Identification of fibrillogenic regions in human triosephosphate isomerase

Abstract

Background. Amyloid secondary structure relies on the intermolecular assembly of polypeptide chains through main-chain interaction. According to this, all proteins have the potential to form amyloid structure, nevertheless, in nature only few proteins aggregate into toxic or functional amyloids. Structural characteristics differ greatly among amyloid proteins reported, so it has been difficult to link the fibrillogenic propensity with structural topology. However, there are ubiquitous topologies not represented in the amyloidome that could be considered as amyloid-resistant attributable to structural features, such is the case of TIM barrel topology. Methods. This work was aimed to study the fibrillogenic propensity of human triosephosphate isomerase (HsTPI) as a model of TIM barrels. In order to do so, aggregation of HsTPI was evaluated under native-like and destabilizing conditions. Fibrillogenic regions were identified by bioinformatics approaches, protein fragmentation and peptide aggregation. Results. We identified four fibrillogenic regions in the HsTPI corresponding to the β3, β6, β7 y α8 of the TIM barrel. From these, the β3-strand region (residues 59-66) was highly fibrillogenic. In aggregation assays, HsTPI under native-like conditions led to amorphous assemblies while under partially denaturing conditions (urea 3.2 M) formed more structured aggregates. This slightly structured aggregates exhibited residual cross-β structure, as demonstrated by the recognition of the WO1 antibody and ATR-FTIR analysis. Discussion. Despite the fibrillogenic regions present in HsTPI, the enzyme maintained under native-favoring conditions displayed low fibrillogenic propensity. This amyloid-resistance can be attributed to the three-dimensional arrangement of the protein, where β-strands, susceptible to aggregation, are protected in the core of the molecule. Destabilization of the protein structure may expose inner regions promoting β-aggregation, as well as the formation of hydrophobic disordered aggregates. Being this last pathway kinetically favored over the thermodynamically more stable fibril aggregation pathway.

Keywords: Aggregation; Amyloid; Cross-β; Fibrillogenesis; Triosephosphate isomerase.

Figure 1. Aggregation kinetics followed by (A) ThT fluorescence and (B) by turbidimetry at 405 nm of HsTPI_n (dashed line) and HsTPI_urea (solid line). (C) TEM images of HsTPI aggregates at the final time point of aggregation.

Scale bars are 1 muM.

Figure 2. Secondary structure of HsTPI aggregates.

(A) Second-derivative ATR-FTIR spectra in the amide I region of salted-out HsTPI (dotted line), HsTPI_n (dashed line) and HsTPI_urea (solid line). (B) Dot-blot assay of HsTPI aggregates with the WO1 antibody confirming cross-β structure.

Figure 3. Identification of potential fibrillogenic regions in HsTPI.

(A) Consensus of different prediction methods of fibrillogenic regions. The β-strands are shown in green, the α-helices in yellow and the chameleonic sequences are underlined. Aspartic residues are in red, indicating the potential sites for acid hydrolysis. Prediction hits are shown in the corresponding line of the predictor. (B) List of the potential fibrillogenic regions selected for peptide aggregation assay. All β-strands were selected as well as the three more significant chameleonic sequences of the protein.

Figure 4. Peptide aggregation.

(A) ThT fluorescence intensities at 485 nm of aggregates of peptides at final time point of incubation. (B) Congo red birefringence assay of the β3 (solid black line), β6 (dashed line), β7 (dotted-dashed line) and CS-3 (dotted line) aggregates. A maximal peak at 540 nm is shown in aggregates compared with Congo red alone (solid gray line).

Figure 5. TEM images of peptides aggregates.

The scale bar are 1 µm.

Figure 6. Acid hydrolysis of HsTPI.

(A) Tricine SDS-PAGE of hydrolyzed HsTPI. (B) Aggregation kinetics of the hydrolyzed fragments of HsTPI followed by ThT fluorescence. (C) TEM image of the amyloid fibrils formed by fragmented HsTPI; the scale bars is 1 µM. (D) Tricine SDS-PAGE of the enriched fragment upon aggregation. (E) MS/MS spectrum of the triply charged precursor ion at m/z 1048.84 identifies the amyloid fragment as the sequence DPKIAVAAENCYKVTNGAFTGEISPGMIKD, which corresponds to residues 57–85 of HsTPI.