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A molecular chaperone breaks the catalytic cycle that generates toxic Aβ oligomers.

Cohen SI, Arosio P, Presto J, Kurudenkandy FR, Biverstål H, Dolfe L, Dunning C, Yang X, Frohm B, Vendruscolo M, Johansson J, Dobson CM, Fisahn A, Knowles TP, Linse S - Nat. Struct. Mol. Biol. (2015)

Bottom Line: Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers.We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates.These results reveal that molecular chaperones can help maintain protein homeostasis by selectively suppressing critical microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge, Cambridge, UK.

ABSTRACT
Alzheimer's disease is an increasingly prevalent neurodegenerative disorder whose pathogenesis has been associated with aggregation of the amyloid-β peptide (Aβ42). Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers. Here we show that a molecular chaperone, a human Brichos domain, can specifically inhibit this catalytic cycle and limit human Aβ42 toxicity. We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates. We verify that this mechanism occurs in living mouse brain tissue by cytotoxicity and electrophysiology experiments. These results reveal that molecular chaperones can help maintain protein homeostasis by selectively suppressing critical microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation.

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Brichos interacts with fibrillar but not monomeric Aβ42(a) Images using TEM with a nano-gold conjugated secondary antibody against anti-Brichos antibodies show that the chaperone binds to Aβ42 fibrils. (b) SPR analysis verifies the specific binding to fibrils and allows determination of the association (kon ≈ 5.1 × 103 M−1s−1) and dissociation (koff ≈ 2.1 × 10−4 s−1) rate constants, implying an apparent equilibrium dissociation constant KD ≈ 40 nM. No binding was observed to the monomer or to control in the absence of Aβ42.
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Figure 3: Brichos interacts with fibrillar but not monomeric Aβ42(a) Images using TEM with a nano-gold conjugated secondary antibody against anti-Brichos antibodies show that the chaperone binds to Aβ42 fibrils. (b) SPR analysis verifies the specific binding to fibrils and allows determination of the association (kon ≈ 5.1 × 103 M−1s−1) and dissociation (koff ≈ 2.1 × 10−4 s−1) rate constants, implying an apparent equilibrium dissociation constant KD ≈ 40 nM. No binding was observed to the monomer or to control in the absence of Aβ42.

Mentions: The kinetic data with and without pre-formed seed fibrils suggest a mechanism for inhibition of aggregation involving the non-covalent association of Brichos with Aβ42 fibrils. To probe this mechanism further, we used transmission electron microscopy (TEM) with nano-gold conjugated anti-Brichos antibodies. The resulting images (Fig. 3a) provide a striking visualization of the nature of the interactions and clearly reveal specific binding of Brichos along the fibril surface. The interactions between the chaperone and the fibrils were also studied using surface plasmon resonance (SPR) measurements. The data (Fig. 3b) reveal that Brichos has a high affinity for the fibrils, whereas no binding was observed to SPR sensor surfaces functionalised with monomeric Aβ42 or to reference surfaces lacking any Aβ42; the absence of detectable binding to monomeric Aβ42 (Fig. 3b) verifies that the mode of action of Brichos is through interactions with the fibrillar species rather than with soluble monomers. These measurements also provide estimates for the association (kon ≈ 5.1 × 103M−1s−1) and dissociation (koff ≈ 2.1 × 10−4 s−1) rate constants for the binding of the chaperone to Aβ42 fibrils (KD ≈ 40 nM). Remarkably, using these rate constants we are able to predict quantitatively (see Supplementary Note for a complete derivation of the rate equations) and accurately the experimental kinetic profiles at each of the intermediate concentrations of Brichos shown in Fig. 1c (the predictions are shows as thin dotted lines). Moreover, the rate constants predict that under the conditions used to study the effects of pre-formed seed fibrils in Fig. 2b, the maximum level of dissociation of Brichos from the seeds prepared in the presence of the chaperone will only be ca. 68% after two hours, a finding consistent with the observation (Fig. 2b) of a partially reduced catalytic effect of fibrils formed in the presence of Brichos even after several hours.


