The Amyloid β Peptide (Aβ1-40) Is Thermodynamically Soluble at Physiological Concentrations

Sengupta, Parijat; Garai, Kanchan; Sahoo, Bankanidhi; Shi, Yuan; Callaway, David J.E.; Maiti, Sudipta

doi:10.1021/bi0341410

Cited by 159 publications

(144 citation statements)

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“…The equilibrium monomer concentration observed in these experiments was substantially less than 100 nM, and lower than concentrations previously reported by several other laboratories (25)(26)(27). It is unlikely that small amounts of monomer are being lost in our system because a standard curve calibrated by amino acid analysis was linear down to 100 nM and had a zero intercept.…”

Section: Discussioncontrasting

confidence: 55%

“…More importantly, however, the number of filaments in these fibrils changed frequently along the length of the fibril. Therefore, it is likely that the internal structure of the fibrils examined in this work differs from fibrils generated in other labs that exhibit lower thermodynamic stabilities (25)(26)(27), and those that yield narrow MPL distributions and narrow NMR spectra (7,28).…”

mentioning

confidence: 90%

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Intrinsic Linear Heterogeneity of Amyloid β Protein Fibrils Revealed by Higher Resolution Mass-per-length Determinations

Komatsu

Feingold-Link

Sharp

et al. 2010

Journal of Biological Chemistry

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Amyloid ␤ proteins spontaneously form fibrils in vitro that vary in their thermodynamic stability and in morphological characteristics such as length, width, shape, longitudinal twist, and the number of component filaments. It is vitally important to determine which variant best represents the type of fibril that accumulates in Alzheimer disease. In the present study, the nature of morphological variation was examined by darkfield and transmission electron microscopy in a preparation of seeded amyloid ␤ protein fibrils that formed at relatively low protein concentrations and exhibited remarkably high thermodynamic stability. The number of filaments comprising these fibrils changed frequently from two to six along their length, and these changes only became apparent when massper-length (MPL) determinations are made with sufficient resolution. The MPL results could be reproduced by a simple stochastic model with a single adjustable parameter. The presence of more than two primary filaments could not be discerned by transmission electron microscopy, and they had no apparent relationship to the longitudinal twist of the fibrils. However, the pitch of the twist was strongly affected by the pH of the negative stain. We conclude that highly stable amyloid fibrils may form in which a surprising amount of intrinsic linear heterogeneity may be obscured by MPL measurements of insufficient resolution, and by the negative stains used for imaging fibrils by electron microscopy.Amyloid fibrils are filamentous aggregates formed by many different proteins in various diseases both in and out of the central nervous system (1). Fibrils formed by 40-residue (A␤40) and 42-residue (A␤42) amyloid ␤ (A␤) 2 proteins comprise the amyloid plaques of Alzheimer disease. Considerable effort has been expended to determine how A␤ proteins are folded within fibrils, yet the models emerging from these efforts differ significantly (1-8). Contributing to uncertainty about the internal structure of fibrils is a growing appreciation that A␤ fibrils are polymorphic when examined by transmission EM, cryo-EM, and AFM (7, 9 -19).Two morphological features have particularly important implications with regard to the internal structure of amyloid fibrils. One feature is a periodic narrowing observed in many transmission EM studies suggesting that fibrils twist around their long axis. The other feature is the fibril MPL, measured by dark field or tilted-beam EM. From this measure, the number of filaments comprising the fibril may be determined. We follow the terminology conventions defined by Kodali et al. (12) with the exception that "protofilament" is shortened simply to "filament."Fibrils with different morphologies can be created by varying fibril growth conditions such as temperature, buffer composition, agitation, and protein concentrations. Once formed, fibrils with a given morphology induce monomeric proteins to form "next generation" fibrils with the same morphology (10). Despite attempts to use identical conditions of formation, different laborat...

