Nanoimaging for prion related diseases

Proceedings of the National Academy of Sciences, 2005

There is a hypothesis that dangerous diseases such as bovine spongiform encephalopathy, Creutzfeldt-Jakob, Alzheimer's, fatal familial insomnia, and several others are induced by propagation of wrong or misfolded conformations of some vital proteins. If for some reason the misfolded conformations were acquired by many such protein molecules it might lead to a ''conformational'' disease of the organism. Here, a theoretical model of the molecular mechanism of such a conformational disease is proposed, in which a metastable (or misfolded) form of a protein induces a similar misfolding of another protein molecule (conformational autocatalysis). First, a number of amino acid sequences composed of 32 aa have been designed that fold rapidly into a well defined native-like ␣-helical conformation. From a large number of such sequences a subset of 14 had a specific feature of their energy landscape, a well defined local energy minimum (higher than the global minimum for the ␣-helical fold) corresponding to ␤-type structure. Only one of these 14 sequences exhibited a strong autocatalytic tendency to form a ␤-sheet dimer capable of further propagation of protofibrillike structure. Simulations were done by using a reduced, although of high resolution, protein model and the replica exchange Monte Carlo sampling procedure. molecular dynamics ͉ Monte Carlo ͉ replica exchange Monte Carlo T he biological function of a protein can be performed only if the protein molecules adopt a precisely folded conformation (the native structure). A deviation from that structure, i.e., a misfolded conformation of a particular protein molecule either makes it inactive or active in another direction, in many cases (however, not always) harmful or even destructive for the host organism. Alternative folding or refolding (1, 2), frequently followed by a large-scale aggregation (3) of proteins may lead to bovine spongiform encephalopathy, Creutzfeldt-Jakob (4), Alzheimer's, fatal familial insomnia, and several other dangerous diseases (5, 6). The misfolded structures that are associated with the prion diseases contain a larger fraction of a ␤-type structure than the native structure does (7, 8). The ␤-rich fragments of such proteins easily associate and subsequently form the amyloid fibrils (9). Interestingly, it appears that under extreme conditions of pH (9-11), solvent composition, high pressure (12), etc. almost all globular proteins can be converted into the amyloid form . Fortunately, at physiological conditions such aggregation is relatively rare. Experimental data indicate that the internal part of amyloid fibrils is highly ordered, although the detailed structure is not known. The leading hypothesis is that the fibrils are composed from protofibrils that have regular ␤-sheet type structure . Alternative molecular models postulate a ␤-helical structure (8, 15).

Journal of Biological Chemistry, 2005

Protein conformational transition from ␣-helices to ␤-sheets precedes aggregation of proteins implicated in many diseases, including Alzheimer and prion diseases. Direct characterization of such transitions is often hindered by the complicated nature of the interaction network among amino acids. A recently engineered small protein-like peptide with a simple amino acid composition features a temperature-driven ␣-helix to ␤-sheet conformational change. Here we studied the conformational transition of this peptide by molecular dynamics simulations. We observed a critical temperature, below which the peptide folds into an ␣-helical coiledcoil state and above which the peptide misfolds into ␤-rich structures with a high propensity to aggregate. The structures adopted by this peptide during low temperature simulations have a backbone root mean square deviation less than 2 Å from the crystal structure. At high temperatures, this peptide adopts an amyloid-like structure, which is mainly composed of coiled anti-parallel ␤-sheets with the cross-␤-signature of amyloid fibrils. Most strikingly, we observed conformational conversions in which an ␣-helix is converted into a ␤-strand by proximate stable ␤-sheets with exposed hydrophobic surfaces and unsaturated hydrogen bonds. Our study suggested a possible generic molecular mechanism of the templatemediated aggregation process, originally proposed by Prusiner (Prusiner, S. B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13363-13383) to account for prion infectivity.

