Disulfide-Bond Formation in the Transthyretin Mutant Y114C Prevents Amyloid Fibril Formation in Vivo and in Vitro^,

Abstract

The Y114C mutation in human transthyretin (TTR) is associated with a particular form of familial amyloidotic polyneuropathy. We show that vitreous aggregates ex vivo consist of either regular amyloid fibrils or disordered disulfide-linked precipitates that maintain the ability to bind Congo red. Furthermore, we demonstrate in vitro that the ATTR Y114C mutant exists in three forms: one unstable but nativelike tetrameric form, one highly aggregated form in which a network of disulfide bonds is formed, and one fibrillar form. The disulfide-linked aggregates and the fibrillar form of the mutant can be induced by heat induction under nonreduced and reduced conditions, respectively. Both forms are recognized by the amyloid specific antibody MAB(39-44). In a previous study, we have linked exposure of this epitope in TTR to a three-residue shift in beta-strand D. The X-ray crystallographic structure of reduced tetrameric ATTR Y114C shows a structure similar to that of the wild type but with a more buried position of Cys10 and with beta-mercaptoethanol associated with Cys114, verifying the strong tendency for this residue to form disulfide bonds. Combined with the ex vivo data, our in vitro findings suggest that ATTR Y114C can lead to disease either by forming regular unbranched amyloid fibrils or by forming disulfide-linked aggregates that maintain amyloid-like properties but are unable to form regular amyloid fibrils.

Probing Solvent Accessibility of Transthyretin Amyloid by Solution NMR Spectroscopy

“…2) were analyzed by the K2D prediction program (13) and resulted in an estimated ␤-sheet content of 42 and 40%, respectively. This is in agreement with a recently presented crystal structure of TTRY114C showing only subtle differences from the native TTR wild-type (12). TTRY114C fibrils formed at a protein concentration around 20 M at pH 7 (verified by AFM) did not cause significant turbidity, a fact that allows for secondary structure analysis by means of CD spectroscopy together with the K2D program (13).…”

“…Temperature-The mutant protein TTRY114C was expressed in E. coli and purified as a native tetramer (12). As a result of the mutation Tyr 114 3 Cys 114 the tetrameric fold of the protein is destabilized and consequently acquires an enhanced propensity to form amyloid at near physiological pH (pH 7) and ionic strength (100 mM NaCl).…”

“…1B shows a representative AFM image of these fibrils. The cysteine residue in position 10, present in both wild-type TTR and TTRY114C, as well as the additional Cys 114 residue in TTRY114C, are complicating features because of the potential formation of intermolecular disulfide bonds within the fibrils (22) (12). To avoid interference of intermolecular disulfide linkage 1% BME was included during the incubation at 55°C.…”

“…This mutant protein can easily be induced to form fibrils at elevated temperatures under otherwise physiological conditions (12) and provides a representative model system for TTR amyloid. The critical process of dissolving fibrils into NMRdetectable monomers at a rate that is significantly faster than the exchange rate within the monomeric state is accomplished by the addition of SDS as a solubilizing agent.…”

Human Pancreatitis-associated Protein Forms Fibrillar Aggregates with a Native-like Conformation

“…The solvent accessibility of transthyretin amyloid probed by solution NMR in combination with hydrogen/deuterium exchange suggested that strands C and D are dislocated from their native edge region and become a solvent-exposed loop, leaving a new interface involving rest of the native ␤-strands for intermolecular interactions (48). However, these fibrils with native-like structures are formed by either direct stacking or stacking with a cross-␤ spine; they do bind to Congo Red (54,(57)(58)(59). In our study, formation of hPAP fibrillar aggregates was seen at pH levels above 5.0 and was maximal at pH 7.0.…”