Yuriy Sharikov

Mechanisms of Hybrid Oligomer Formation in the Pathogenesis of Combined Alzheimer's and Parkinson's Diseases

Background Misfolding and pathological aggregation of neuronal proteins has been proposed to play a critical role in the pathogenesis of neurodegenerative disorders. Alzheimers disease (AD) and Parkinsons disease (PD) are frequent neurodegenerative diseases of the aging population. While progressive accumulation of amyloid β protein (Aβ) oligomers has been identified as one of the central toxic events in AD, accumulation of α-synuclein (α-syn) resulting in the formation of oligomers and protofibrils has been linked to PD and Lewy body Disease (LBD). We have recently shown that Aβ promotes α-syn aggregation and toxic conversion in vivo, suggesting that abnormal interactions between misfolded proteins might contribute to disease pathogenesis. However the molecular characteristics and consequences of these interactions are not completely clear. Methodology/Principal Findings In order to understand the molecular mechanisms involved in potential Aβ/α-syn interactions, immunoblot, molecular modeling, and in vitro studies with α-syn and Aβ were performed. We showed in vivo in the brains of patients with AD/PD and in transgenic mice, Aβ and α-synuclein co-immunoprecipitate and form complexes. Molecular modeling and simulations showed that Aβ binds α-syn monomers, homodimers, and trimers, forming hybrid ring-like pentamers. Interactions occurred between the N-terminus of Aβ and the N-terminus and C-terminus of α-syn. Interacting α-syn and Aβ dimers that dock on the membrane incorporated additional α-syn molecules, leading to the formation of more stable pentamers and hexamers that adopt a ring-like structure. Consistent with the simulations, under in vitro cell-free conditions, Aβ interacted with α-syn, forming hybrid pore-like oligomers. Moreover, cells expressing α-syn and treated with Aβ displayed increased current amplitudes and calcium influx consistent with the formation of cation channels. Conclusion/Significance These results support the contention that Aβ directly interacts with α-syn and stabilized the formation of hybrid nanopores that alter neuronal activity and might contribute to the mechanisms of neurodegeneration in AD and PD. The broader implications of such hybrid interactions might be important to the pathogenesis of other disorders of protein misfolding.

Dynamics of α-synuclein aggregation and inhibition of pore-like oligomer development by β-synuclein

Accumulation of α‐synuclein resulting in the formation of oligomers and protofibrils has been linked to Parkinsons disease and Lewy body dementia. In contrast, β‐synuclein (β‐syn), a close homologue, does not aggregate and reduces α‐synuclein (α‐syn)‐related pathology. Although considerable information is available about the conformation of α‐syn at the initial and end stages of fibrillation, less is known about the dynamic process of α‐syn conversion to oligomers and how interactions with antiaggregation chaperones such as β‐synuclein might occur. Molecular modeling and molecular dynamics simulations based on the micelle‐derived structure of α‐syn showed that α‐syn homodimers can adopt nonpropagating (head‐to‐tail) and propagating (head‐to‐head) conformations. Propagating α‐syn dimers on the membrane incorporate additional α‐syn molecules, leading to the formation of pentamers and hexamers forming a ring‐like structure. In contrast, β‐syn dimers do not propagate and block the aggregation of α‐syn into ring‐like oligomers. Under in vitro cell‐free conditions, α‐syn aggregates formed ring‐like structures that were disrupted by β‐syn. Similarly, cells expressing α‐syn displayed increased ion current activity consistent with the formation of Zn2+‐sensitive nonselective cation channels. These results support the contention that in Parkinsons disease and Lewy body dementia, α‐syn oligomers on the membrane might form pore‐like structures, and that the beneficial effects of β‐synuclein might be related to its ability to block the formation of pore‐like structures.

Role of α-synuclein penetration into the membrane in the mechanisms of oligomer pore formation.

Parkinson’s disease (PD) and dementia with Lewy bodies are common disorders of the aging population and characterized by the progressive accumulation of α‐synuclein (α‐syn) in the central nervous system. Aggregation of α‐syn into oligomers with a ring‐like appearance has been proposed to play a role in toxicity. However, the molecular mechanisms and the potential sequence of events involved in the formation of pore‐like structures are unclear. We utilized computer modeling and cell‐based studies to investigate the process of oligomerization of wild‐type and A53T mutant α‐syn in membranes. The studies suggest that α‐syn penetrates the membrane rapidly, changing its conformation from α‐helical towards a coiled structure. This penetration facilitates the incorporation of additional α‐syn monomers in the complex, and the subsequent displacement of phospholipids and the formation of oligomers in the membrane. This process occurred more rapidly, and with a more favorable energy of interaction, for mutant A53T compared with wild‐type α‐syn. After 4 ns of simulation of the protein–membrane model, α‐syn had penetrated through two‐thirds of the membrane. By 9 ns, the penetration of the annular α‐syn oligomers can result in the formation of pore‐like structures that fully perforate the lipid bilayer. Experimental incubation of recombinant α‐syn in synthetic membranes resulted in the formation of similar pore‐like complexes. Moreover, mutant (A53T) α‐syn had a greater tendency to accumulate in neuronal membrane fractions in cell cultures, resulting in greater neuronal permeability, as demonstrated with the calcein efflux assay. These studies provide a sequential molecular explanation for the process of α‐syn oligomerization in the membrane, and support the role of formation of pore‐like structures in the pathogenesis of the neurodegenerative process in PD.

