Biophysical processes underlying cross-seeding in amyloid aggregation and implications in amyloid pathology

Abstract

Abnormal aggregation of proteins into filamentous aggregates commonly associates with many diseases, such as Alzheimer's disease, Parkinson's disease and type-2 diabetes. These filamentous aggregates, also known as amyloids, can propagate their abnormal structures to either the same precursor molecules (seeding) or other protein monomers (cross-seeding). Cross-seeding has been implicated in the abnormal protein aggregation and has been found to facilitate the formation of physiological amyloids. It has risen to be an exciting area of research with a high volume of published reports. In this review article, we focus on the biophysical processes underlying the cross-seeding for some of the most commonly studied amyloid proteins. Here we will discuss the relevant literature related to cross-seeded polymerization of amyloid-beta, human islet amyloid polypeptide (hIAPP, or also known as amylin) and alpha-synuclein. SEVI (semen-derived enhancer of viral infection) amyloid formation by the cross-seeding between the bacterial curli protein and PAP_248-286 is also briefly discussed.

Fig. 1.

Hypothetical cross-seeding models via “template-assisted” and “conformational selection and population shift” mechanisms. The figure was adapted with permission from reference [54].

Fig. 2.

Hypothetical β-sheet-driven cross-seeding models via (a) β-structures, (b) dry-interfaced β-sheets, and (c) β-strand-loop-β-strands

Fig. 3.

Electron micrographs of hIAPP, Aβ₄₂ and Aβ₄₂-hIAPP samples showing (A) fibril-like hIAPP (17 ± 0.5 nm in width, 202 ± 9.9 nm in length, n = 77) and spherical (B) Aβ₄₂ oligomers (6 ± 0.2 nm in diameter, n = 207). (C) Aβ₄₂-hIAPP formed large amorphous aggregates. Analysis of fibril (D) length and (E) diameter demonstrated that Aβ₄₂-hIAPP (14 ± 0.6 nm in diameter, 539 ± 22.7 nm in length, n = 100) was significantly different from hIAPP (mean ± SEM, **p < 0.005, ***p < 0.001). Aβ₄₂-hIAPP mixtures are large amorphous structures that are distinctly different from either spherical Aβ₄₂ oligomers or fibril-like hIAPP. Scale bar (A) 200 nm, (B) 20 nm, (C) 500 nm/μm. The figure was adapted with permission from Reference [74].

Fig. 4.

The aggregation kinetics of Aβ₄₂-IAPP was followed by ¹H NMR. (A–C) Time-lapse ¹H NMR spectra of 25 μM Aβ₄₂ co-incubated with 25 μM human-IAPP or rat-IAPP. NMR samples were prepared using 10 mM NaPi buffer, pH = 7.4 containing 10% D₂O. Spectra were recorded on a 500 MHz NMR spectrometer at 25 ^◦C. (D–F) Intensity decay of ¹H NMR signal calculated from the spectra shown in (A–C) for 2, 24, and 120 hours incubations. The integrated intensities of the selected regions highlighted in (A–C) were analyzed using MestReNova. The figure was adapted with permission from reference [74].

Fig. 5.

Cross-seeding of PAP_248–286 with bacterial Curli results in longer SEVI fibrils. Transmission electron microscope (TEM) images of SEVI fibrils formed in the absence of curli (A) and in the presence of 5 mol% CsgA (B) and CsgB (C) fibrils. TEM images of CsgA (D) and CsgB (E) fibrils. (F) Aspect ratios of individual fibrils grown with and without curli nucleation/cross-seeding (n= 47, 23 and 37 for SEVI, SEVI+CsgA, and SEVI+CsgB respectively). Fibrils were grown at a concentration of 440 μM PAP_248–286 at 37 ^◦C with 1400 rpm orbital shaking for 7 days. P values were calculated using a two-tailed unpaired Student t-test against the control sample. The figure was adapted with permission from reference [40].

Fig. 6.

