole 3‐propionic acid gave some inhibition, and indole aldehyde and tryptophan showed no significant inhibition. Although indoles did not protect the HEWL native state from conformational changes, they were effective in diminishing HEWL amyloid fibril formation, delaying both the nucleation and elongation phases. Disaggregation of previously formed HEWL amyloid fibrils was also enhanced by indole 3‐acetic acid. Various medium conditions, such as the presence of different anions and alcoholic cosolvents, were explored to gain an insight into possible mechanisms. These observations, taken together, suggest that the indole ring is likely to play the main role in inhibition and that the side chain hydroxyl group may contribute positively, in contrast to the side chain carbonyl and intervening methylene groups.

N-truncated amyloid β (Aβ) 4-42 forms stable aggregates and induces acute and long-lasting behavioral deficits

N-truncated Aβ4-42 is highly abundant in Alzheimer disease (AD) brain and was the first Aβ peptide discovered in AD plaques. However, a possible role in AD aetiology has largely been neglected. In the present report, we demonstrate that Aβ4-42 rapidly forms aggregates possessing a high aggregation propensity in terms of monomer consumption and oligomer formation. Short-term treatment of primary cortical neurons indicated that Aβ4-42 is as toxic as pyroglutamate Aβ3-42 and Aβ1-42. In line with these findings, treatment of wildtype mice using intraventricular Aβ injection induced significant working memory deficits with Aβ4-42, pyroglutamate Aβ3-42 and Aβ1-42. Transgenic mice expressing Aβ4-42 (Tg4-42 transgenic line) developed a massive CA1 pyramidal neuron loss in the hippocampus. The hippocampus-specific expression of Aβ4-42 correlates well with age-dependent spatial reference memory deficits assessed by the Morris water maze test. Our findings indicate that N-truncated Aβ4-42 triggers acute and long-lasting behavioral deficits comparable to AD typical memory dysfunction.

Intrinsically disordered proteins: from sequence and conformational properties toward drug discovery.

Structural disorder of functional proteins under physiological conditions is widespread within eukaryotic proteomes. The lack of stable tertiary and secondary structure offers a variety of functional advantages to intrinsically disordered proteins (IDPs): their malleability of interaction with different partners, specific but low‐affinity binding, and their fine modulation by post‐translational modifications. IDPs are therefore central players in key processes such as cell‐cycle control and signal‐transduction pathways, and impairment of their function is associated with many disease states such as cancer and neurodegenerative disorders. Fascinating progress in the experimental characterization of IDPs has been made in the last decade, especially in NMR spectroscopy and small‐angle X‐ray scattering as well as in single‐molecule techniques. It has been accompanied by the development of powerful computational tools to translate experimental results in explicit ensemble representations of IDPs. With the aid of bioinformatics tools, these advances have paved the way to targeting IDP interactions in rational drug‐discovery projects.

Effect of Zinc Binding on β-Amyloid Structure and Dynamics: Implications for Aβ Aggregation

Assembly of β-amyloid (Aβ) peptide into toxic oligomers is widely believed to initiate Alzheimers disease pathogenesis. Under in vitro physiological conditions, zinc (Zn(II)) can bind to Aβ and redirect its assembly from amyloid fibrillar toward less toxic amorphous aggregation. Propensity of Aβ to go toward a specific form of aggregate state is determined by structural and dynamical properties of the initial monomeric as well as the aggregate state. Here we probe the structural and dynamical impact of binding of Zn(II) to monomeric Aβ40 using NMR spectroscopy. To obtain further support for the importance of intrinsic dynamics in the aggregation precursor, (15)N relaxation measurements were also performed for Aβ42, the more fibrillar aggregation-prone variant of Aβ. The combined data suggest that, upon Zn(II)-binding to the N-terminus of Aβ40, a relatively rigid turnlike structure is induced at residues Val(24)-Lys(28) whereas the residues flanking this region become more mobile on the picosecond-to-nanosecond timescale. This is in contrast to the increased rigidity of Aβ42 at the C-terminus, and proposed to be linked to the higher propensity of Zn(II)-bound peptide to form amorphous aggregates with less entropic penalties than their fibrillar counterparts.

Methylation of K9 in histone H3 directs alternative modes of highly dynamic interaction of heterochromatin protein hHP1β with the nucleosome.