Mechanism of alpha-synuclein oligomerization and membrane interaction: theoretical approach to unstructured proteins studies.

Misfolding and oligomerization of unstructured proteins is involved in the pathogenesis of Parkinsons disease (PD), Alzheimers disease, Huntingtons disease, and other neurodegenerative disorders. Elucidation of possible conformations of these proteins and their interactions with the membrane is necessary to understand the molecular mechanisms of neurodegeneration. We developed a strategy that makes it possible to elucidate the molecular mechanisms of alpha-synuclein aggregation-a key molecular event in the pathogenesis of PD. This strategy can be also useful for the study of other unstructured proteins involved in neurodegeneration. The results of these theoretical studies have been confirmed with biochemical and electrophysiological studies. Our studies provide insights into the molecular mechanism for PD initiation and progression, and provide a useful paradigm for identifying possible therapeutic interventions through computational modeling.

Recognition of dextran-superparamagnetic iron oxide nanoparticle conjugates (Feridex) via macrophage scavenger receptor charged domains.

Dextran-coated superparamagnetic iron oxide nanoparticles (dextran-SPIO conjugates) offer the attractive possibility of enhancing MRI imaging sensitivity so that small or diffuse lesions can be detected. However, systemically injected SPIOs are rapidly removed by macrophages. We engineered embryonic cells (HEK293T) to express major macrophage scavenger receptor (SR) subtypes including SR-AI, MARCO, and endothelial receptor collectin-12. These SRs possess a positively charged collagen-like (CL) domain and they promoted SPIO uptake, while the charge neutral lipoprotein receptor SR-BI did not. In silico modeling indicated a positive net charge on the CL domain and a net negative charge on the cysteine-rich (CR) domain of MARCO and SR-AI. In vitro experiments revealed that CR domain deletion in SR-AI boosted uptake of SPIO 3-fold, while deletion of MARCOs CR domain abolished this uptake. These data suggest that future studies might productively focus on the validation and further exploration of SR charge fields in SPIO recognition.

An All-Atom Model of the Structure of Human Copper Transporter 1

Human copper transporter 1 (hCTR1) is the major high affinity copper influx transporter in mammalian cells that also mediates uptake of the cancer chemotherapeutic agent cisplatin. A low resolution structure of hCTR1 determined by cryoelectron microscopy was recently published. Several protein structure simulation techniques were used to create an all-atom model of this important transporter using the low resolution structure as a starting point. The all-atom model provides new insights into the roles of specific residues of the N-terminal extracellular domain, the intracellular loop, and C-terminal region in metal ion transport. In particular, the model demonstrates that the central region of the pore contains four sets of methionine triads in the intramembranous region. The structure confirms that two triads of methionine residues delineate the intramembranous region of the transporter, and further identifies two additional methionine triads that are located in the extracellular N-terminal part of the transporter. Together, the four triads create a structure that promotes stepwise transport of metal ions into and then through the intramembranous channel of the transporter via transient thioether bonds to methionine residues. Putative copper-binding sites in the hCTR1 trimer were identified by a program developed by us for prediction of metal-binding sites. These sites correspond well with the known effects of mutations on the ability of the protein to transport copper and cisplatin.

MAPAS: a tool for predicting membrane-contacting protein surfaces.