A molecular chaperone breaks the catalytic cycle that generates toxic Aβ oligomers.

Cohen SI, Arosio P, Presto J, Kurudenkandy FR, Biverstål H, Dolfe L, Dunning C, Yang X, Frohm B, Vendruscolo M, Johansson J, Dobson CM, Fisahn A, Knowles TP, Linse S - Nat. Struct. Mol. Biol. (2015)

Brichos interacts with fibrillar but not monomeric Aβ42(a) Images using TEM with a nano-gold conjugated secondary antibody against anti-Brichos antibodies show that the chaperone binds to Aβ42 fibrils. (b) SPR analysis verifies the specific binding to fibrils and allows determination of the association (kon ≈ 5.1 × 103 M−1s−1) and dissociation (koff ≈ 2.1 × 10−4 s−1) rate constants, implying an apparent equilibrium dissociation constant KD ≈ 40 nM. No binding was observed to the monomer or to control in the absence of Aβ42.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4595974&req=5

Figure 3: Brichos interacts with fibrillar but not monomeric Aβ42(a) Images using TEM with a nano-gold conjugated secondary antibody against anti-Brichos antibodies show that the chaperone binds to Aβ42 fibrils. (b) SPR analysis verifies the specific binding to fibrils and allows determination of the association (kon ≈ 5.1 × 103 M−1s−1) and dissociation (koff ≈ 2.1 × 10−4 s−1) rate constants, implying an apparent equilibrium dissociation constant KD ≈ 40 nM. No binding was observed to the monomer or to control in the absence of Aβ42.
Mentions: The kinetic data with and without pre-formed seed fibrils suggest a mechanism for inhibition of aggregation involving the non-covalent association of Brichos with Aβ42 fibrils. To probe this mechanism further, we used transmission electron microscopy (TEM) with nano-gold conjugated anti-Brichos antibodies. The resulting images (Fig. 3a) provide a striking visualization of the nature of the interactions and clearly reveal specific binding of Brichos along the fibril surface. The interactions between the chaperone and the fibrils were also studied using surface plasmon resonance (SPR) measurements. The data (Fig. 3b) reveal that Brichos has a high affinity for the fibrils, whereas no binding was observed to SPR sensor surfaces functionalised with monomeric Aβ42 or to reference surfaces lacking any Aβ42; the absence of detectable binding to monomeric Aβ42 (Fig. 3b) verifies that the mode of action of Brichos is through interactions with the fibrillar species rather than with soluble monomers. These measurements also provide estimates for the association (kon ≈ 5.1 × 103M−1s−1) and dissociation (koff ≈ 2.1 × 10−4 s−1) rate constants for the binding of the chaperone to Aβ42 fibrils (KD ≈ 40 nM). Remarkably, using these rate constants we are able to predict quantitatively (see Supplementary Note for a complete derivation of the rate equations) and accurately the experimental kinetic profiles at each of the intermediate concentrations of Brichos shown in Fig. 1c (the predictions are shows as thin dotted lines). Moreover, the rate constants predict that under the conditions used to study the effects of pre-formed seed fibrils in Fig. 2b, the maximum level of dissociation of Brichos from the seeds prepared in the presence of the chaperone will only be ca. 68% after two hours, a finding consistent with the observation (Fig. 2b) of a partially reduced catalytic effect of fibrils formed in the presence of Brichos even after several hours.

Bottom Line: Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers.We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates.These results reveal that molecular chaperones can help maintain protein homeostasis by selectively suppressing critical microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge, Cambridge, UK.

ABSTRACT
Alzheimer's disease is an increasingly prevalent neurodegenerative disorder whose pathogenesis has been associated with aggregation of the amyloid-β peptide (Aβ42). Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers. Here we show that a molecular chaperone, a human Brichos domain, can specifically inhibit this catalytic cycle and limit human Aβ42 toxicity. We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates. We verify that this mechanism occurs in living mouse brain tissue by cytotoxicity and electrophysiology experiments. These results reveal that molecular chaperones can help maintain protein homeostasis by selectively suppressing critical microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation.

Show MeSH
Related in: MedlinePlus