Understanding and controlling the aggregation behavior of amyloid proteins is a field of intense research. Theoretically, nearly all proteins can form large aggregates through non-covalent bonds in a beta-sheet structure, with proteins linking without branching. This is possible because such aggregates are thermodynamically stable. Upon forming amyloid aggregates, numerous amino acids (approximately 50–60%) within the protein establish hydrogen bonds with neighboring proteins, resulting in significant enthalpic stabilization. This key feature distinguishes amyloid aggregation from amorphous aggregation, where interactions between proteins are irregular and limited in number.
As amyloid aggregation progresses, fibers ranging from hundreds of nanometers to several micrometers form. These large fibers precipitate out of solution, releasing surrounding water molecules and increasing entropy. Thus, amyloid fiber formation is a thermodynamically spontaneous process. However, as previously mentioned, this spontaneous process involves overcoming several barriers. The height of these barriers determines whether amyloid aggregation is challenging or straightforward.
Thermodynamics is crucial for understanding the spontaneity of chemical reactions. Accordingly, introductory chemistry textbooks at universities first focus on atomic structure and quantum mechanics before extensively covering thermodynamics and reaction spontaneity. Unfortunately, kinetics, which addresses reaction rates, is often introduced briefly at the end of these courses. However, many chemical processes in the real world are governed by kinetics, which provides insights into how fast reactions occur and reveals the mechanisms underlying these reactions.
To illustrate, returning home after work is typically a natural, spontaneous process. However, the mode of transport—bus, subway, car, or taxi—can significantly affect travel time. Traffic congestion may slow down buses, while walking to a distant subway station could delay subway users. Similarly, in chemical reactions, pathways chosen based on kinetics impact the reaction rate. By studying reaction pathways, scientists can either accelerate or inhibit reactions.
Amyloid fiber formation via aggregation follows several steps, which can be summarized into four key stages:
- Protein-Protein Interaction:
Proteins must encounter each other within a confined space. This is unlikely to occur naturally in the human body and represents the primary barrier. In laboratory experiments, to facilitate amyloid fiber formation, researchers artificially bypass this step by increasing protein concentration. - Disruption of Protein Structures:
Native tertiary and quaternary structures must break down. Hydrophobic regions within proteins interact to form oligomer-based aggregation nuclei. Recent studies suggest these oligomers exhibit high cellular toxicity. - Fiber Growth:
Once aggregation nuclei form, they act as focal points, binding surrounding proteins to grow into fibers. Fiber elongation may occur incrementally as monomers attach to the nuclei or via merging with preformed oligomers. These mechanisms often coexist. - Formation of Large Aggregates:
Interactions between fibers lead to massive aggregates, resulting in precipitation. Amyloid fibers are highly heterogeneous, even when derived from identical proteins. Differences in progression rates and pathways contribute to this heterogeneity, with nucleation typically being slower than fiber elongation.
Insulin, often used as a model protein for studying protein folding, behaves like an amyloid protein under specific conditions, such as high temperatures. While insulin rarely forms amyloid fibers in vivo, instances of fibrous fatty tissue formation and skin necrosis have been observed in diabetic patients receiving regular insulin injections. However, the protein’s well-characterized structure and controllable fiber formation make it an ideal research model. Under acidic conditions (pH ~2), insulin’s quaternary structure destabilizes, and monomers exist. The tertiary structure, dominated by alpha-helices, remains intact but unfolds at elevated temperatures, forming beta-sheet-rich fibers. To facilitate amyloid fiber formation in insulin, researchers overcome two primary barriers: destabilizing the quaternary structure using acidic conditions and altering the tertiary structure at elevated temperatures (100 – 131°F (37 – 55°C)). Without these adjustments, amyloid fiber formation would be impractical in laboratory settings.
The amyloid aggregation of α-synuclein, a disordered protein involved in neuronal processes, is closely associated with Parkinson’s disease. α-Synuclein forms amyloid-based aggregates known as Lewy bodies in the brains of Parkinson’s patients. The NAC (non-amyloid-β component) region, a highly hydrophobic segment of α-synuclein, drives amyloid aggregation. When α-synuclein interacts with lipid membranes via electrostatic forces, its helical structure stabilizes and protects the NAC region. However, defects in the lipid membrane promote protein interactions and aggregation, lowering the barriers for amyloid fiber formation. Calcium ions (Ca²⁺), abundant in neuronal terminals, further facilitate α-synuclein aggregation. Ca²⁺ interactions reduce the prevalence of conformations shielding the NAC region, increasing its exposure and promoting hydrophobic interactions between proteins. Additionally, Ca²⁺ ions strengthen interactions between fibers, contributing to larger aggregates and Lewy body formation. Copper ions (Cu²⁺) similarly influence α-synuclein aggregation, forming strong coordination bonds that expose the NAC region. This accelerates nucleation, depleting monomers required for fiber elongation and resulting in shorter, highly toxic amyloid aggregates. These findings highlight the importance of understanding amyloid aggregation kinetics and pathways for developing strategies to mitigate protein misfolding disorders.
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References
1. Lee et al. Anal Chem 2014, 86 (3), 1909-1916
2. Lee et al. Angew Chem Int Ed 2014, 126 (29), 7591-7595
3. Choi et al. Biophys J 2014, 107 (8), 1939-1949
4. Choi et al. Sci Rep 2017, 7, 5710
5. Han et al. Sci Rep 2018, 8, 1895
6. Choi et al. Angew Chem Int Ed 2018, 57 (12), 3099-3103
7. Choi et al. Biochim Biophys Acta Biomembr 2018, 1860 (9), 1854-1862
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