Journal of Neurochemistry, 2007

Prion diseases or transmissible spongiform encephalopathies (TSEs) are infectious and fatal neurodegenerative disorders in humans and animals. Pathological features of TSEs include the conversion of cellular prion protein (PrP C ) into an altered disease-associated conformation generally designated PrP Sc , abnormal deposition of PrP Sc aggregates, and spongiform degeneration of the brain. The molecular steps leading to PrP C aggregation are unknown. Here, we have utilized an inducible oligomerization strategy to test if, in the absence of any infectious prion particles, the encounter between PrP C molecules may trigger its aggregation in neuronal cells. A chimeric PrP C composed of one (Fv1) or two (Fv2) modified FK506-binding protein (Fv) fused with PrP C were created, and transfected in N2a cells. Similar to PrP C , Fv1-PrP and Fv2-PrP were glycosylated, displayed normal localization, and anti-apoptotic function. When cells were treated with the dimeric Fv ligand AP20187, to induce dimerization (Fv1) or oligomerization (Fv2) of PrP C , both dimerization and oligomerization of PrP C resulted in the de novo production, release and deposition of extracellular PrP aggregates. Aggregates were insoluble in non-ionic detergents and partially resistant to proteinase K. These findings demonstrate that homologous interactions between PrP C molecules may constitute a minimal and sufficient molecular event leading to PrP C aggregation and extracellular deposition.

FEBS Journal, 2008

Journal of Biological Chemistry

2010

Using a recently developed mesoscopic theory of protein dielectrics, we have calculated the salt bridge energies, total residue electrostatic potential energies, and transfer energies into a low dielectric amyloidlike phase for 12 species and mutants of the prion protein. Salt bridges and self energies play key roles in stabilizing secondary and tertiary structural elements of the prion protein. The total electrostatic potential energy of each residue was found to be invariably stabilizing. Residues frequently found to be mutated in familial prion disease were among those with the largest electrostatic energies. The large barrier to charged group desolvation imposes regional constraints on involvement of the prion protein in an amyloid aggregate, resulting in an electrostatic amyloid recruitment profile that favours regions of sequence between alpha helix 1 and beta strand 2, the middles of helices 2 and 3, and the region N-terminal to alpha helix 1. We found that the stabilization due to salt bridges is minimal among the proteins studied for disease-susceptible human mutants of prion protein.

Biophysical Journal, 2005

Although the cellular monomeric form of the benign prion protein is now well characterized, a model for the monomer of the misfolded conformation (PrP Sc ) remains elusive. PrP Sc quickly aggregates into highly insoluble fibrils making experimental structural characterization very difficult. The tendency to aggregation of PrP Sc in aqueous solution implies that the monomer fold must be hydrophobic. Here, by using molecular dynamics simulations, we have studied the cellular mouse prion protein and its D178N pathogenic mutant immersed in a hydrophobic environment (solution of CCl 4 ), to reveal conformational changes and/or local structural weaknesses of the prion protein fold in unfavorable structural and thermodynamic conditions. Simulations in water have been also performed. Although observing in general a rather limited conformation activity in the nanosecond timescale, we have detected a significant weakening of the antiparallel b-sheet of the D178N mutant in CCl 4 and to a less extent in water. No weakening is observed for the native prion protein. The increase of b-structure in the monomer, recently claimed as evidence for misfolding to PrP Sc , has been also observed in this study irrespective of the thermodynamic or structural conditions, showing that this behavior is very likely an intrinsic characteristic of the prion protein fold.

Journal of Proteome Research, 2006

Misfolding and self-assembly of proteins in nanoaggregates of different sizes and morphologies (nanoensembles, primary nanofilaments, nanorings, filaments, protofibrils, fibrils, etc.) is a common theme unifying a number of human pathologies termed protein misfolding diseases. Recent studies highlight increasing recognition of the public health importance of protein misfolding diseases, including various neurodegenerative disorders and amyloidoses. It is understood now that the first essential elements in the vast majority of neurodegenerative processes are misfolded and aggregated proteins. Altogether, the accumulation of abnormal protein nanoensembles exerts toxicity by disrupting intracellular transport, overwhelming protein degradation pathways, and/or disturbing vital cell functions. In addition, the formation of inclusion bodies is known to represent a major problem in the production of recombinant therapeutic proteins. Formulation of these therapeutic proteins into delivery systems and their in vivo delivery are often complicated by protein association. Thus, protein folding abnormalities and subsequent events underlie a multitude of human pathologies and difficulties with protein therapeutic applications. The field of medicine therefore can be greatly advanced by establishing a fundamental understanding of key factors leading to misfolding and selfassembly responsible for various protein folding pathologies. This article overviews protein misfolding diseases and outlines some novel and advanced nanotechnologies, including nanoimaging techniques, nanotoolboxes and nanocontainers, complemented by appropriate ensemble techniques, all focused on the ultimate goal to establish etiology and to diagnose, prevent, and cure these devastating disorders.

