Abstract

The premelanosomal protein (PMEL17) forms functional amyloid fibrils involved in melanin biosynthesis. Multiple PMEL17 isoforms are produced, two of which arise from excision of a cryptic intron within the amyloid-forming repeat (RPT) domain, leading to long (lRPT) and short (sRPT) isoforms with 10 and 7 imperfect repeats, respectively. Both lRPT and sRPT isoforms undergo similar pH-dependent mechanisms of amyloid formation and fibril dissolution. Here, using human PMEL17, we tested the hypothesis that the minor, but more aggregation-prone, sRPT facilitates amyloid formation of lRPT. We observed that cross-seeding by sRPT fibrils accelerates the rate of lRPT aggregation, resulting in propagation of an sRPT-like twisted fibril morphology, unlike the rodlike structure that lRPT normally adopts. This templating was specific, as the reversed reaction inhibited sRPT fibril formation. Despite displaying ultrastructural differences, self- and cross-seeded lRPT fibrils had a similar β-sheet structured core, revealed by Raman spectroscopy, limited-proteolysis, and fibril disaggregation experiments, suggesting the fibril twist is modulated by N-terminal residues outside the amyloid core. Interestingly, bioinformatics analysis of PMEL17 homologs from other mammals uncovered that long and short RPT isoforms are conserved among members of this phylogenetic group. Collectively, our results indicate that the short isoform of RPT serves as a “nucleator” of PMEL17 functional amyloid formation, mirroring how bacterial functional amyloids assemble during biofilm formation. Whereas bacteria regulate amyloid assembly by using individual genes within the same operon, we propose that the modulation of functional amyloid formation in higher organisms can be accomplished through alternative splicing.

Highlights

  • The premelanosomal protein (PMEL17) forms functional amyloid fibrils involved in melanin biosynthesis

  • We have shown here that a minor isoform of the human PMEL RPT domain can cross-seed and accelerate the aggregation of the major PMEL RPT isoform in vitro, which mirrors functional amyloid assembly in bacteria

  • The cross-seeded fibrils display morphological features unique to shorter RPT isoform (sRPT), yet the amyloid core is unaffected, which suggests that N-terminal residues outside of the amyloid core are responsible for defining the macroscopic fibril architecture

Read more

Summary

Introduction

The premelanosomal protein (PMEL17) forms functional amyloid fibrils involved in melanin biosynthesis. Self- and cross-seeded lRPT fibrils had a similar ␤-sheet structured core, revealed by Raman spectroscopy, limited-proteolysis, and fibril disaggregation experiments, suggesting the fibril twist is modulated by N-terminal residues outside the amyloid core. We performed cross-seeding experiments, where preformed fibril seeds were added to soluble protein to measure the effect on aggregation kinetics, and the resulting fibrillar materials were characterized by Trp fluorescence, Raman spectroscopy, as well as transmission EM (TEM).

Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call