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Protein fibrillation lag times during kinetic inhibition
Rodrigo S Pagano1, Máximo López Medus1, Gabriela E Gómez2
1Structural Cell Biology Laboratory, Fundación Instituto Leloir and Instituto de Investigaciones Bioquímicas de Buenos Aires (IIBBA-CONICET), Buenos Aires, Argentina.
Abstract:
Protein aggregation is linked to more than 30 human pathologies, including Alzheimer's and Parkinson's diseases. Since small oligomers that form at the beginning of the fibrillation process probably are the most toxic elements, therapeutic strategies involving fibril fragmentation could be detrimental. An alternative approach, named kinetic inhibition, aims to prevent fibril formation by using small ligands that stabilize the parent protein. The factors that govern fibrillation lag times during kinetic inhibition are largely unknown, notwithstanding their importance for designing effective long-term therapies. Inhibitor-bound species are not likely to be incorporated into the core of mature fibrils, although their presence could alter the kinetics of the fibrillation process. For instance, inhibitor-bound species may act as capping elements that impair the nucleation process and/or fibril growth. Here, we address this issue by studying the effect of two natural inhibitors on the fibrillation behavior of lysozyme at neutral pH. We analyzed a set of 79 fibrillation curves obtained in lysozyme alone and a set of 37 obtained in the presence of inhibitors. We calculated the concentrations of the relevant species at the beginning of the curves using the inhibitor-binding constants measured under the same experimental conditions. We found that inhibitor-bound protein species do not affect fibrillation onset times, which are mainly determined by the concentration of unbound protein species present in equilibrium. In this system, knowledge of the fibrillation kinetics and inhibitor affinities suffices to predict the effect of kinetic inhibitors on fibrillation lag times. In addition, we developed a new methodology to better estimate fibrillation lag times from experimental curves.
Insights
Kinetic inhibition prevents protein aggregation linked to diseases like Alzheimer's. This study shows that unbound protein concentration, not inhibitor-bound species, determines fibrillation onset, aiding therapeutic design.
Area of Science:
- Biochemistry
- Molecular Biology
- Disease Pathogenesis
Background:
- Protein aggregation is implicated in over 30 human diseases, including neurodegenerative disorders like Alzheimer's and Parkinson's.
- Small oligomers formed during early fibrillation stages are considered highly toxic, making fibril fragmentation a potentially detrimental therapeutic strategy.
- Kinetic inhibition, which uses ligands to stabilize proteins and prevent aggregation, offers an alternative therapeutic approach, but factors governing its effectiveness, particularly fibrillation lag times, remain poorly understood.
Purpose of the Study:
- To investigate the impact of natural inhibitors on lysozyme fibrillation kinetics at neutral pH.
- To determine whether inhibitor-bound protein species influence fibrillation lag times and onset.
- To develop a predictive model for kinetic inhibitor efficacy based on protein-ligand interactions and fibrillation dynamics.
Main Methods:
- Analysis of 79 fibrillation curves for lysozyme alone and 37 curves with two natural inhibitors.
- Calculation of species concentrations at fibrillation onset using experimentally determined inhibitor-binding constants.
- Development of a novel methodology for more accurate estimation of fibrillation lag times from experimental data.
Main Results:
- Inhibitor-bound protein species were found to have no significant effect on fibrillation onset times.
- Fibrillation lag times were primarily determined by the concentration of unbound protein species in equilibrium.
- The study demonstrated that fibrillation kinetics and inhibitor affinities are sufficient to predict the impact of kinetic inhibitors on lag times.
Conclusions:
- Kinetic inhibition is a viable strategy for preventing toxic protein aggregation.
- The concentration of free, unbound protein is the key determinant of fibrillation lag time, not the presence of inhibitor-bound species.
- Accurate prediction of kinetic inhibitor efficacy is achievable through understanding fibrillation kinetics and inhibitor binding affinities, paving the way for improved therapeutic design.
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