title: Dissecting the structure, thermodynamic stability, and aggregation properties of the A25T transthyretin (A25T-TTR) variant involved in leptomeningeal amyloidosis: identifying protein partners that co-aggregate during A25T-TTR fibrillogenesis in cerebrospinal fluid.
abstract: Deposition of amorphous aggregates and fibrils of transthyretin (TTR) in leptomeninges and subarachnoid vessels is a characteristic of leptomeningeal amyloidosis (LA), a currently untreatable cerebral angiopathy. Herein, we report the X-ray structure of the A25T homotetramer of TTR, a natural mutant described in a patient with LA. The structure of A25T-TTR is indistinguishable from that of wild-type TTR (wt-TTR), indicating that the difference in amyloidogenicity between A25T-TTR and wt-TTR cannot be ascribed to gross structural differences. Using pressure-induced dissociation of the tetramer, we show that A25T-TTR is 3 kcal/mol less stable than L55P-TTR, the most aggressive mutant of TTR described to date.  After incubation for 15 days at 37 °C (pH 7.3), A25T-TTR forms mature amyloid fibrils. To mimic the environment in which TTR aggregates, we investigated aggregation in cerebrospinal fluid (CSF). Unlike L55P-TTR, A25T-TTR rapidly forms amyloid aggregates in CSF that incorporated several protein partners. Utilizing a proteomics methodology, we identified 19 proteins that copurified with A25T-TTR amyloid fibrils. We confirmed the presence of proteins previously identified to be associated with TTR aggregates in biopsies of TTR amyloidosis patients, such as clusterin, apolipoprotein E, and complement proteins. Moreover, we identified novel proteins, such as blood coagulation proteins. Overall, our results revealed the in vitro characterization of TTR aggregation in a biologically relevant environment, opening new avenues of investigation into the molecular mechanisms of LA.
authors.affiliation: Instituto de Bioquímica Médica, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590 Brazil.
authors.forename: Estefania P C
authors.initials: EP
authors.lastname: Azevedo
authors.forename: Humberto M
authors.initials: HM
authors.lastname: Pereira
authors.forename: Richard C
authors.initials: RC
authors.lastname: Garratt
authors.forename: Jeffery W
authors.initials: JW
authors.lastname: Kelly
authors.forename: Debora
authors.initials: D
authors.lastname: Foguel
authors.forename: Fernando L
authors.initials: FL
authors.lastname: Palhano
mesh_terms: D019943:Amino Acid Substitution; D000682:Amyloid / Q000134:cerebrospinal fluid* / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D001057:Apolipoproteins E / Q000737:chemistry / Q000378:metabolism; D001779:Blood Coagulation Factors / Q000737:chemistry / Q000378:metabolism; D016657:Cerebral Amyloid Angiopathy / Q000134:cerebrospinal fluid* / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002555:Cerebrospinal Fluid / Q000737:chemistry* / Q000378:metabolism; D051152:Clusterin / Q000737:chemistry / Q000378:metabolism; D003165:Complement System Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006874:Hydrostatic Pressure; D007700:Kinetics; D008578:Meninges / Q000473:pathology; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D011228:Prealbumin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D013816:Thermodynamics
chemical_list: D000682:Amyloid; D001057:Apolipoproteins E; D001779:Blood Coagulation Factors; C495242:CLU protein, human; D051152:Clusterin; D050505:Mutant Proteins; D011228:Prealbumin; D011994:Recombinant Proteins; D003165:Complement System Proteins