Thermodynamic Modulation of Light Chain Amyloid Fibril Formation

Abstract

To obtain further insight into the pathogenesis of amyloidosis and develop therapeutic strategies to inhibit fibril formation we investigated: 1) the relationship between intrinsic physical properties (thermodynamic stability and hydrogen-deuterium (H-D) exchange rates) and the propensity of human immunoglobulin light chains to form amyloid fibrils in vitro; and 2) the effects of extrinsically modulating these properties on fibril formation. An amyloid-associated protein readily formed amyloid fibrils in vitro and had a lower free energy of unfolding than a homologous nonpathological protein, which did not form fibrils in vitro. H-D exchange was much faster for the pathological protein, suggesting it had a greater fraction of partially folded molecules. The thermodynamic stabilizer sucrose completely inhibited fibril formation by the pathological protein and shifted the values for its physical parameters to those measured for the nonpathological protein in buffer alone. Conversely, urea sufficiently destabilized the nonpathological protein such that its measured physical properties were equivalent to those of the pathological protein in buffer, and it formed fibrils. Thus, fibril formation by light chains is predominantly controlled by thermodynamic stability; and a rational strategy to inhibit amyloidosis is to design high affinity ligands that specifically increase the stability of the native protein.

Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

“…5,6,9 Additionally, under physiological conditions, antibody light chains and associated variable light chains have been shown to possess the ability to form amyloid, and have been implicated in free light chain amyloidosis, a neurodegenerative disorder. 10 Antibody aggregation is influenced by formulation conditions, such as pH, buffer, and excipient. 4,7 Techniques have been detailed to quantitatively measure antibody aggregation.…”

Kinetics and Energetics of Assembly, Nucleation, and Growth of Aggregates and Fibrils for an Amyloidogenic Protein

“…Thermodynamic and Kinetic Data Analyses-The linear extrapolation method was used to calculate the fraction of native protein (f N ) present during pressure-and urea-induced unfolding (19,21). From data for urea-induced dissociation and unfolding at atmospheric pressure, the free energy of dissociation in buffer A alone, ⌬G d (buffer A), was obtained by extrapolation of the plot of ⌬G d versus [urea] …”

“…Transmission Electron Microscopy (TEM) and CR Staining of Precipitated Protein-CR binding to precipitates was assessed as previously described (19,20). TEM was performed as described (13) after DTT was removed from the aggregates by centrifugation and resuspension with 1 volume of buffer A.…”

“…Aggregate pellets were also resuspended in 7 M guanidine HCl in buffer A, incubated for 1 h, and analyzed by size-exclusion HPLC. ThT assay for protein fibrils was performed as described (13,19). FT-IR spectroscopy was used to determine the secondary structure of native SMA (20 mg/ml in buffer A), pure SMA amyloid fibrils (generated by incubation at atmospheric pressure by agitation for 96 h and concentrated 20-fold), and the insoluble aggregates formed under high pressure after washing three times with buffer A and concentrating 20-fold (13,19).…”

Current and New Perspectives on the Molecular and Cellular Mechanisms of Amyloid Formation and Toxicity in Light Chain Amyloidosis

“…The germline protein was more thermodynamically stable than its amyloidogenic counterpart, and although it was able to form fibrils, its fibril formation kinetics were significantly slower than AL-09. Additionally, fibril formation of AL protein BIF and MM protein GAL (also of the I O18/O8 germline) was compared at 37°C, but only BIF formed fibrils (Kim et al, 2000). Because the 6a germline is expressed almost exclusively in AL patients (it is one of the last germline genes screened in the process of selection) and is not expressed in the normal LC repertoire (Abraham et al, 2003;Comenzo et al, 2001;Prokaeva et al, 2007), del Pozo Yauner et al hypothesized that this germline would be as unstable as AL proteins.…”