Reixach et al. study the aggregation and cytotoxicity of mutant and wild-type (WT) forms of Transthyretin (TTR), one of the 23 known human proteins involved in sporadic and familial amyloidoses. It was previously found by Kelly and coworkers that aggregation of the homotetrameric TTR involves a monomeric intermediate in vitro, mutants that cause monomerization of the protein form amyloid fibrils more readily than WT TTR, and small molecules that stabilize the native homotetramer can prevent aggregation. In this work, the authors used a human neuroblastoma cell line and four previously designed variants of TTR (WT TTR, V30M TTR, WT-M TTR and V30M-M TTR, the latter two being multi-point mutants that cause monomerization of the protein) to correlate the toxicity (measured using a MTT assay) with the aggregation state of the protein (characterized by size exclusion chromatography). They incubated known concentrations (  Read more

Reixach et al. study the aggregation and cytotoxicity of mutant and wild-type (WT) forms of Transthyretin (TTR), one of the 23 known human proteins involved in sporadic and familial amyloidoses. It was previously found by Kelly and coworkers that aggregation of the homotetrameric TTR involves a monomeric intermediate in vitro, mutants that cause monomerization of the protein form amyloid fibrils more readily than WT TTR, and small molecules that stabilize the native homotetramer can prevent aggregation. In this work, the authors used a human neuroblastoma cell line and four previously designed variants of TTR (WT TTR, V30M TTR, WT-M TTR and V30M-M TTR, the latter two being multi-point mutants that cause monomerization of the protein) to correlate the toxicity (measured using a MTT assay) with the aggregation state of the protein (characterized by size exclusion chromatography). They incubated known concentrations (<1 mg/ml) of each protein mutant with the cell line, and found that V30M TTR is toxic and causes apoptosis, but WT TTR does not. The monomeric mutants are have higher cytotoxicity but pre-formed amyloid fibrils of V30M TTR and large (>100 kDa) aggregates of the monomeric mutant WT-M TTR did not cause any cell death. Compounds that prevented fibrillization of TTR in vitro were found to inhibit cytotoxicity. These observations indicate that monomers and/or small soluble oligomers of TTR may induce cell death, while fibrils may be relatively benign. The TTR species appearing in solution under cell culture conditions were monitored by size exclusion. They found that the monomeric mutants heavily aggregate on the time scales of the cytotoxicity assay, while, curiously, the toxic mutant V30M TTR retains its tetrameric state. These studies provide solid evidence for a general model of aggregation-induced cytotoxicity in which misfolded proteins rapidly oligomerize into small prefibrillar species, which are responsible for cell death, by as yet unknown mechanism.

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