To the editor: Many important biological processes, from serum phospholipid metabolism to amyloid disease, involve formation of protein-membrane complexes. Thus, tools for identifying membranecontacting features in a protein structure are very important. However, few algorithmic approaches for membrane-contacting surface prediction have yet been reported1,2. We developed a program and web-based tool called MAPAS, or membrane-associated-proteins assessment (http://cancertools.sdsc.edu/MAPAS/pro2.html). MAPAS uses a set of algorithmic scoring functions to predict whether a given protein structure can form strong membrane contacts and to define the regions of the protein surface that most likely form such contacts (Supplementary Methods online). The MAPAS input window (Supplementary Fig. 1 online) accepts Protein Data Bank (PDB) protein identifiers or a pasted file in pdb format. The MAPAS algorithm is based on the assumption that membrane-contacting protein surfaces have a specific distribution of membranephilic surface residues in a plane. This planar region would contact the membrane (the explicit assumption is that, on the scale of proteins, the cell membrane can be considered as a plane). These residues must provide the necessary binding energy to keep the protein at the membrane surface. MAPAS (i) identifies the planar surfaces that encompass a given protein, and (ii) scores them according to their membranephilic properties. To provide a measure of membranephilicity, we estimated the relative tendency of individual residues to bind to a phospholipid bilayer. We calculated scoring functions using a semi-empiric approach based on steered molecular dynamics (Supplementary Figs. 2–4 and Supplementary Table 1 online) and Poisson-Boltzmann calculations (Supplementary Methods). MAPAS accepts a protein’s three-dimensional structure as input and identifies all planes encompassing the protein structure (Fig. 1a) then calculates all residues that lie in the layer of a given thickness (Supplementary Fig. 5 online). Then MAPAS sorts the planar protein surfaces based on their membranephilic character. The output window displays rotatable three-dimensional presentations of submitted proteins with their possible membrane–contacting surfaces indicated (see for example, Supplementary Figs. 6 and 7 online). We validated the performance of MAPAS with several known membrane-contacting proteins (Fig. 1b and Supplementary Tables 2 and 3 online). MAPAS can predict membrane-contacting proteins, membrane-associated proteins and the membrane-contacting surfaces of proteins including transmembrane proteins (Supplementary Discussion online). Nevertheless, as with all prediction programs, MAPAS can yield false positive and false negative predictions. One possible source of error is the fact that coordinates of proteins listed in PDB as membrane-contacting do not include the membrane–contacting regions, either because they are disordered or because they are engineered out of the protein to permit crystallization. Another problem is the relatively small area of membrane contact found in some proteins. Our tests show that MAPAS is reliable when the number of membrane-contacting residues is at least 5 (data not shown). With fewer residues in the membrane-contacting zone the statistical error increases. Note: Supplementary information is available on the Nature Methods website.

Structural Diversity of Alzheimer's Disease Amyloid-β Dimers and Their Role in Oligomerization and Fibril Formation

Alzheimers disease (AD) is associated with the formation of toxic amyloid-β (Aβ)42 oligomers, and recent evidence supports a role for Aβ dimers as building blocks for oligomers. Molecular dynamics simulation studies have identified clans for the dominant conformations of Aβ42 forming dimers; however, it is unclear if a larger spectrum of dimers is involved and which set(s) of dimers might evolve to oligomers verse fibrils. Therefore, for this study we generated multiple structural conformations of Aβ42, using explicit all-atom molecular dynamics, and then clustering the different structures based on key conformational similarities. Those matching a selection threshold were then used to model a process of oligomerization. Remarkably, we showed a greater diversity in Aβ dimers than previously described. Depending on the clan family, different types of Aβ dimers were obtained. While some had the tendency to evolve into oligomeric rings, others formed fibrils of diverse characteristics. Then we selected the dimers that would evolve to membranephilic annular oligomers. Nearly one third of the 28 evaluated annular oligomers had the dimer interfaces between the neighboring Aβ42 monomers with possible salt bridges between the residue K28 from one side and either residue E22 or D23 on the other. Based on these results, key amino acids were identified for point mutations that either enhanced or suppressed the formation and toxicity of oligomer rings. Our studies suggest a greater diversity of Aβ dimers. Understanding the structure of Aβ dimers might be important for the rationale design of small molecules that block formation of toxic oligomers.

Molecular Determinants of α-Synuclein Mutants’ Oligomerization and Membrane Interactions

Parkinsons disease (PD) is associated with the formation of toxic α-synuclein oligomers that can penetrate the cell membrane. Familial forms of PD are caused by the point mutations A53T, A30P, E46K, and H50Q. Artificial point mutations E35K and E57K also increase oligomerization and pore formation. We generated structural conformations of α-synuclein and the above-mentioned mutants using molecular dynamics. We elucidated four main regions in these conformers contacting the membrane and found that the region including residues 39-45 (Zone2) may have maximum membrane penetration. E57K mutant had the highest rate of interaction with the membrane, followed by A53T, E46K, and E35K mutants and wild type (wt) α-synuclein. The mutant A30P had the smallest percentage of conformers that contact the membrane by Zone 2 than all other mutants and wt α-synuclein. These results were confirmed experimentally in vitro. We identified the key amino acids that can interact with the membrane (Y38, E62, and N65 (first hydrophilic layer); E104, E105, and D115 (second hydrophilic layer), and V15 and V26 (central hydrophobic layer)) and the residues that are involved in the interprotein contacts (L38, V48, V49, Q62, and T64). Understanding the molecular interactions of α-synuclein mutants is important for the design of compounds blocking the formation of toxic oligomers.

Molecular mechanism of pore creation in bacterial membranes by amyloid proteins

This study explores the mechanism of pore creation in cellular membranes by MccE92 bacterial proteins. The results of this study are then compared with the mechanism of alpha-synuclein (aS)-based pore formation in mammalian cells, and its role in Parkinsons disease.