RESUMO
Light chain amyloidosis is the most common form of systemic amyloidosis. This disease is caused by the formation and deposition of amyloid fibers made from immunoglobulin light chains. Environmental conditions such as pH and temperature can affect protein structure and induce the development of these fibers. Several studies have shed light on the native state, stability, dynamics, and final amyloid state of these proteins; however, the initiation process and the fibril formation pathway remain poorly understood structurally and kinetically. To study this, we analyzed the unfolding and aggregation process of the 6aJL2 protein under acidic conditions, with temperature changes, and upon mutation, using biophysical and computational techniques. Our results suggest that the differences in amyloidogenicity displayed by 6aJL2 under these conditions are caused by traversing different aggregation pathways, including unfolded intermediates and the formation of oligomers.
Assuntos
Amiloidose , Cadeias Leves de Imunoglobulina , Humanos , Cadeias Leves de Imunoglobulina/química , Amiloide/química , Amiloidose/metabolismo , Proteínas Amiloidogênicas/genética , MutaçãoRESUMO
Light-chain amyloidosis (AL) is the most common systemic amyloidosis and is caused by the deposition of mainly insoluble immunoglobulin light chain amyloid fibrils in multiple organs, causing organ failure and eventually death. The germ-line λ6a has been implicated in AL, where a single point mutant at amino acid 24 (6aJL2-R24G) has been observed in around 25% of patient samples. Structural analysis has shown only subtle differences between both proteins; nevertheless, 6aJL2-R24G is more prone to form amyloid fibrils. To improve our understanding of the role of protein flexibility in amyloid fibril formation, we have used a combination of solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations to complement the structural insight with dynamic knowledge. Fast timescale dynamics (ps-ns) were equivalent for both proteins, but suggested exchange events for some residues. Even though most of the intermediate dynamics (µs-ms) occurred at a similar region for both proteins, the specific characteristics are very different. A minor population detected in the dispersion experiments could be associated with the formation of an off-pathway intermediate that protects from fiber formation more efficiently in the germ-line protein. Moreover, we found that the hydrogen bond patterns for both proteins are similar, but the lifetime for the mutant is significantly reduced; as a consequence, there is a decrease in the stability of the tertiary structure that extends throughout the protein and leads to an increase in the propensity to form amyloid fibers.
Assuntos
Amiloidose/metabolismo , Humanos , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/metabolismo , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
AL amyloidosis is the most common amyloid systemic disease and it is characterized by the deposition of immunoglobulin light chain amyloid fibers in different organs, causing organ failure. The immunoglobulin light chain germinal line 6a has been observed to over-express in AL patients, moreover, it was observed that, out of these amyloidogenic proteins, 25% present a mutation of an Arg to Gly in position 24. In vitro studies have shown that this mutation produces proteins with a higher amyloid fiber propensity. It was proposed that this difference was due, in part, to the formation of a non-canonical structural element. In order to get a more detailed understanding of the structural and dynamic properties that govern the amyloid fibers formation process, we have determined the solution structure by NMR for the two constructs, showing that the difference in amyloid fibril formation is not due to sequence or structure.