PLOS ONE, 2017

Proteins associated with neurodegenerative diseases are highly pleiomorphic and may adopt an all-α-helical fold in one environment, assemble into all-β-sheet or collapse into a coil in another, and rapidly polymerize in yet another one via divergent aggregation pathways that yield broad diversity of aggregates' morphology. A thorough understanding of this behaviour may be necessary to develop a treatment for Alzheimer's and related disorders. Unfortunately, our present comprehension of folding and misfolding is limited for want of a physicochemical theory of protein secondary and tertiary structure. Here we demonstrate that electronic configuration and hyperconjugation of the peptide amide bonds ought to be taken into account to advance such a theory. To capture the effect of polarization of peptide linkages on conformational and H-bonding propensity of the polypeptide backbone, we introduce a function of shielding tensors of the C α atoms. Carrying no information about side chain-side chain interactions, this function nonetheless identifies basic features of the secondary and tertiary structure, establishes sequence correlates of the metamorphic and pHdriven equilibria, relates binding affinities and folding rate constants to secondary structure preferences, and manifests common patterns of backbone density distribution in amyloidogenic regions of Alzheimer's amyloid β and tau, Parkinson's α-synuclein and prions. Based on those findings, a split-intein like mechanism of molecular recognition is proposed to underlie dimerization of Aβ, tau, αS and PrP C , and divergent pathways for subsequent association of dimers are outlined; a related mechanism is proposed to underlie formation of PrP Sc fibrils. The model does account for: (i) structural features of paranuclei, off-pathway oligomers, non-fibrillar aggregates and fibrils; (ii) effects of incubation conditions, point mutations, isoform lengths, small-molecule assembly modulators and chirality of solid-liquid interface on the rate and morphology of aggregation; (iii) fibril-surface catalysis of secondary nucleation; and (iv) self-propagation of infectious strains of mammalian prions.

Prion

Protein misfolding and assembly into ordered, self-templating aggregates (amyloid) has emerged as a novel mechanism for regulating protein function. For a subclass of amyloidogenic proteins known as prions, this process induces transmissible changes in normal cellular physiology, ranging from neurodegenerative disease in animals and humans to new traits in fungi. The severity and stability of these altered phenotypic states can be attenuated by the conformation or amino-acid sequence of the prion, but in most of these cases, the protein retains the ability to form amyloid in vitro. Thus, our ability to link amyloid formation in vitro with its biological consequences in vivo remains a challenge. In two recent studies, we have begun to address this disconnect by assessing the effects of the cellular environment on traits associated with the misfolding of the yeast prion Sup35. Remarkably, the effects of quality control pathways and of limitations on protein transfer in vivo amplify th...

Biophysical Journal, 2003

We extend our previous stochastic cellular automata-based model for two-dimensional (areal) aggregation of prion proteins on neuronal surfaces. The new anisotropic model allows us to simulate both strong b-sheet and weaker attachment bonds between proteins. Constraining binding directions allows us to generate aggregate structures with the hexagonal lattice symmetry found in recently observed in vitro experiments. We argue that these constraints on rules may correspond to underlying steric constraints on the aggregation process. We find that monomer-dominated growth of the areal aggregate is too slow to account for some observed doubling-time-to-incubation-time ratios inferred from data, and so consider aggregation dominated by relatively stable but noninfectious oligomeric intermediates. We compare a kinetic theory analysis of oligomeric aggregation to spatially explicit simulations of the process. We find that with suitable rules for misfolding of oligomers, possibly due to water exclusion by the surrounding aggregate, the resulting oligomeric aggregation model maps onto our previous monomer aggregation model. Therefore it can produce some of the same attractive features for the description of prion incubation time data. We propose experiments to test the oligomeric aggregation model.

