Multi-organ structural homogeneity of amyloid fibrils in ATTRv-T60A amyloidosis patients, revealed by Cryo-EM

Abstract

ATTR amyloidosis is a degenerative disorder characterized by the systemic deposition of the protein transthyretin. These amyloid aggregates of transthyretin (ATTR) can deposit in different parts of the body causing diverse clinical manifestations. Our laboratory aims to investigate a potential relationship between the different genotypes, organ of deposition, clinical phenotypes, and the structure of ATTR fibrils. Using cryo-electron microscopy, we have recently described how the neuropathic related mutations ATTRv-I84S and ATTRv-V122∆ can drive structural polymorphism in ex vivo fibrils. Here we question whether the mutation ATTRv-T60A, that commonly triggers cardiac and neuropathic symptoms, has a similar effect. To address this question, we extracted and determined the structure of ATTR-T60A fibrils from multiple organs (heart, thyroid, kidney, and liver) from the same patient and from the heart of two additional patients. We have found a consistent conformation among all the fibril structures, acquiring the "closed-gate morphology" previously found in ATTRwt and others ATTRv related to cardiac or mixed manifestations. The closed-gate morphology is composed by two segments of the protein that interact together forming a polar channel, where the residues glycine 57 to isoleucine 68 act as a gate of the polar cavity. Our study indicates that ATTR-T60A fibrils present in peripheral organs adopt the same structural conformation in all patients, regardless of the organ of deposition.

Figure 1.. Cryo-EM structures of cardiac fibrils from three different ATTRv-T60A patients.

3D class and models of the structures resolved from the fibrils of a) Patient 1 and b) Patient 2. Although different interlayer distance and crossover were calculated, we can see they share the same morphology composed by two fragments of transthyretin: N-terminal (proline 11 to lysine 35) and C terminal (Threonine 123 to glycine 57/Leucine 58). c) The 3D class obtained from patient 3 and the morphology distinguished in the density map indicate that these fibrils acquired the same morphology previously described for patient 1 and 2. The density map obtained from this patient had not resolution enough to construct the model.

Figure 2.. Identification of amyloid deposits by histological staining of the non-cardiac organs from Patient 3.

General cellular staining with hematoxylin and eosin (top) and specific amyloid staining with Congo red (middle) and Thioflavin S, Th-S, (bottom) was done for every tissue. a) Congo red stained amyloid deposition closed to the blood walls and Th-S showed the deposits found in the adipose tissue of thyroid. b) Slices corresponding to the same areas were stained with the different dyes. The amyloid deposits found in adipose tissue are indicated. c) Staining of a cross-section slice of the liver show the amyloid-specific staining of the Glisson capsule.

Figure 3.. Structure of the fibrils extracted from the thyroid, kidney, and liver of the patient 3.

3D map and models of the fibrils from a) thyroid, b) kidney, and c) liver of ATTRv-T60A fibrils from patient 3 shows the same single morphology for the three of them. Crossover and interlayer distances are indicated. The resulting morphology corresponds to the “closed-gate” fold, in agreement with the structure obtained in the previously analyzed cardiac fibrils.

Figure 4.. Molecular characterization of the ATTRv-T60A fibrils analyzed in this study.

a) Western-blot of the different fibrils show the presence of the bands corresponding to the full-length monomeric transthyretin and the c-fragment, according to type A fibrils. b) T-test comparison of the ratio C-fragment/full-length does not indicate differences in the cleavage of the ATTRv-T60A fibrils depending on the individual nor the organ. c) Cardiac and non-cardiac ATTRv-T60A fibrils seeds the aggregation of MTTR, generating the typical ThT sigmoidal curve, except for that from thyroid. d) TEM images after seeding reaction. The presence of fibrils confirmed the success of the seeding reaction.