Mutations can cause light chains to be too stable or too unstable to form amyloid fibrils

Abstract

Light chain (AL) amyloidosis is an incurable human disease, where the amyloid precursor is a misfolding-prone immunoglobulin light-chain. Here, we identify the role of somatic mutations in the structure, stability and in vitro fibril formation for an amyloidogenic AL-12 protein by restoring four nonconservative mutations to their germline (wild-type) sequence. The single restorative mutations do not affect significantly the native structure, the unfolding pathway, and the reversibility of the protein. However, certain mutations either decrease (H32Y and H70D) or increase (R65S and Q96Y) the protein thermal stability. Interestingly, the most and the least stable mutants, Q96Y and H32Y, do not form amyloid fibrils under physiological conditions. Thus, Q96 and H32 are key residues for AL-12 stability and fibril formation and restoring them to the wild-type residues preclude amyloid formation. The mutants whose equilibrium is shifted to either the native or unfolded states barely sample transient partially folded states, and therefore do not form fibrils. These results agree with previous observations by our laboratory and others that amyloid formation occurs because of the sampling of partially folded states found within the unfolding transition (Blancas-Mejia and Ramirez-Alvarado, Ann Rev Biochem 2013;82:745-774). Here we provide a new insight on the AL amyloidosis mechanism by demonstrating that AL-12 does not follow the established thermodynamic hypothesis of amyloid formation. In this hypothesis, thermodynamically unstable proteins are more prone to amyloid formation. Here we show that within a thermal stability range, the most stable protein in this study is the most amyloidogenic protein.

Aggregation of Full-length Immunoglobulin Light Chains from Systemic Light Chain Amyloidosis (AL) Patients Is Remodeled by Epigallocatechin-3-gallate

“…More recent studies have painted a more differentiated picture. Mutations that alter the interface in AL-LC dimers may alter amyloid formation without altering V L monomer stability (44,62).…”

Recruitment of Light Chains by Homologous and Heterologous Fibrils Shows Distinctive Kinetic and Conformational Specificity

“…27,28,36 $$y=\frac{A_1-A_2}{1+{\mathrm{e}}^{false(x-x_{0}false)/\mathrm{normald}\mathrm{normalx}}}+A_2$$ where A 1 is the initial fluorescence value, A 2 is the final fluorescence value, x 0 is the midpoint (or t 50 value), and dx is defined as the time constant.…”

Differential recruitment efficacy of patient-derived amyloidogenic and myeloma light chain proteins by synthetic fibrils—A metric for predicting amyloid propensity

“…Our theoretical understanding of LC amyloidogenesis comes almost exclusively from in vitro studies of fibrillogenesis using recombinant VL fragments of κ4 [20, 36–39], λ6 [21, 40, 41] and κ1[18, 19, 42–44] LC subgroups produced in E. coli . Fibril formation from these components often requires harsh conditions such as low pH, agitation, or the presence of chaotropes, indicating the need for a denaturing milieu to initiate protein misfolding and, consequently, fibril formation.…”

“…Fibril formation from these components often requires harsh conditions such as low pH, agitation, or the presence of chaotropes, indicating the need for a denaturing milieu to initiate protein misfolding and, consequently, fibril formation. Although there are rare exceptions [19, 45], these studies demonstrated a general inverse correlation between VL folding free energy and the propensity for in vitro fibrillogenesis, such that less stably folded VL domains more readily form fibrils. Thus, de novo fibrillogenesis from soluble VL domains can be rapid and requires misfolding of the VL, allowing structured self-association into thermodynamically stable multimers that act as templates, or seeds, for the recruitment of additional VL domains.…”