Single-Molecule Fluorescence Reveals the Oligomerization and Folding Steps Driving the Prion-like Behavior of ASC

Abstract

Single-molecule fluorescence has the unique ability to quantify small oligomers and track conformational changes at a single-protein level. Here we tackled one of the most extreme protein behaviors, found recently in an inflammation pathway. Upon danger recognition in the cytosol, NLRP3 recruits its signaling adaptor, ASC. ASC start polymerizing in a prion-like manner and the system goes in "overdrive" by producing a single micron-sized "speck." By precisely controlling protein expression levels in an in vitro translation system, we could trigger the polymerization of ASC and mimic formation of specks in the absence of inflammasome nucleators. We utilized single-molecule spectroscopy to fully characterize prion-like behaviors and self-propagation of ASC fibrils. We next used our controlled system to monitor the conformational changes of ASC upon fibrillation. Indeed, ASC consists of a PYD and CARD domains, separated by a flexible linker. Individually, both domains have been found to form fibrils, but the structure of the polymers formed by the full-length ASC proteins remains elusive. For the first time, using single-molecule Förster resonance energy transfer, we studied the relative positions of the CARD and PYD domains of full-length ASC. An unexpectedly large conformational change occurred upon ASC fibrillation, suggesting that the CARD domain folds back onto the PYD domain. However, contradicting current models, the "prion-like" conformer was not initiated by binding of ASC to the NLRP3 platform. Rather, using a new method, hybrid between Photon Counting Histogram and Number and Brightness analysis, we showed that NLRP3 forms hexamers with self-binding affinities around 300nM. Overall our data suggest a new mechanism, where NLRP3 can initiate ASC polymerization simply by increasing the local concentration of ASC above a supercritical level.

SARS-CoV-2 proteases cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species and the search for reservoir hosts

“…71 human proteins were cloned for expression as GFP-fusions in a cell-free expression system based on the eukaryotic organism of Leishmania tarentolae (LTE). This system produces full-length proteins up to 200 kDa with minimal truncations, minimal protein aggregation [36] and was previously used by our group to study the behaviour of various apoptotic proteins such as MyD88 [37], MAL [38] or ASC and NLRP3 [39].…”

Effects of Pathological Mutations on the Prion-Like Polymerisation of MyD88

“…By controlling the concentration of DNA priming the system, we can tune the final expression levels of proteins and coexpress proteins at controlled ratios. We have tested this combination on a variety of biological systems over the years, and demonstrated that the flexibility of cell-free protein expression is a great asset to study protein aggregation and prion-like fibrillation 15,[18][19][20][21] .…”

“…To evaluate the size of the oligomers and assemblies more precisely, we used Photon Counting Histograms (PCH) analysis to determine the stoichiometry of the complexes. We have described the PCH method in detail in previous publications 15,19,22 , and will provide a brief description here of the principle and analysis. To perform Photon Counting, the sample (expressed at the highest DNA loading) is diluted so that individual peaks can be fully resolved.…”

“…Intriguingly, many adaptors with prion-like properties contain two domains, both with selfassembly properties. For example with ASC, the PYD and CARD domains can both form filaments separately, and although the structure of the full-length ASC protein upon polymerisation is still unknown 15,16 . Similarly, for MyD88, both DD-Myddosome and TIR filament structures have been solved but the interplay between the two domains and their contribution to both processes is unknown.…”

Editorial Overview: Single-Molecule Approaches to Difficult Challenges in Folding and Dynamics

“…Thus, both chaperones appear to play an active role in protein folding instead of just shielding unfolded protein from aggregating. Gambin and coworkers used single-molecule fluorescence microscopy to investigate polymerization of ASC, a protein involved in inflammation signaling [23]. They observed that ASC alone can polymerize into a fibril-like structure in a high ASC concentration.…”