An enigmatic anion may stitch together amyloid fibrils

Amyloid fibrils appear in many neurodegenerative diseases, and scientists are eager to understand how they form and spread. But amyloids—clumped aggregations of misfolded proteins—can be difficult to study. Certain proteins can adopt multiple distinct amyloid shapes, and recent research has shown that the fibrils that researchers grow in laboratories to study are shaped differently than fibrils isolated from the brains of people with neurodegenerative diseases.

In α-synuclein fibrils from people with multiple system atrophy, for example, researchers have identified a structure not observed in lab-grown fibrils. In the fibrils from brains, electron density maps suggest a molecule distinct from α-synuclein is present but it can’t be identified (Nature 2020, DOI: 10.1038/s41586-020-2317-6). In a study published today, researchers in Ursula Jakob’s lab at the University of Michigan argue that the mystery density is most likely polyphosphate (PLOS Biol. 2024, DOI: 10.1371/journal.pbio.3002650).

Polyphosphate is “still highly enigmatic,” Jakob says. Despite its simple structure—it’s a linear chain of anionic phosphate groups—researchers do not know exactly how human cells make polyphosphate, and they have limited insight into its biological roles. Jakob’s group has previously found that adding polyphosphate to protein solutions accelerates fibril formation and makes for stabler fibrils. In cultured cells, adding polyphosphate reduces cell death from amyloids (Cold Spring Harbor Perspect. Biol. 2019, DOI: 10.1101/cshperspect.a034041;Mol. Cell 2016, DOI: 10.1016/j.molcel.2016.07.016) Counterintuitively, stable fibril structures may be beneficial, Jakob says: stabilized fibrils are less likely to shed small chunks of misfolded proteins called oligomers, which scientists think can spread proteopathic misfolding between cells.

Now Jakob’s team has found that structural models and molecular docking simulations predict that polyphosphate will nestle into exactly the same pocket in the structure of brain-derived fibrils that the real mystery density occupies. The researchers suspect that the negatively charged polyphosphate helps overcome electrostatic repulsion from three positively charged amino acids that rub shoulders in the packed fibril structure. When the group mutated any of these three amino acids in purified protein experiments, the mutant synucleins packed together much faster and showed much less dependence on polyphosphate to form fibrils.

“This interaction resembles a thread passing through multiple layers of fabric, holding them together tightly,” says Zongchao Jia, an expert on polyphosphate-protein interactions at Queen’s University in Canada. “In this analogy, polyP serves as the thread… effectively adhering the fibril layers into a stable, cohesive structure.” Jia finds the experimental evidence compelling but thinks constraints on the molecular dynamics simulation made it predict an unlikely conformation for polyphosphate.

In a written statement, Sjors Scheres and Michel Goedert, senior authors on the study that found the unidentified density in the first place, say the paper raises an interesting possible identity for the mystery molecule, but “convincing proof for this hypothesis is not provided.” They would like to see data confirming that polyphosphate is present in patient-derived samples.

Jakob says that her lab is working on such experiments but that they remain technically challenging. Finding polyphosphate in patient-derived fibrils would be convincing—but the starting material is notoriously sticky and impure, making mass spectrometry inconclusive, and so far her team has not been able to detect polyphosphate in fibrils using specific binding probes.

James Morrissey of the University of Michigan, who discovered a role for polyphosphate in blood clotting, writes, “We are still in the early days of investigating the biological roles of polyphosphate.” He says that future improvements in detecting the enigmatic molecule could help researchers get more certainty about whether it truly participates in amyloid formation in the brain.