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Section: Discussioncontrasting

confidence: 55%

mentioning

confidence: 90%

Intrinsic Linear Heterogeneity of Amyloid β Protein Fibrils Revealed by Higher Resolution Mass-per-length Determinations

Komatsu

Feingold-Link

Sharp

et al. 2010

Journal of Biological Chemistry

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“…There are two reasons for this simplification. First, the low protein concentrations found in vivo 19,20 will greatly favor monomeric pathways relative to the high concentration systems studied in vitro. 21 Second, the reduced complexity of monomeric growth makes the first-order process an obvious starting point.…”

Section: Introductionmentioning

confidence: 99%

Kinetic theory of amyloid fibril templating

Schmit

2013

The Journal of Chemical Physics

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The growth of amyloid fibrils requires a disordered or partially unfolded protein to bind to the fibril and adapt the same conformation and alignment established by the fibril template. Since the H-bonds stabilizing the fibril are interchangeable, it is inevitable that H-bonds form between incorrect pairs of amino acids which are either incorporated into the fibril as defects or must be broken before the correct alignment can be found. This process is modeled by mapping the formation and breakage of H-bonds to a one-dimensional random walk. The resulting microscopic model of fibril growth is governed by two timescales: the diffusion time of the monomeric proteins, and the time required for incorrectly bound proteins to unbind from the fibril. The theory predicts that the Arrhenius behavior observed in experiments is due to off-pathway states rather than an on-pathway transition state. The predicted growth rates are in qualitative agreement with experiments on insulin fibril growth rates as a function of protein concentration, denaturant concentration, and temperature. These results suggest a templating mechanism where steric clashes due to a single mis-aligned molecule prevent the binding of additional molecules.

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“…By itself, resveratrol is a potent anti-oxidant which is thought to be responsible for the French Paradox [65]; relating to the low incidence of cardiovascular disease in a French population with high intake of saturated fats. In addition to its demonstrated capacity to inhibit (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) fibril formation in vitro [16], resveratrol is also capable of promoting intracellular uptake and degradation of through a proteosome-dependent mechanism [66] and protecting against -induced neurotoxicity [67,68], possibly through activation of the NAD-dependent histone deacetylase, SIRT1 [69]. In the present study using the ELISA-based assay, catechin showed no inhibitory activity during the early stages of oligomerization, whereas rosmarinic acid demonstrated some activity at the highest concentration tested.…”

Section: Discussionmentioning

confidence: 99%

“…Questions have arisen asking if these in vitro reactions are truly reflective of oligomerization in vivo since it is unlikely that localized brain levels of peptide would ever reach these concentrations [22][23][24][25].…”

Section: Introductionmentioning

confidence: 99%

A Sensitive Aβ Oligomerization Assay for Identification of Small Molecule Inhibitors

Gonzales¹,

Orlando²

2009

TOBIOTJ

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Amyloid deposits found in Alzheimer's disease result from aggregation of peptide which leads to loss of synaptic function, chronic microglial activation and cognitive impairment. Because of this, identification of small molecule inhibitors of aggregation as potential therapeutics is a topic of current interest. The majority of inhibitor screening approaches rely on in vitro assays that lack the necessary sensitivity to distinguish low-molecular weight oligomers from larger, more advanced-stage fibrillar structures. Differentiating between these two structures is of vital concern since recent studies indicate that small, early-stage oligomers are the most neurotoxic form of peptide aggregate. To address this limitation, we have explored the adaptability of a recently described ELISA-based assay for discovery of small molecule inhibitors of oligomerization. Results show that this assay is highly sensitive as it is able to quantify oligomers with as little as 80 nM input peptide. In addition, data were obtained re-confirming the function of curcumin as a potent inhibitor of aggregation (IC 50 = 2 μM) and defining its inhibitor:peptide functional stoichiometry. Further examination of other known anti-aggregation compounds showed that this assay is able to discriminate between inhibitors of early-stage, low-molecular weight oligomers and later-stage, high-molecular weight fibrillar structures. These findings indicate that this new ELISA-based assay is capable of identifying novel small molecule inhibitors that function during the initial stages of peptide assembly.

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The Amyloid β Peptide (Aβ₁_-₄₀) Is Thermodynamically Soluble at Physiological Concentrations

Cited by 159 publications

References 40 publications

Intrinsic Linear Heterogeneity of Amyloid β Protein Fibrils Revealed by Higher Resolution Mass-per-length Determinations

Intrinsic Linear Heterogeneity of Amyloid β Protein Fibrils Revealed by Higher Resolution Mass-per-length Determinations

Kinetic theory of amyloid fibril templating

A Sensitive Aβ Oligomerization Assay for Identification of Small Molecule Inhibitors

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