Study Of Expression, Puirfication And Stability Of Transthyretin Wild-type, V30A And V30M Mutants

Transthyretin（TTR）is a55-kDa homotetrameric protein. It is mainly expressedin the liver, choroid plexus and retinal pigment epithelial cells and thensecreted intothe plasma and cerebrospinal fluid.The function of TTR is to transport a thyroidhormone （L-thyroxine, T₄） and retinal, in the latter case through binding toretinol-binding protein. Single point mutations in the transthyretin gene destabilizethe native TTR, favoring dissociation of tetramers into unfolded monomeric specieswhich can self-assemble into insoluble amyloid fibrils which subsequently causeamyloid diseases and neurodegenerative diseases such as familial amyloidticcardiomyopathy （FAC），senile systemic amyloidosis （SSA），and familial amyloidticpolyneuropathy （FAP）.Recently, we found a Chinese FAP family involving a rare TTR Val30to Alasubstitution. To clarify whether TTR V30A mutation is the pathogenic factor of thisFAP family, we compared the impacts of V30A and V30M mutations on TTRstructural stability and amyloid fibril formation.Firstly, we successfully constructed an E. coli system （pQE30vector and M15strain） to express the TTR wild-type, V30A and V30M variants, which were furtherpurified by Ni-NTA system.Secondly, we evaluated the quaternary, tertiary structure stabilities and amyloidfibril formation of the wild-type TTR and the V30A and V30M variants under thedenature pressure. The results showed that in the pressure of pH or urea, the V30Aand V30M, especially the V30A mutation significantly destablized the quaternaryand tertiary structure of TTR, and promoted the amyloid fibril formation.Thirdly，results from kinetic studies demonstrated that the V30A mutant had thefastest rates of tetramer dissociation, unfolded monomeric species and amyloid fibrilformation compared to the wild-type TTR and the V30M variant. It suggests that the V30A mutation maybe the causal factor of the FAP disease in this Chinese family.Last of all, the treatment for FAP disease mainly include liver transplantationand drug therapy of small molecule inhibitors. Therefore the finding of smallmolecule inhibitors which can stabilize the TTR tetramer and inhibit the amyloidformation will be helpful for the relief of FAP symptoms.We observed the impactsof160drugs on TTR amyloid fibril formation by OD₄₀₀nm optical density analysis.The results showed that four nonsteroidal anti-inflammatory drugs in the centralnervous system including ibuprofen, antipyrine, aspirin and probenecid significantlyinhibited the amyloid fibril formation.These results may provide a theoretical basisfor the development of TTR inhibitors and drug treatment of FAP.