Conserved features of intermediates in amyloid assembly determine their benign or toxic states

Krishnan, Rajaraman; Goodman, Jessica L.; Mukhopadhyay, Samrat; Pacheco, Chris D.; Lemke, Edward A.; Deniz, Ashok A.; Lindquist, Susan

doi:10.1073/pnas.1209527109

Cited by 120 publications

(134 citation statements)

References 35 publications

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“…In cases of extensive OPA, increasing the protein concentration may even prolong the duration of the lag phase and decrease the amyloid aggregation rate. This angle of approach is, to our knowledge, totally original because the main focus has been on the detection and morphological/ toxicological characterization of the amorphous precipitates (4,7,8,9,14). The new possibility of estimating the relative amounts of off-pathway and amyloid aggregates accentuates the need for cataloguing the deleterious species in each disease.…”

Section: Discussionmentioning

confidence: 99%

What Can the Kinetics of Amyloid Fibril Formation Tell about Off-pathway Aggregation?

Crespo

Villar‐Alvarez

Taboada

et al. 2016

Journal of Biological Chemistry

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Some of the most prevalent neurodegenerative diseases are characterized by the accumulation of amyloid fibrils in organs and tissues. Although the pathogenic role of these fibrils has not been completely established, increasing evidence suggests offpathway aggregation as a source of toxic/detoxicating deposits that still remains to be targeted. The present work is a step toward the development of off-pathway modulators using the same amyloid-specific dyes as those conventionally employed to screen amyloid inhibitors. We identified a series of kinetic signatures revealing the quantitative importance of off-pathway aggregation relative to amyloid fibrillization; these include nonlinear semilog plots of amyloid progress curves, highly variable end point signals, and half-life coordinates weakly influenced by concentration. Molecules that attenuate/intensify the magnitude of these signals are considered promising off-pathway inhibitors/promoters. An illustrative example shows that amyloid deposits of lysozyme are only the tip of an iceberg hiding a crowd of insoluble aggregates. Thoroughly validated using advanced microscopy techniques and complementary measurements of dynamic light scattering, CD, and soluble protein depletion, the new analytical tools are compatible with the high-throughput methods currently employed in drug discovery.

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Section: Discussionmentioning

confidence: 99%

What Can the Kinetics of Amyloid Fibril Formation Tell about Off-pathway Aggregation?

Crespo

Villar‐Alvarez

Taboada

et al. 2016

Journal of Biological Chemistry

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“…6–9 Of note, a variety of amyloid proteins and peptides with different primary structures form oligomers with similar quaternary structures. 8,10 These structures are more stable than their monomeric and smaller oligomeric precursors, but less stable than their ultimate fibrillar products. 11,12 From a structure-function perspective, toxic oligomers would be predicted to have relatively well-organized structures that interact with cellular membranes, receptors, or other proteins.…”

Section: Introductionmentioning

confidence: 99%

Amyloid β-Protein C-Terminal Fragments: Formation of Cylindrins and β-Barrels

et al. 2016

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In order to evaluate potential therapeutic targets for treatment of amyloidoses such as Alzheimer’s disease (AD), it is essential to determine the structures of toxic amyloid oligomers. However, for the amyloid β-protein peptide (Aβ), thought to be the seminal neuropathogenetic agent in AD, its fast aggregation kinetics and the rapid equilibrium dynamics among oligomers of different size pose significant experimental challenges. Here we use ion-mobility mass spectrometry, in combination with electron microscopy, atomic force microscopy, and computational modeling, to test the hypothesis that Aβ peptides can form oligomeric structures resembling cylindrins and β-barrels. These structures are hypothesized to cause neuronal injury and death through perturbation of plasma membrane integrity. We show that hexamers of C-terminal Aβ fragments, including Aβ(24-34), Aβ(25-35) and Aβ(26-36), have collision cross-sections similar to those of cylindrins. We also show that linking two identical fragments head-to-tail using di-glycine increases the proportion of cylindrin-sized oligomers. In addition, we find that larger oligomers of these fragments may adopt β-barrel structures and that β-barrels can be formed by folding an out-of-register β-sheet, a common type of structure found in amyloid proteins.

