Dynamics in the Unfolded State of β2-microglobulin Studied by NMR

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Nanoparticles present enormous surface areas and are found to enhance the rate of protein fibrillation by decreasing the lag time for nucleation. Protein fibrillation is involved in many human diseases, including Alzheimer's, Creutzfeld-Jacob disease, and dialysis-related amyloidosis. Fibril formation occurs by nucleationdependent kinetics, wherein formation of a critical nucleus is the key rate-determining step, after which fibrillation proceeds rapidly. We show that nanoparticles (copolymer particles, cerium oxide particles, quantum dots, and carbon nanotubes) enhance the probability of appearance of a critical nucleus for nucleation of protein fibrils from human ␤2-microglobulin. The observed shorter lag (nucleation) phase depends on the amount and nature of particle surface. There is an exchange of protein between solution and nanoparticle surface, and ␤2-microglobulin forms multiple layers on the particle surface, providing a locally increased protein concentration promoting oligomer formation. This and the shortened lag phase suggest a mechanism involving surface-assisted nucleation that may increase the risk for toxic cluster and amyloid formation. It also opens the door to new routes for the controlled self-assembly of proteins and peptides into novel nanomaterials.amyloid ͉ nanotoxicology ͉ surface-assisted nucleation

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Nucleation of protein fibrillation by nanoparticles

Linse

Cabaleiro-Lago

Xue

et al. 2007

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795

769

“…Using this approach, we have studied the assembly of ␤ 2 -microglobulin (␤ 2 m) into amyloid-like fibrils at low pH (15,16). Under these conditions, fibril formation occurs both rapidly and quantitatively, without visible formation of amorphous aggregates, providing an ideal system with which to develop theories of nucleated amyloid assembly.…”

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confidence: 99%

Systematic analysis of nucleation-dependent polymerization reveals new insights into the mechanism of amyloid self-assembly

Xue

Homans

Radford

2008

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428

523

Self-assembly of misfolded proteins into ordered fibrillar aggregates known as amyloid results in numerous human diseases. Despite an increasing number of proteins and peptide fragments being recognised as amyloidogenic, how these amyloid aggregates assemble remains unclear. In particular, the identity of the nucleating species, an ephemeral entity that defines the rate of fibril formation, remains a key outstanding question. Here, we propose a new strategy for analyzing the self-assembly of amyloid fibrils involving global analysis of a large number of reaction progress curves and the subsequent systematic testing and ranking of a large number of possible assembly mechanisms. Using this approach, we have characterized the mechanism of the nucleationdependent formation of ␤2-microglobulin (␤2m) amyloid fibrils. We show, by defining nucleation in the context of both structural and thermodynamic aspects, that a model involving a structural nucleus size approximately the size of a hexamer is consistent with the relatively small concentration dependence of the rate of fibril formation, contrary to expectations based on simpler theories of nucleated assembly. We also demonstrate that fibril fragmentation is the dominant secondary process that produces higher apparent cooperatively in fibril formation than predicted by nucleated assembly theories alone. The model developed is able to explain and predict the behavior of ␤2m fibril formation and provides a rationale for explaining generic properties observed in other amyloid systems, such as fibril growth acceleration and pathway shifts under agitation.AIC model comparison analysis ͉ amyloid fibril formation ͉ fibril brittleness ͉ global analysis S elf-assembly of misfolded forms of normally soluble and functional proteins or peptides into amyloid fibrils results in numerous human diseases (1). Understanding how amyloid self-assembly occurs, therefore, is of paramount importance for a molecular interpretation of amyloidosis and for the development of therapies against amyloid disease. Over the past decade, advances have been made toward a more complete description of amyoid fibril formation, including the determination of increasingly refined models of fibril structures (reviewed in ref. 1) and the identification of amyloid precursors and oligomeric states reviewed in ref. 2, one or more of which could be the culprits of cytotoxicity associated with several amyloid diseases (e.g., ref.3). However, the molecular events occurring during the self-assembly process itself remain obscure because of the heterogeneity and the complexity of the early association events.Amyloid fibril self-assembly reactions are generally accepted as a form of nucleated polymerization (4). These reactions are characterized by an initial lag phase where little or no change in fibril concentration can be detected. This is followed by an elongation phase where a large mass percentage of the starting protein material is converted into fibrils. A shared feature among amyloid formation and other nuclea...

“…A key observation is that the apoSOD monomer has an intrinsic weakness in the form of labile edge strands protecting the hydrophobic center of the major ␤-sheet. Upon local unfolding of these protective strands the structure of SOD is analogous to that of the ␤ 2 -microglobulin intermediate implicated in dialysis-related amyloidosis (23,24) and the high-energy intermediate of the D67H lysozyme mutant triggering non-neuropathic systemic amyloidoses (25). It is thus apparent that ALS and fibril deposition conditions share a similar disease-provoking precursor species, despite fundamental differences in the amount, morphology, and cellular location of aggregated material depositing during the pathogenesis.…”

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confidence: 99%

Folding of Cu/Zn superoxide dismutase suggests structural hotspots for gain of neurotoxic function in ALS: Parallels to precursors in amyloid disease

Nordlund

Oliveberg

2006

131

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease linked to misfolding of the ubiquitous enzyme Cu͞Zn superoxide dismutase (SOD). In contrast to other protein-misfolding disorders with similar neuropathogenesis, ALS is not always associated with the in vivo deposition of protein aggregates. Thus, under the assumption that all protein-misfolding disorders share at primary level a similar disease mechanism, ALS constitutes an interesting disease model for identifying the yet-mysterious precursor states from which the cytotoxic pathway emerges. In this study, we have mapped out the conformational repertoire of the apoSOD monomer through analysis of its folding behavior. The results allow us to target the regions of the SOD structure that are most susceptible to unfolding locally under physiological conditions, leading to the exposure of structurally promiscuous interfaces that are normally hidden in the protein's interior. The structure of this putative ALS precursor is strikingly similar to those implicated in amyloid disease.neurodegenerative disease ͉ protein folding ͉ transition state A n increasing number of severe neurodegenerative disorders have been linked to misfolding and subsequent aggregation of proteins (1). In some cases the protein aggregates deposit in the form of highly ordered amyloid fibrils (2), whereas in other cases they appear more granular or even amorphous (3). Even so, the main cause of neurotoxic function seems not to be the macroscopically deposited protein itself but rather a mysterious precursor species in the aggregation pathway (1, 4-6). In this perspective, it is notable that there are also disease cases where in vivo depositions of the disease-associated proteins are hard to find and sometimes are not even observable. Characteristic examples are found among the sporadic cases of CreuzfeldtJacob disease and amyotropic lateral sclerosis (ALS) (7). It is further apparent that the ALS-associated enzyme Cu͞Zn superoxide dismutase (SOD) has relatively low propensities to aggregate in vitro: SOD can be produced and analyzed by standard protocols despite being destabilized to the extent that it is half unfolded in physiological buffer at 25°C (8). For comparison there are few nondisease proteins that would resist aggregation under these conditions. The question then arises, do the disease cases with and without in vivo deposition of protein aggregates share at the primary level the same toxicity mechanism? We have previously observed that ALS-provoking SOD mutations produce a common shift of the folding equilibrium toward unfolded and partly folded monomers, implicating that the noxious side reaction emerges from the early folding events. The observation is in full agreement with a series of reports pointing at the immature apoSOD monomer as precursor for cytotoxicity (8)(9)(10)(11)(12)(13)(14)(15). Accompanying the shift of the folding equilibrium, the ALS-associated SOD mutations indicate a quantitative relation between decreased protein stability, net charge, and diseas...