Reixach N, Deechongkit S, Jiang X, Kelly JW, Buxbaum JN.
Tissue damage in the amyloidoses: Transthyretin monomers and nonnative oligomers are the major cytotoxic species in tissue culture.
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2817-22.
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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
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.