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“…More recently, mass spectrometry coupled with ion mobility spectrometry (IM-MS) experiments have provided information regarding the size and shape of the lowmolecular-weight oligomeric forms of Aβ and other amyloidoic proteins (6, 7). A variety of biophysical methods have been used to study the oligomers extensively (2,(7)(8)(9)(10)(11)(12)(13)(14).Despite such studies, the ability to measure the rates of formation of the oligomers remains difficult. Recently, Lee et al (15) reported a method suitable for monitoring the full time course of aggregation showing distinct phases corresponding to oligomerization and fibrillization using Cys-Cys-Aβ and FlAsH dye binding to tetracysteine motifs arising from self-association of Aβ.…”

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confidence: 99%

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confidence: 99%

Quantitative analysis of the time course of Aβ oligomerization and subsequent growth steps using tetramethylrhodamine-labeled Aβ

Garai

Frieden

2013

Proc. Natl. Acad. Sci. U.S.A.

125

171

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Although amyloid β (Aβ) is a critical player in the pathology of Alzheimer's disease, there is currently little Information on the rate and extent of formation of oligomers that lead to the presence of Aβ fibrils observed in amyloid plaques. Here we describe a unique method to monitor the full time course of Aβ aggregation. In this method, Aβ is labeled with tetramethylrhodamine at a lysine residue on the N-terminal end. During aggregation, the fluorescence is quenched in a time-dependent manner in three distinct phases: an early oligomerization phase, an intermediate phase, and a growth phase. The oligomerization phase can be characterized as a monomer-dimer-trimer process for which we have determined the rate and equilibrium constants. The rate constants differ markedly between Aβ 1-42 and Aβ 1-40 , with Aβ 1-42 showing a greater oligomerization propensity. The intermediate phase reflects slow clustering and reorganization of the oligomers, whereas the growth phase ultimately results in the formation of fibrillar material. The data are consistent with a conformational change being an important rate-limiting step in the overall aggregation process. The rates of all phases are highly sensitive to temperature and pH, with the pH-dependent data indicating important roles for lysine and histidine residues. From the temperaturedependent data, activation energies of oligomerization and fibrillization are estimated to be 5.5 and 12.1 kCal/mol, respectively. The methodologies presented here are simple and can be applied to other amyloidogenic peptides or proteins.oligomer formation | fluorescence quenching | kinetics of aggregation | nucleation A lzheimer's disease (AD), the most prevalent form of neurodegenerative diseases, is characterized by deposition of senile plaques in the brain. These plaques contain aggregates of the amyloid β (Aβ) peptides Aβ 1-42 and Aβ 1-40 . In vitro, both peptides self-assemble into soluble oligomers and insoluble fibrils in a time-dependent manner. There has been extensive structure-function characterization of the in vitro aggregation process using a large number of available methods (1). The most widely used method follows changes in the fluorescence of Thioflavin T (ThT). This dye is particularly useful because of the large fluorescence increase observed during the final phase of aggregation coincident with the formation of β-strand structure. This assay, however, does not report on intermediates in the aggregation process and therefore cannot detect soluble oligomers that lack well-defined β-strand structure. It is critical to understand the nature of these small oligomers, because recent experiments suggest that they may be the major cytotoxic species for AD (2-5).Data in the literature present a highly complex picture of the oligomer formation, starting from dimers to spherical oligomers and linear protofibrils comprised of a large number of monomers. Biophysical characterization of the low-molecular-weight oligomers, however, has been difficult because of their small size and meta...

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Conserved features of intermediates in amyloid assembly determine their benign or toxic states

Cited by 120 publications

References 35 publications

What Can the Kinetics of Amyloid Fibril Formation Tell about Off-pathway Aggregation?

What Can the Kinetics of Amyloid Fibril Formation Tell about Off-pathway Aggregation?

Amyloid β-Protein C-Terminal Fragments: Formation of Cylindrins and β-Barrels

Quantitative analysis of the time course of Aβ oligomerization and subsequent growth steps using tetramethylrhodamine-labeled Aβ

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