Publications

Amyloid fibrils, which are aggregates of misfolded proteins characterized by 13-sheet-rich structures, are implicated in several neurodegenerative and systemic pathologies, including Alzheimer's and Parkinson's diseases and type II diabetes mellitus. Traditional amyloid markers, such as Congo Red and Thioflavin T, are widely used for amyloid detection but present limitations, particularly in cellular assays, due to spectral interference and aggregation inhibition. This study investigates YAT2150, a novel fluorescent dye with enhanced amyloid-binding specificity and sensitivity, as a potential alternative to conventional dyes. We evaluated YAT2150's efficacy for detecting amyloid aggregates in both in vitro and in cellula assays. First, we compared its fluorescence intensity and binding specificity to that of Thioflavin Tin amyloid fibril assays, demonstrating that YAT2150 exhibits high affinity and selectivity for amyloid structures, with minimal interference from non-aggregated proteins. Furthermore, we explored YAT2150's utility in Escherichia colt as a model system for studying protein aggregation and amyloid formation in a procaryotic cellular context. Our findings indicate that YAT2150 effectively labels amyloid-like inclusion bodies in E. colt producing a robust fluorescence signal with low background noise. These results suggest that YAT2150 is a promising new tool for amyloid research, offering greater sensitivity compared to traditional dyes, even in complex cellular environments.

JTD Keywords: Aggregation, Amyloid, Amyloid inhibitor, Bacterial inclusion-bodies, Detect, In-vitro, Inclusion bodies, Kinetics, Model, Parameters, Prion, Screening, Silico, Yat2150

The formation of aggregates by misfolded proteins is thought to be inherently toxic, affecting cell fitness. This observation has led to the suggestion that selection against protein aggregation might be a major constraint on protein evolution. The precise fitness cost associated with protein aggregation has been traditionally difficult to evaluate. Moreover, it is not known if the detrimental effect of aggregates on cell physiology is generic or depends on the specific structural features of the protein deposit. In bacteria, the accumulation of intracellular protein aggregates reduces cell reproductive ability, promoting cellular aging. Here, we exploit the cell division defects promoted by the intracellular aggregation of Alzheimer's-disease-related amyloid β peptide in bacteria to demonstrate that the fitness cost associated with protein misfolding and aggregation is connected to the protein sequence, which controls both the in vivo aggregation rates and the conformational properties of the aggregates. We also show that the deleterious impact of protein aggregation on bacterial division can be buffered by molecular chaperones, likely broadening the sequential space on which natural selection can act. Overall, the results in the present work have potential implications for the evolution of proteins and provide a robust system to experimentally model and quantify the impact of protein aggregation on cell fitness.

JTD Keywords: Amyloid fibrils, Chaperones, Escherichia coli, Inclusion bodies, Protein aggregation

The accumulation of aggregated protein in the cell is associated with the pathology of many diseases and constitutes a major concern in protein production. Intracellular aggregates have been traditionally regarded as nonspecific associations of misfolded polypeptides. This view is challenged by studies demonstrating that, in vitro, aggregation often involves specific interactions. However, little is known about the specificity of in vivo protein deposition. Here, we investigate the degree of in vivo co-aggregation between two self-aggregating proteins, A beta A2 amyloid peptide and foot-and-mouth disease virus VP1 capsid protein, in prokaryotic cells. In addition, the ultrastructure of intracellular aggregates is explored to decipher whether amyloid fibrils and intracellular protein inclusions share structural properties. The data indicate that in vivo protein aggregation exhibits a remarkable specificity that depends on the establishment of selective interactions and results in the formation of oligomeric and fibrillar structures displaying amyloid-like properties. These features allow prokaryotic A beta A2 intracellular aggregates to act as effective seeds in the formation of A beta A2 amyloid fibrils. overall, our results suggest that conserved mechanisms underlie protein aggregation in different organisms. They also have important implications for biotechnological and biomedical applications of recombinant polypeptides.

JTD Keywords: Protein aggregation, Inclusion bodies, Conformational diseases, Amyloid fibrils, Protein folding