Studies of an unusual transthyretin protein (TTR GLU51_SER52DUP) associated with familial amyloidosis
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Transthyretin-related amyloidosis (ATTR) is a disease involving the formation of a misfolded transthyretin (TTR) protein and resulting insoluble aggregates that deposit in extracellular regions of various tissues and organs. There are hereditary forms of the disease, referred to as ATTRm, and more than 100 TTR amyloid-forming mutants have been reported. The major goal of this work was to analyze the biochemical and biophysical properties of a unique and recently identified TTR mutant protein, TTR Glu51_Ser52dup, found in a patient with ATTRm. Unlike other single nucleotide replacements that have been described as amyloidogenic, the gene abnormality in the present case is the first identification of a TTR duplication mutation. The patient with TTR Glu51_Ser52dup exhibited an extremely aggressive form of ATTRm; clinical symptoms included peripheral neuropathy at baseline evaluation and rapid disease progression to early death from pneumonia and congestive heart failure. We hypothesized that the TTR Glu51_Ser52dup variant would be less stable than the wild-type protein and similar in stability to another highly amyloidogenic mutant, TTR L55P; moreover, the highly unstable nature of this TTR variant would provide a basis for understanding the extremely aggressive clinical phenotype observed in this case. Using Escherichia coli (E. coli) as an expression system and an appropriately modified expression vector, we produced histidine-tagged recombinant human TTR Glu51_Ser52dup protein in high yield and purified to homogeneity. Structural and stability studies were performed by circular dichroism (CD) spectroscopy and SDS-PAGE analysis. We demonstrated that TTR Glu51_Ser52dup was less stable than the wild-type or L55P proteins when measured under different types of denaturing conditions, including thermal and chemical stress. The presence of diflunisal, a drug that stabilizes tetrameric TTR and is currently approved for treatment of ATTRm, was also investigated; our results indicated that diflunisal stabilized the TTR Glu51_Ser52dup protein. Collectively, the data obtained from these studies suggest that Glu51_Ser52dup is one of the least stable and most amyloidogenic TTR variant described to date. Future investigations are necessary to determine which specific structural elements of the protein destabilize the TTR tetramer, and precisely characterize the binding of small molecules, including diflunisal, to the protein.