Self-assembly of human latexin into amyloid-like oligomers

Abstract

Background: In conformational disorders, it is not evident which amyloid aggregates affect specific molecular mechanisms or cellular pathways, which cause disease because of their quantity and mechanical features and which states in aggregate formation are pathogenic. Due to the increasing consensus that prefibrillar oligomers play a major role in conformational diseases, there is a growing interest in understanding the characteristics of metastable polypeptide associations.

Results: Here, we show that human latexin, a protein that shares the same fold with cystatin C, assembles into stable spherical amyloid-like oligomers that bind thioflavin-T and congo red similarly to common amyloid structures but do not evolve into fibrils. Latexin self-assembly correlates with the formation of a mostly denaturated state rather than with the population of partially structured intermediates during the unfolding process. The results suggest that unfolding of alpha-helix 3 might be involved in the transition of latexin toward amyloidotic species, supporting the notion of the protective role of the native protein structure against polymerization.

Conclusion: Overall the data herein indicate that latexin could be a good model for the study of the structural and sequential determinants of oligomeric assemblies in protein aggregation processes.

Figure 1

Conformational properties of latexin. A) CD spectrum of latexin at increasing urea concentrations. B) Overlay of the urea denaturation curve monitored by intrinsic fluorescence and by far UV-CD. The difference in the two transition midpoints is usually associated with the presence of an intermediate state whose population is theoretically calculated from the normalized unfolding curves. C) 3D structure of latexin (2BO9). The five triptophans (red) are located in the C-terminal subdomain (light blue and green) and the region corresponding to the main hot spot is located in the alpha-helix 3 (green).

Figure 2

SDS-PAGE electrophoresis of latexin in various concentrations of urea. A) Isolated recombinant latexin was incubated at 1 mg/ml for 24 hours at room temperature at increasing urea concentrations. The incubation mixtures were analyzed by electrophoresis in 12% SDS-PAGE. B) Latexin incubated at 6 M urea for 24 hours at room temperature and after the urea was removed and incubated for 12 hours.

Figure 3

Latexin secondary structural changes after 24 hour incubation in urea monitored by CD. Far-UV circular dichroism spectra were measured at 25°C for samples at 0 M urea incubated 24 hours; 6 M urea incubated 24 hours; 6 M urea not incubated.

Figure 4

Binding of amyloid dyes by latexin. A) Changes in the fluorescence emission of Th-T and in congo red absorbance upon binding to latexin (0.1 mg/ml) after 24 hours incubations at various concentrations of urea and at 25°C. B) Th-T fluorescence at 485 nm at various latexin concentrations. (Inset) Fluorescence emission spectra of th-T at various latexin concentrations.

Figure 5

Representative images of latexin protein incubated at various urea concentrations for more than one day. A) Representative EM images of latexin samples incubated during: (1) 24 hours at 0 M urea; (2) 24 hours at 6 M urea; (3) 24 hours at 9 M urea. B) EM images of latexin samples incubated at 6 M urea during: (1) 24 hours; (2) one week; (3) one month. The scale bar represents 500 nm.

Figure 6

Aggregation profile of human latexin. The aggregation profile was generated using the program AGGRESCAN. The region that displays the highest aggregation propensity, T134-W149, is framed. The regular secondary structure elements shown, correspond to the human latexin structure, cylinders stand for α-helices and arrows for β-strands.

Figure 7

Analysis of VLHLAWVA peptide self-assembly. A) An electron micrograph of the VLHLAWVA microcristals. The scale bar represents 1 μm. B) CD spectrum of VLHLAWVA after 0 hours of incubation and 24 hours at 25°C in buffer P. C) Thioflavin-T fluorescence of stained material after one day incubation in buffer P. (1) Phase contrast microscopy. (2) Fluorescence microscopy under UV-light. D) FTIR spectrum of the non-preincubated peptide in buffer P and the peptide incubated in buffer P 24 hours. The positions of the spectral components are estimated from the second derivate analysis.