Oligomerization Profile of Human Transthyretin Variants with Distinct Amyloidogenicity

Abstract

One of the molecular hallmarks of amyloidoses is ordered protein aggregation involving the initial formation of soluble protein oligomers that eventually grow into insoluble fibrils. The identification and characterization of molecular species critical for amyloid fibril formation and disease development have been the focus of intense analysis in the literature. Here, using photo-induced cross-linking of unmodified proteins (PICUP), we studied the early stages of oligomerization of human transthyretin (TTR), a plasma protein involved in amyloid diseases (ATTR amyloidosis) with multiple clinical manifestations. Upon comparison, the oligomerization processes of wild-type TTR (TTRwt) and several TTR variants (TTRV30M, TTRL55P, and TTRT119M) clearly show distinct oligomerization kinetics for the amyloidogenic variants but a similar oligomerization mechanism. The oligomerization kinetics of the TTR amyloidogenic variants under analysis showed a good correlation with their amyloidogenic potential, with the most amyloidogenic variants aggregating faster (TTRL55P > TTRV30M > TTRwt). Moreover, the early stage oligomerization mechanism for these variants involves stepwise addition of monomeric units to the growing oligomer. A completely different behavior was observed for the nonamyloidogenic TTRT119M variant, which does not form oligomers in the same acidic conditions and even for longer incubation times. Thorough characterization of the initial steps of TTR oligomerization is critical for better understanding the origin of ATTR cytotoxicity and developing novel therapeutic strategies for the treatment of ATTR amyloidosis.

Multifunctional Therapeutic Cyclodextrin-Appended Dendrimer Complex for Treatment of Systemic and Localized Amyloidosis

“…In Figures 3A and 4A, small aggregates of several hundred nm in size were observed by CDE treatment. Because TTR has been reported to form up to octameric structures, 50 nm or less, as soluble oligomers, 27 the aggregates observed by CDE treatment are considered to be TTR amyloid fibrils (different from oligomers). According to the Th-T assay, both CDE and CDE/shRNA significantly reduced the amount of TTR V30M amyloid fibrils, but while CDE/shRNA disrupted approximately 55% of fibrils, CDE alone only disrupted approximately 36% (Figure 4B).…”

Molecular Mechanisms of Cardiac Amyloidosis

“…Dasari et al showed that wild-type TTR tends to form linear oligomers, while the G53A TTR variant forms annular oligomers with pore-like structures [ 48 ]. Frangolho et al compared the oligomerization processes of wild-type TTR and several TTR variants (V30M, L55P and T119M) and found distinct oligomerization kinetics but a similar oligomerization mechanism [ 49 ]. The oligomerization kinetics of the wild-type TTR and TTR variants showed a good correlation with their amyloidogenic potential, and the most amyloidogenic variants aggregated faster (L55P > V30M > wild-type).…”

Transthyretin Misfolding, A Fatal Structural Pathogenesis Mechanism

“…This observation indicated that the protofibril might be formed by the mechanism of adding 15-nm oligomers to the end of the protofibril [ 73 ]. More recently, Frangolho et al used photo-induced crosslinking experiments to capture up to octameric species during acid-induced TTR aggregation at pH 3.6 [ 74 ]. They found that amyloidogenic variants of TTR showed faster oligomerization rates, and also provided evidence supporting sequential monomeric addition to oligomers.…”