During the biogenesis of melanin in melanocytes, pigmented cells found mostly in the skin and eyes, the pre-melanosomal protein (Pmel17) undergoes processing and forms amyloid fibrils that serve as a scaffold for melanin polymerization within melanosomes, the acidic organelle in which melanin is made. In our work, we have studied the mechanism of amyloid formation of the Pmel repeat (RPT) domain, composed of ten 13 imperfect repeats, rich in Pro, Thr, Ser, and Glu residues. Interestingly, alternative splicing leads to multiple isoforms of Pmel17, two of which differ in the length of the RPT region and are expressed independently. While both RPT isoforms form amyloid fibrils in acidic conditions similar to those found in early melanosomes, the shorter isoform (sRPT) aggregates significantly faster and forms morphologically-distinct fibrils as compared to the longer isoform (lRPT), even though sRPT lacks the residues identified to be within the amyloid core of lRPT (Δrepeats 5-8). To understand how the two isoforms might influence each other, cross-seeding experiments were performed and their kinetics were monitored by intrinsic tryptophan fluorescence. The addition of sRPT fibrils (“seeds”) led to shortened lag times of lRPT aggregation as compared to the unseeded control. Strikingly, in the reverse reaction, <i>i.e</i>. the addition of lRPT seeds to soluble sRPT, we found that lRPT inhibits the maturation phase of sRPT fibrils, which we have described previously. Visualizing the morphology of cross-seeded fibrils by transmission electron microscopy revealed that sRPT seeds alter the microscopic features of lRPT to a twisted fibril, while lRPT seeds had no apparent influence on sRPT. Secondary structural characterization using circular dichroism and Raman microspectroscopy as well as limited-protease digestions, all suggest that lRPT adopts a distinct fibril conformation when cross-seeded with sRPT. Overall, this work reveals a unique interplay between RPT isoforms and suggests sRPT may serve as a “nucleator” of lRPT amyloid formation during melanogenesis, similar to what has been described for other functional amyloids.

Interplay between two isoforms of the functional amyloid Pmel17 repeat domain

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.