Experimental methods for studying amyloid cross-interactions

Abstract

Interactions between amyloid proteins represent the cornerstone of various pathogenic pathways, including prion conversion and co-development of distinct kinds of systemic amyloidosis. Various experimental methodologies provide insights into the effects of such cross-interactions on amyloid self-assembly, which range from acceleration to complete inhibition. Here, we present a comprehensive review of experimental methods most commonly used to study amyloid cross-interactions both in vitro and in vivo, such as fluorescence-based techniques, high-resolution imaging, and spectroscopic methods. Although each method provides distinct information on amyloid interactions, we highlight that no method can fully capture the complexity of this process. In order to achieve an exhaustive portrayal, it is necessary to employ a hybrid strategy combining different experimental techniques. A core set of fluorescence methods (e.g., thioflavin T) and high-resolution imaging techniques (e.g., atomic force microscopy or Cryo-EM) are required to verify the lack of self-assembly or alterations in fibril morphology. At the same time, immuno-electron microscopy, mass spectrometry, or solid-state NMR can confirm the presence of heterotypic fibrils.

Keywords: cross‐interactions; cross‐seeding; fibril polymorphism; high‐resolution microscopy; thioflavin T.

FIGURE 1

Overview of amyloid fibrillation and stability across assembly states. (a) Schematic representation of the three stages of amyloid fibrillation. The fibrillation process begins with the lag phase, where proteins exist in a dynamic equilibrium between soluble monomers and small aggregates, with minimal thioflavin T (ThT) fluorescence intensity observed. During the nucleation phase, these small aggregates cluster into a stable fibril nucleus, triggering a noticeable increase in ThT fluorescence as fibrils form. The elongation phase follows, characterized by the rapid addition of monomers to the growing fibrils and a corresponding sharp rise in ThT fluorescence intensity, indicating the formation of mature fibrils. Finally, in the plateau phase, fibril growth slows as the system approaches equilibrium, with ThT fluorescence intensity stabilizing as the concentration of remaining monomers diminishes. (b) The varying stability levels across distinct assembly states for heterotypic and homotypic amyloid fibrils. The assembly states range from stable, permanent interactions, marked as irreversible and characterized by strong intermolecular bonds between monomers, to transient strong interactions, which require specific environmental factors or conformational changes for bond dissociation. The transient weak interactions represent a reversible equilibrium, where bonds between monomers form and break spontaneously. Monomer populations of different proteins are shown in gray and red, with double arrows indicating the reversibility of the process. External factors that trigger dissociation are noted, with red lightning bolts denoting cases where a specific trigger is necessary for dissociation.

FIGURE 2

Diverse approaches and their relevance to amyloid aggregation analysis. Relevance of diverse experimental methods to four crucial aspects of amyloid aggregation: Fibrillation kinetics, secondary structure, fibril morphology, and fibril homogeneity. Each method is positioned according to the specific aspect(s) it can effectively examine, highlighting the complementary nature of these techniques. This visual representation underscores the importance of integrating multiple approaches to comprehensively understand amyloid aggregation and cross‐interactions.