Physical Chemistry Chemical Physics, 2013

Protein misfolding and aggregation are relevant to many fields. Recently, their investigation has experienced a revival as a central topic in the research of numerous human diseases, including Parkinson's and Alzheimer's. Much has been learned from ensemble biochemical approaches, but the inherently heterogeneous nature of the underlying processes has obscured many important details. Single-molecule techniques offer unique capabilities to study heterogeneous systems, while providing high temporal and structural resolution to characterize them. In this Perspective, we give an overview of the single-molecule assays that have been applied to protein misfolding and aggregation, which are mainly based on fluorescence and force spectroscopy. We describe some of the technical challenges involved in studying aggregation at the single-molecule level and discuss what has been learned about aggregation mechanisms from the different approaches.

Biochimica et biophysica acta, 2018

Prion (PrP) diseases are neurodegenerative diseases characterized by the formation of β-sheet rich, insoluble and protease resistant protein deposits (called PrP) that occur throughout the brain. Formation of synthetic or in vitro PrP can occur through on-pathway toxic oligomers. Similarly, toxic and infectious oligomers identified in cell and animal models of prion disease indicate that soluble oligomers are likely intermediates in the formation of insoluble PrP. Despite the critical role of prion oligomers in disease progression, little is known about their structure. In order, to obtain structural insight into prion oligomers, we generated oligomers by shaking-induced conversion of recombinant, monomeric prion protein PrP (spanning residues 90-231). We then obtained two-dimensional solution NMR spectra of the PrP monomer, a 40% converted oligomer, and a 94% converted oligomer. Heteronuclear single-quantum correlation (H-N) studies revealed that, in comparison to monomeric PrP, th...

Journal of Proteome Research, 2006

Misfolding and self-assembly of proteins in nanoaggregates of different sizes and morphologies (nanoensembles, primary nanofilaments, nanorings, filaments, protofibrils, fibrils, etc.) is a common theme unifying a number of human pathologies termed protein misfolding diseases. Recent studies highlight increasing recognition of the public health importance of protein misfolding diseases, including various neurodegenerative disorders and amyloidoses. It is understood now that the first essential elements in the vast majority of neurodegenerative processes are misfolded and aggregated proteins. Altogether, the accumulation of abnormal protein nanoensembles exerts toxicity by disrupting intracellular transport, overwhelming protein degradation pathways, and/or disturbing vital cell functions. In addition, the formation of inclusion bodies is known to represent a major problem in the production of recombinant therapeutic proteins. Formulation of these therapeutic proteins into delivery systems and their in vivo delivery are often complicated by protein association. Thus, protein folding abnormalities and subsequent events underlie a multitude of human pathologies and difficulties with protein therapeutic applications. The field of medicine therefore can be greatly advanced by establishing a fundamental understanding of key factors leading to misfolding and selfassembly responsible for various protein folding pathologies. This article overviews protein misfolding diseases and outlines some novel and advanced nanotechnologies, including nanoimaging techniques, nanotoolboxes and nanocontainers, complemented by appropriate ensemble techniques, all focused on the ultimate goal to establish etiology and to diagnose, prevent, and cure these devastating disorders.

Nanomedicine: Nanotechnology, Biology and Medicine, 2005

Misfolding and self assembly of proteins in nano-aggregates of different sizes and morphologies (nano-ensembles, primarily nanofilaments and nano-rings) is a complex phenomenon that can be facilitated, impeded, or prevented, by interactions with various intracellular metabolites, intracellular nanomachines controlling protein folding and interactions with other proteins. A fundamental understanding of molecular processes leading to misfolding and self-aggregation of proteins involved in various neurodegenerative diseases will provide critical information to help identify appropriate therapeutic routes to control these processes. An elevated propensity of misfolded protein conformation in solution to aggregate with the formation of various morphologies impedes the use of traditional physical chemical approaches for studies of misfolded conformations of proteins. In our recent alternative approach, the protein molecules were tethered to surfaces to prevent aggregation and AFM force spectroscopy was used to probe the interaction between protein molecules depending on their conformations. It was shown that formation of filamentous aggregates is facilitated at pH values corresponding to the maximum of rupture forces. In this paper, a novel surface chemistry was developed for anchoring of amyloid β (Aβ) peptides at their N-terminal moieties. The use of the site specific immobilization procedure allowed to measure the rupture of Aβ -Aβ contacts at single molecule level. The rupture of these contacts is accompanied by the extension of the peptide chain detected by a characteristic elasto-mechanical component of the forcedistance curves. Potential applications of the nanomechanical studies to understanding the mechanisms of development of protein misfolding diseases are discussed.