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 Estefania P. C. Azevedo, † Humberto M. Pereira, ‡ Richard C. Garratt, ‡ Jeffery W. Kelly, § Debora Foguel,* ,† and Fernando L. Palhano* ,†,§ † Instituto de Bioquímica Me ́ dica, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590 Brazil ‡ Centro de Biotecnologia Molecular Estrutural, Instituto de Física de Sa ̃ o Carlos, Universidade de Sã o Paulo, Sã o Paulo, 13560-970 Brazil § Departments of Chemistry and Molecular and Experimental Medicine and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, United States * S Supporting Information 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. P rotein misfolding diseases include a broad range of pathologies in which proteins fail to fold properly and are degraded or cease to remain in their folded state once folded, populating partially denatured states. 1 At sufficient concentrations, partially denatured states can lead to protein aggregation, resulting in a spectrum of quaternary structures, including oligomers, protofibrils, and fibrils. 2 Many degener- ative diseases are associated with protein cross-β-sheet quaternary structure, or amyloid fibril, formation termed the amyloidoses. 3 Amyloid deposits can be detected using Congo red birefringence or thioflavin T fluorescence and are often associated with glycosaminoglycans, the amyloid P component, and other proteins. Amyloid filaments often share an in-register parallel cross-β quaternary structure in which the strands are oriented perpendicular to the fibril axis. Amyloid fibrils usually consist of multiple interacting filaments that are at least two β- sheets thick. 4-6 Some fibrils are formed by a highly ordered triangular core, which is in turn formed by three parallel cross-β sheets. 7 On the basis of the generic nature of these assemblies, it has been proposed that any protein, regardless of its amino acid sequence, can form amyloid fibrils if subjected to appropriate solution conditions. 8,9 Transthyretin (TTR) is a 54 kDa homotetrameric protein composed of identical 127-residue subunits exhibiting a predominantly β-sheet tertiary structure. 10 TTR is secreted into the human bloodstream by the liver and into the cerebrospinal fluid (CSF) by the choroid plexus. Each TTR monomer is composed of one α-helix and eight β-strands Received: August 29, 2011 Revised: November 3, 2011 Published: November 17, 2011 Article pubs.acs.org/biochemistry © 2011 American Chemical Society 11070 dx.doi.org/10.1021/bi201365r | Biochemistry 2011, 50, 11070-11083