A peptide strategy for inhibiting different protein aggregation pathways.

Protein aggregation correlates with many human diseases. Protein aggregates differ in structure and shape. Strategies to develop effective aggregation inhibitors that reach the clinic failed so far. Here, we developed a family of peptides targeting early aggregation stages for both amorphous and fibrillar aggregates of proteins unrelated in sequence and structure. They act on dynamic precursors before mechanistic differentiation takes place. Using peptide arrays, we first identified peptides inhibiting the amorphous aggregation of a molten globular, aggregation-prone mutant of the Axin tumor suppressor. Optimization revealed that the peptides activity did not depend on their sequences but rather on their molecular determinants: a composition of 20-30% flexible, 30-40% aliphatic and 20-30% aromatic residues, a hydrophobicity/hydrophilicity ratio close to 1, and an even distribution of residues of different nature throughout the sequence. The peptides also suppressed fibrillation of Tau, a disordered protein that forms amyloids in Alzheimer's disease, and slowed down that of Huntingtin Exon1, an amyloidogenic protein in Huntington's disease, both entirely unrelated to Axin. Our compounds thus target early aggregation stages of different aggregation mechanisms, inhibiting both amorphous and amyloid aggregation. Such cross-mechanistic, multi-targeting aggregation inhibitors may be lead compounds for developing drug candidates against various protein aggregation diseases.

Amyloid Aggregation Is Potently Slowed Down by Osmolytes Due to Compaction of Partially Folded State.

Amyloid aggregation is a key process in amyloidoses and neurodegenerative diseases. Hydrophobicity is one of the major driving forces for this type of aggregation, as an increase in hydrophobicity generally correlates with aggregation susceptibility and rate. However, most experimental systems in vitro and prediction tools in silico neglect the contribution of protective osmolytes present in the cellular environment. Here, we assessed the role of hydrophobic mutations in amyloid aggregation in the presence of osmolytes. To achieve this goal, we used the model protein human muscle acylphosphatase (mAcP) and mutations to leucine that increased its hydrophobicity without affecting its thermodynamic stability. Osmolytes significantly slowed down the aggregation kinetics of the hydrophobic mutants, with an effect larger than that observed on the wild-type protein. The effect increased as the mutation site was closer to the middle of the protein sequence. We propose that the preferential exclusion of osmolytes from mutation-introduced hydrophobic side-chains quenches the aggregation potential of the ensemble of partially unfolded states of the protein by inducing its compaction and inhibiting its self-assembly with other proteins. Our results suggest that including the effect of the cellular environment in experimental setups and predictive softwares, for both mechanistic studies and drug design, is essential in order to obtain a more complete combination of the driving forces of amyloid aggregation.

Osmolytes and crowders regulate aggregation of the cancer-related L106R mutant of the Axin protein.

Protein aggregation is involved in a variety of diseases, including neurodegenerative diseases and cancer. The cellular environment is crowded by a plethora of cosolutes comprising small molecules and biomacromolecules at high concentrations, which may influence the aggregation of proteins in vivo. To account for the effect of cosolutes on cancer-related protein aggregation, we studied their effect on the aggregation of the cancer-related L106R mutant of the Axin protein. Axin is a key player in the Wnt signaling pathway, and the L106R mutation in its RGS domain results in a native molten globule that tends to form native-like aggregates. This results in uncontrolled activation of the Wnt signaling pathway, leading to cancer. We monitored the aggregation process of Axin RGS L106R in vitro in the presence of a wide ensemble of cosolutes including polyols, amino acids, betaine, and polyethylene glycol crowders. Except myo-inositol, all polyols decreased RGS L106R aggregation, with carbohydrates exerting the strongest inhibition. Conversely, betaine and polyethylene glycols enhanced aggregation. These results are consistent with the reported effects of osmolytes and crowders on the stability of molten globular proteins and with both amorphous and amyloid aggregation mechanisms. We suggest a model of Axin L106R aggregation in vivo, whereby molecularly small osmolytes keep the protein as a free soluble molecule but the increased crowding of the bound state by macromolecules induces its aggregation at the nanoscale. To our knowledge, this is the first systematic study on the effect of osmolytes and crowders on a process of native-like aggregation involved in pathology, as it sheds light on the contribution of cosolutes to the onset of cancer as a protein misfolding disease and on the relevance of aggregation in the molecular etiology of cancer.

Rapid oligomer formation of human muscle acylphosphatase induced by heparan sulfate.

Many human diseases are caused by the conversion of proteins from their native state into amyloid fibrils that deposit in the extracellular space. Heparan sulfate, a component of the extracellular matrix, is universally associated with amyloid deposits and promotes fibril formation. The formation of cytotoxic prefibrillar oligomers is challenging to study because of its rapidity, transient appearance and the heterogeneity of species generated. The process is even more complex with agents such as heparan sulfate. Here we have used a stopped-flow device coupled to turbidometry detection to monitor the rapid conversion of human muscle acylphosphatase into oligomers with varying heparan sulfate and protein concentrations. We also analyzed mutants of the 15 basic amino acids of acylphosphatase, identifying the residues primarily involved in heparan sulfate-induced oligomerization of this protein and tracing the process with unprecedented molecular detail.