Molecular crowding accelerates aggregation of α-synuclein by altering its folding pathway.

Intracellular macromolecular crowding can lead to increased aggregation of proteins, especially those that lack a natively folded conformation. Crowding may also be mimicked by the addition of polymers like polyethylene glycol (PEG) in vitro. α-Synuclein is an intrinsically disordered protein that exhibits increased aggregation and amyloid fibril formation in a crowded environment. Two hypotheses have been proposed to explain this observation. One is the excluded volume effect positing that reduced water activity in a crowded environment leads to increased effective protein concentration, promoting aggregation. An alternate explanation is that increased crowding facilitates conversion to a non-native form increasing the rate of aggregation. In this work, we have segregated these two hypotheses to investigate which one is operating. We show that mere increase in concentration of α-synuclein is not enough to induce aggregation and consequent fibrillation. In vitro, we find a complex relationship between PEG concentrations and aggregation, in which smaller PEGs delay fibrillation; while, larger ones promote fibril nucleation. In turn, while PEG600 did not increase the rate of aggregation, PEG1000 did and PEG4000 and PEG12000 slowed it but led to a higher overall fibril burden in the latter to cases. In cells, PEG4000 reduces the aggregation of α-synuclein but in a way specific to the cellular environment/due to cellular factors. The aggregation of the similarly sized, globular lysozyme does not increase in vitro when at the same concentrations with either PEG8000 or PEG12000. Thus, natively disordered α-synuclein undergoes a conformational transition in specific types of crowded environment, forming an aggregation-prone conformer.

Oligomers, fibrils and aggregates formed by alpha-synuclein: role of solution conditions.

The classical Hofmeister series orders ions into kosmotropes and chaotropes, based on their interaction with the solvent, water. The role of protein is mostly ignored probably because most of the proteins studied are natively folded and broadly follow this classification pattern. Recent reports suggest that the interaction of ions is different with solvent molecules of proximal layer and bulk. Intrinsically disordered proteins (IDPs) differ from globular proteins in the fraction of polar vis-à-vis hydrophobic amino acids and the absence of distinct secondary and tertiary structures. The kosmotrope, ammonium sulphate, increases the compactness of the polypeptide conformation, with differing effects for globular proteins and IDPs. For globular proteins, lowered flexibility corresponds to a more stable native structure. Using oligomer-specific and aggregation-specific antibodies and comparing with fibrillation results, we show for alpha-synuclein, an IDP, ammonium sulphate-induced compaction results in the formation of the aggregation-prone hydrophobic core, which combines with other similar moieties to form the fibrillar 'seed'. SEC-HPLC and SAXS analysis show the presence of the threshold oligomers. In the presence of the aggregation suppressor, arginine too, an oligomer is formed. This oligomer, however, is 'dead', and does not move further along the aggregation pathway. Thus, alpha-synuclein undergoes compaction in the presence of protein stabilisers, with differing consequences. In case of the chaotropes, KSCN and urea, aggregation of alpha-synuclein is partially inhibited. However, the amounts and types of aggregates formed are different in the two cases. Thus, the classical catalogue of molecules into protein stabilisers and destabilisers requires a relook for IDPs.Communicated by Ramaswamy H. Sarma.

Harmine Acts as an Indirect Inhibitor of Intracellular Protein Aggregation.

Protein aggregation and oxidative stress are two pathological hallmarks of a number of protein misfolding diseases, including Huntington's disease (HD). Whether protein aggregation precedes elevation of oxidative stress or follows it remains ambiguous. We have investigated the role of harmine, a beta-carboline alkaloid, in aggregation of a mutant huntingtin fragment (103Q-htt) in a yeast model of HD. We observed that harmine was able to decrease intracellular aggregation of 103Q-htt, and this reduction was higher than that observed with trehalose, a conventional protein stabilizer. The presence of harmine also decreased prion formation. Decreased protein aggregation was accompanied by reduction in oxidative stress. However, harmine had no effect on aggregation of the mutant huntingtin fragment in vitro. Thus, based on experimental data, we conclude that the antioxidant harmine lowers aggregation-induced elevation in oxidative stress, which slows down intracellular protein aggregation.