Nucleobindin 1 binds to multiple types of pre-fibrillar amyloid and inhibits fibrillization

Abstract

During amyloid fibril formation, amyloidogenic polypeptides misfold and self assemble into soluble pre-fibrillar aggregates, i.e., protofibrils, which elongate and mature into insoluble fibrillar aggregates. An emerging class of chaperones, chaperone-like amyloid binding proteins (CLABPs), has been shown to interfere with aggregation of particular misfolded amyloid peptides or proteins. We have discovered that the calcium-binding protein nuclebindin-1 (NUCB1) is a novel CLABP. We show that NUCB1 inhibits aggregation of islet-amyloid polypeptide associated with type 2 diabetes mellitus, a-synuclein associated with Parkinson's disease, transthyretin V30M mutant associated with familial amyloid polyneuropathy, and Aβ42 associated with Alzheimer's disease by stabilizing their respective protofibril intermediates. Kinetic studies employing the modeling software AmyloFit show that NUCB1 affects both primary nucleation and secondary nucleation. We hypothesize that NUCB1 binds to the common cross-β-sheet structure of protofibril aggregates to "cap" and stabilize soluble macromolecular complexes. Transmission electron microscopy and atomic force microscopy were employed to characterize the size, shape and volume distribution of multiple sources of NUCB1-capped protofibrils. Interestingly, NUCB1 prevents Aβ42 protofibril toxicity in a cellular assay. NUCB1-stabilized amyloid protofibrils could be used as immunogens to prepare conformation-specific antibodies and as novel tools to develop screens for anti-protofibril diagnostics and therapeutics.

Figure 1. Kinetic analysis of mtNUCB1 inhibition of aggregation mechanisms.

(A–D) Aggregation of 2.5 μM Aβ42 and (E–G) 10 μM Aβ42 in presence of different mtNUCB1 concentrations was measured by Thio-T assay and analysed by AmyloFit. (A,E) For each Aβ42 concentration, the aggregation plateau values were normalized to 1 and the rate constant parameters for primary nucleation, (B,F) elongation and (C,G) secondary nucleation were individually fitted in parallel to global fitting of the other two, and the fitted (lines) and the experimental (circles) data were compared. In each panel, the insets show the fitting residuals over time obtained from the respective analysis. (D) Fitted rate constants for secondary and primary nucleation are shown for low and (H) high Aβ42 concentrations, respectively, as a function of mtNUCB1 equivalent to Aβ42 concentration. The rate constants are normalized relative to the values in the absence of mtNUCB1.

Figure 2. The mtNUCB1: Aβ42 ratio determines Aβ42 monomer and soluble aggregates concentration.

Samples containing 10 μM Aβ42 and 0, 1, 2.5, 5, 10, 15, 25, and 30 μM mtNUCB1 were incubated for 24 h at 37 °C in quiescent conditions. (A) Quantification of monomer concentration. At the end of the reaction, the monomer concentration was measured. Samples were ultracentrifuged and the supernatant fraction was separated with SEC. Eluted fractions of monomer were pooled and the Aβ42 content was measured by indirect ELISA. (B) Quantification of soluble aggregates concentration. A portion of the supernatant sample was probed with indirect ELISA using the anti-oligomer antibody A11 to detect small soluble aggregates. **p < 0.01, ***p < 0.001 vs 5 μM mtNUCB1. ^#p < 0.05, ^###p < 0.001 vs 10 μM. One-way ANOVA followed by Tukey’s post-hoc comparison.

Figure 3. mtNUCB1 stabilizes short Aβ42 protofibrils.

(A) Representative EM images of 10 μM Aβ42, 10 μM mtNUCB1, and 10 μM Aβ42+10 μM mtNUCB1 incubated at 37 °C for 24 h. All samples were incubated with both mouse anti-Aβ 6E10 and rabbit anti-NUCB1 antibodies and successively with the 6-nm gold-conjugated anti-mouse antibody and 12-nm gold-conjugated anti-rabbit antibody. Panel shows three 100 × 100 nm images per group. Arrowheads indicate 12-nm gold particles (mtNUCB1) and small arrows indicate 6-nm gold particles (Aβ42). (B) Representative EM images of mtNUCB1-Aβ42 protofibrils purified with SEC and incubated with both mouse anti-Aβ 6E10 and rabbit anti-NUCB1 antibodies and successively with the 6-nm gold-conjugated anti-mouse antibody and the 12-nm gold-conjugated anti-rabbit antibody. The panel shows twelve 100 × 100 nm images with arrowheads indicating 12-nm gold particles (mtNUCB1) and small arrows indicating 6-nm gold particles (Aβ42). (C) Composite of representative mtNUCB1-Aβ42 protofibrils (n = 47) selected based on the volumetric analysis of the sample imaged by AFM. Integrated xy scale bar is 40 nm; the colorimetric scale bar indicates the height of the particles.

Figure 4. mtNUCB1 has pan amyloid chaperone-like activity.

(A) hIAPP (30 μM) kinetics of aggregation was tested in absence or in presence of equimolar concentrations of mtNUCB1, or the control protein BSA, and 10 μM Thio-T at 25 °C in quiescent conditions, over 24 h. (B) α-synuclein (100 μM) aggregation was measured as end point fluorescence during incubation in absence or in presence of 10 μM mtNUCB1 or BSA, and 10 μM Thio-T, at 37 °C in shaking conditions, for 3 days. (C) The transthyretin V30M mutant (10 μM) aggregation was tested in absence or in presence of equimolar concentrations of mtNUCB1 or BSA, and 10 μM Thio-T, at 37 °C in quiescent conditions, over 24 h. (D) Composite of representative mtNUCB1- hIAPP, (E) mtNUCB1-α-synuclein and (F) mtNUCB1-V30M protofibrils purified by SEC and imaged by AFM. For each amyloid, representative protofibrils were selected based on the sample distribution of volumes. Integrated xy scale bar is 40 nm; colorimetric scale bar indicates the height of the particles.

Figure 5. mtNUCB1 protects against Aβ42-induced toxicity.

(A) Dose-dependent protective effect of mtNUCB1 against Aβ42-induced cell toxicity. PC12 cells were treated with 100 nM Aβ42 in the presence of mtNUCB1 or the control protein BSA. The cell viability was tested by MTT assay and indicates that mtNUCB1 has an IC₅₀ of 1.4 μM. Inset shows the dose-dependent toxicity experienced by PC12 cells to Aβ42 as measured in an MTT cell viability assay and indicates an EC₅₀ of 100 nM.