Proposed cross-seeded mechanisms of SEVI formation. Top row: Epitaxial Heteronucleation. (A) Binding of the PAP_248–286 monomers (red) to the bacterial curli seed (blue) induces β-sheet formation of PAP_248–286 (red). (B) Fibril growth proceeds epitaxially from the seeding nucleus to form fully matured amyloid fibrils. Bottom row: Possible mechanism for non-epitaxial heteronucleation. (C) A nucleus of the SEVI amyloid fibril forms independently of the curli fibril. (D) Growth of the SEVI fibril initially proceeds slowly due to unfavorable interactions between fibril subunits. (E) Lateral attachment of the nascent SEVI fibril to curli fibrils reduces repulsion between fibril subunits thereby enhancing the rate of fibrillogenesis. The curli seed may be incorporated into the final SEVI fibrils or may detach to catalyze further fibril extension events. The figure was adapted with permission from reference [40].

Fig. 7.

Schematic diagram of α-synuclein multimerization and Lewy body formation. For simplicity a single cell is shown. Homo- and hetero- multimers of α-synuclein are formed in both physiological (green) and pathological conditions (pink-red). Different shapes depict the structural heterogeneity of the pathogenic species. Cross-seeds are colored in blue and brown. Normally, α-synuclein is natively unfolded protein that adopts different conformations. This structural plasticity facilitates binding to many different proteins allowing α-synuclein to perform different functions. α-Synuclein also binds to vesicle membranes as a α-helical monomer. Multiple different stressors favor conformations that are prone to pathogenic multimerization. These factors include protein mutations, abnormal PTMs, metabolites, change of lipid composition, environmental pollutants (pesticides), and reactive oxygen species. Other triggers of multimerization are impaired ubiquitin-proteasome system (UPS) and chaperone function, as well as, dysregulation of autophagy (APH) and mitochondrial oxidative stress. The pathogenic multimerization results in off-pathway oligomers, or on- pathway oligomers. The on- pathway amyloid oligomers further grow into β-sheet-rich amyloid fibrils. Additionally some types of lipids can cross-seed the abnormal multimerization of α-synuclein (on- or off- pathway oligomers). Similar to lipids, other aggregation-prone proteins (tau, bacterial, Aβ, IAPP, prion etc) can cross-seed the multimerization of α-synuclein converting native α-synuclein and other cellular proteins into pathogenic entities. The end product of α-synuclein multimerization is Lewy bodies, which are large cellular inclusions with complex structures containing other multiple other components in addition to α-synuclein. The role of the non-synuclein components that can cross-seed α-synuclein multimerization and facilitate LB formation are poorly understood.

Fig. 8.

A phase diagram of nucleation-dependent amyloid formation. A typical phase diagram that explains the solubility and supersaturation-limited amyloid formation. Each region of the phase diagram designate the distinct phase properties of the amyloid proteins. These regions are labeled and separated by curves (labeled as 1, 2 and 3) which control the aggregation behaviors. The (thermodynamic) solubility curve in saturation is labeled as curve-1 (blue) and the (kinetic) solubility curve (or a metastability of supersaturation curve) is curve-2 (magenta). Curve-3 (red) is the amorphous aggregation curve. Strength of supersaturation is guided with two arrows. x and y axes indicate the concentration of proteins and external factors such as the concentration of salt, crowders, organic solvent, temperature, etc, respectively.

Fig. 9.

Phase diagrams showing the effects of cross-seeding on amyloid generation. Promotion (A) and inhibition (B–D) of amyloid generation due to cross-seeding are represented using phase diagrams. Amyloid proteins under given conditions are positioned with closed circles. It is assumed that the addition of cross seeds does not change the position of precursor amyloid proteins in the phase diagram. Curves in the presence of external amyloid seeds, or soluble amyloid proteins, are colored in red for promoting effects and blue for inhibiting effects. Curves prior to cross seeding are colored in gray and their corresponding shift is guided by arrows. Regions with distinct phase properties, curves (black and gray), and x and y axes are the same as explained in Fig. 8.