MIRRAGGE - Minimum Information Required for Reproducible AGGregation Experiments

Abstract

Reports on phase separation and amyloid formation for multiple proteins and aggregation-prone peptides are recurrently used to explore the molecular mechanisms associated with several human diseases. The information conveyed by these reports can be used directly in translational investigation, e.g., for the design of better drug screening strategies, or be compiled in databases for benchmarking novel aggregation-predicting algorithms. Given that minute protocol variations determine different outcomes of protein aggregation assays, there is a strong urge for standardized descriptions of the different types of aggregates and the detailed methods used in their production. In an attempt to address this need, we assembled the Minimum Information Required for Reproducible Aggregation Experiments (MIRRAGGE) guidelines, considering first-principles and the established literature on protein self-assembly and aggregation. This consensus information aims to cover the major and subtle determinants of experimental reproducibility while avoiding excessive technical details that are of limited practical interest for non-specialized users. The MIRRAGGE table (template available in Supplementary Information) is useful as a guide for the design of new studies and as a checklist during submission of experimental reports for publication. Full disclosure of relevant information also enables other researchers to reproduce results correctly and facilitates systematic data deposition into curated databases.

Keywords: amyloid; peptide; phase separation; protein; reproducible data.

FIGURE 1

Amyloid and non-amyloid aggregates. (A) Amyloids have the characteristic fibrillar appearance illustrated in this transmission electron microscopy (TEM) image of negatively stained fibrils from a peptide derived from the SH3 domain of PI3-kinase (SH3-PI3K) (Ventura et al., 2004). The bar corresponds to 200 nm. (B) The ordered β-sheet structure of amyloid fibrils produces the cross-β X-ray diffraction pattern here exemplified for fibrils of a peptide derived from Mot3 yeast prion (Sant’Anna et al., 2016; Fernandez et al., 2017). The blue and red arrows indicate typical reflections at 4.7 and 10.2 Å, respectively. (C) Thioflavin-S (Th-S) is an histological dye that shows fluorescence in presence of amyloid fibrils (example of SH3-PI3K fibrils stained with Th-S) (Ventura et al., 2004). The bar corresponds to 20 μm. (D) Folded and misfolded monomers are in dynamic equilibrium with soluble oligomers and may coexist in solution with different condensates, such as dense liquids and gels, amyloid fibrils and their protofibrillar precursors, crystals and amorphous solids. For simplicity, the more complex cases of fibril polymorphism, multiprotein assemblies or nucleic acid-protein assemblies are not shown, and amyloid fibrils are represented as composed of four protofilament units. (B) Adapted from (Sant’Anna et al., 2016). All copyright permissions have been secured by the authors.

FIGURE 2

Ontological map of protein aggregation. Phase equilibria (solid outlines) are established between the liquid phase (green circle), the dense liquid phase (orange circle) and the solid phase (blue circles). The different species (represented as in Figure 1D) may undergo structural transitions (dashed lines) that originate distinct species within the same stable phase (different shades of color). Visual examples of aggregation pathways are provided, comprising many (arrow 1), some (arrow 2) and no (arrow 3) intermediate states. Off-pathway aggregation occurring in parallel with amyloid fibrillation is represented by a bifurcation (arrow 2′). The outcome of phase separation (black dots) is contingent on concentration, temperature, pH, ionic strength, amino acid sequence, etc., but also on the initial state of the protein, as it determines the point in the green region from where the experiments depart (white dots).

FIGURE 3

Aggregation of human ataxin-3 (UniProt accession code: P54252-2). (A) Schematic representation of the structural organization of ataxin-3, with the N-terminal globular Josephin domain (JD) and the flexible C-terminal tail encompassing the ubiquitin-interacting motifs (UIMs) and the polyQ tract containing 13 glutamine residues (13Q); (B) Representative graph of human ataxin-3 aggregation at 37°C monitored by Th-T fluorescence. (C) TEM image of ataxin-3 protofibrils at the end of the aggregation assay (∼60 h).