Biochemical And Structural Insights Into Transthyretin Amyloid Fibrils Made In Vitro

Cardiac transthyretin amyloidosis（ATTR-CA）is a localized or systemic disease caused by transthyretin（TTR）misfolding and forming amyloid in the heart.The prevelance of ATTR-CA is largely underestimateded due to misdiagnosis.Therefore,it is of great importance to understand the mechanism of the disease in order to detect and diagnose the disease in its early stage.However,few relevant basic scientific research could be found.A lot of questions about the disease are still unclear.For instance,the mechanism of how TTR tetramers dissociate into monomers and aggregate into amyloid fibrils in ATTR-CA patients’heart is unclear.We still don’t have a perfect cell or animal model for ATTR-CA.Withdrawing amyloid fibrils from ATTR-CA patients’heart for research purpose is almost impossible for all laboritaries.Based on the problems mentioned above,this research aims at making TTR amyloid fibrils which have smilar biochemical and structural properties to amyloid fibrils in ATTR-CA patients’heart.The protein could be used for futrue cell or animal modeling.Making this protein lays solid foundation to future mechanism research and drug development for ATTR-CA.（1）Aim:Making TTR amyloid fibrils by purified WT TTR（Wild type TTR）and m TTR（TTR monomer）in vitro through different methods.Analyzing their biochemical and structural properties by different methods.Comparing those properties with TTR amyloid fibrils withdrew from ATTR-CA patients.Finding the one that has the most similar properties to TTR amyloid fibrils in ATTR-CA and utilizing it for future mechanism studies and drug development.（2）Methods:First,this research transfected expression plasmid into bacteria and induced the bateria to produce TTR protein.The expressed TTR protein was further isolated by sonicating,salting out,centrifuging and dialyzing.The TTR protein was finally purified by using Source15Q anion exchange chromatography and Superdex 75 size exclusion chromatography.Secondly,the purified protein was induced to make amyloid fibrils by strong acid,high temperature and mild temperature which mimicked the environment in human’s body in vitro.Thirdly,the biochemical properties of amloid fibrils were analyzed by Th T fluorescence curve,Native PAGE,SDS PAGE,Superose 6 size exclusion chromatography,transmission electron microscopy and dot blot.We also withdrew amyloid fibrils from ATTR-CA patients’transplanted heart and compared them with the amyloid fibrils we made in vitro.Finally,we used solid state nuclear magnetic resonance to analyze the structural properties of the amyloid fibrils made in vitro.（3）Results:First of all,this research successfully made highly purified regular unlabeled and ¹³C¹⁵N labeled WT TTR and m TTR.Secondly,we doubted the classic method of mild acid inducing TTR amyloid fibrils,since the aggregates made at p H 4.4 were amorphous instead of fibrous which is commonly found in ATTR-CA.Thirdly,we found that the morphology of TTR amyloid we made by using p H 2.0 37℃（strong acid）,p H 7.6 65℃（high temperature）and p H 7.6 37℃（mimic human environment）were fibrous.The TTR amyloid made in strong acid had the most amount of b-sheet.The TTR fibrils made at high temperature was the most stabe one.The size distribution and the ability of binding different antibodies were different among three fibrils.Finally,the structure analysis showed that TTR fibrils made at p H 7.6 65℃and p H 7.637℃shared similar structure,showing significantly different structure to p H 2.0 37℃induced amyloid fibrils.p H 2.0 37℃strong acid induced amyloid fibrils had similarities with WT TTR tetramer while p H 7.6 65℃and p H 7.6 37℃induced amyloid fibrils might had more similarities with amyloid fibrils in ATTR-CA.（4）Conclusion:This study suggests that p H 2.0 strong acid,65℃high temperature and 37℃mild temperature can successfully induce WT TTR or m TTR to form TTR amyloid fibrils in vitro.The biochemical and structural properties of each fibril are different.p H 7.6 65℃and p H 7.637℃induced amyloid fibrils might have more similarities with amyloid fibrils in ATTR-CA patients.