Background: Chromatin-HP1 (heterochromatin protein 1) interaction is crucial for heterochromatin assembly. Results: hHP1β uses alternative interfaces to bind nucleosomes depending on histone 3 methylation within a highly dynamic complex. Conclusion: hHP1β explores chromatin for sites of methyl-mark enrichment where it can bind histone 3 tails from adjacent nucleosomes. Significance: We provide a conceptual framework to understand the molecular basis of dynamic interactions regulated by histone modification. Binding of heterochromatin protein 1 (HP1) to the histone H3 lysine 9 trimethylation (H3K9me3) mark is a hallmark of establishment and maintenance of heterochromatin. Although genetic and cell biological aspects have been elucidated, the molecular details of HP1 binding to H3K9me3 nucleosomes are unknown. Using a combination of NMR spectroscopy and biophysical measurements on fully defined recombinant experimental systems, we demonstrate that H3K9me3 works as an on/off switch regulating distinct binding modes of hHP1β to the nucleosome. The methyl-mark determines a highly flexible and very dynamic interaction of the chromodomain of hHP1β with the H3-tail. There are no other constraints of interaction or additional multimerization interfaces. In contrast, in the absence of methylation, the hinge region and the N-terminal tail form weak nucleosome contacts mainly with DNA. In agreement with the high flexibility within the hHP1β-H3K9me3 nucleosome complex, the chromoshadow domain does not provide a direct binding interface. Our results report the first detailed structural analysis of a dynamic protein-nucleosome complex directed by a histone modification and provide a conceptual framework for understanding similar interactions in the context of chromatin.

Long-Range Correlated Dynamics in Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) are involved in a wide variety of physiological and pathological processes and are best described by ensembles of rapidly interconverting conformers. Using fast field cycling relaxation measurements we here show that the IDP α-synuclein as well as a variety of other IDPs undergoes slow reorientations at time scales comparable to folded proteins. The slow motions are not perturbed by mutations in α-synuclein, which are related to genetic forms of Parkinsons disease, and do not depend on secondary and tertiary structural propensities. Ensemble-based hydrodynamic calculations suggest that the time scale of the underlying correlated motion is largely determined by hydrodynamic coupling between locally rigid segments. Our study indicates that long-range correlated dynamics are an intrinsic property of IDPs and offers a general physical mechanism of correlated motions in highly flexible biomolecular systems.

Discovery and structure activity relationship of small molecule inhibitors of toxic β-amyloid-42 fibril formation

Background: Self-aggregation of β-amyloid plays an important role in the pathogenesis of Alzheimer disease. Results: Small molecule inhibitors of β-amyloid fibril formation reduce β-amyloid mediated cell toxicity. Conclusion: Rational design led to the successful development of small molecule inhibitors of β-amyloid oligomerization and toxicity. Significance: Small molecules targeting β-amyloid misfolding may provide new treatments for Alzheimer disease. Increasing evidence implicates Aβ peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aβ aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aβ42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the β-sheet conformation of Aβ42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aβ42. The efficacy of these compounds on inhibiting Aβ fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aβ42 leading to decreased cell toxicity.

Conformational Changes of α-Chymotrypsin in a Fibrillation-Promoting Condition: A Molecular Dynamics Study☆

Amyloid nanofibril formation appears to be a generic property of polypeptide chains. alpha-Chymotrypsin (aCT) was recently driven toward amyloid-like aggregation by the addition of trifluoroethanol (TFE) at intermediate concentrations. In this study we employed a molecular dynamics simulation to investigate the early events in TFE-induced conformational changes of aCT that precede amyloid formation, and compared the results of the simulation with previous experiments. TFE molecules were found to rapidly replace the water molecules closely associated with the protein surface. The gyration radius, together with total and hydrophobic solvent-accessible surface areas of aCT, was significantly increased. In accord with the experimental observations, the extended beta-conformation of backbone was increased. The secondary structural elements of aCT in water and TFE/water mixture showed a reasonable fit, whereas significant deviations were observed for several loops. These alterations originated largely from main-chain rotations at glycine residues. The catalytic active site and S1 binding pocket of the enzyme were also distorted in the TFE/water mixture. The obtained results are suggested to provide more insights into the conformational properties of the amyloid aggregation-prone protein species. Possible mechanisms of TFE-induced alterations in the conformation and dynamics of the protein structure are also discussed.