Mechanisms of Functional Amyloid Formation

National Heart, Lung, And Blood Institute

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Abstract

In our work, we have studied a crucial fibril forming domain termed the repeat domain (RPT, residues 315-444) derived from the human functional amyloid, Pmel17, to gain insights into what may differentiate functional from pathological amyloid. Pmel17 is a transmembrane precursor protein that is proteolytically processed to form intralumenal fibrils in melanosomes upon which melanin is deposited. Pmel17 is highly regulated in vivo, undergoing a series of post-translational and proteolytic modifications whereby the timing and sequence of these events permit amyloid formation. RPT is essential for the amyloid structures observed in melanosomes. Fibrils are formed during the early stages of melanosome development and once formed are responsible for the deposition of the pigment melanin. Since melanin precursors are cytotoxic, sequestering their synthesis on fibrils prevents potential detriment to the organelle. During this review period, we made and characterized a new RPT construct that is tagless to address whether the C-terminal hexahistidine tag influences RPT aggregation. This was achieved by introducing a tobacco etch virus (TEV) protease recognition sequence (ENLYGQG) immediately upstream of a native Pmel17 glycine residue at position 312. Thus, upon purification and cleavage with TEV protease, a native Pmel17 fragment (nRPT) spanning residues 312444 is produced. We characterized the pH-dependent aggregation and disaggregation of native RPT, comparing it to that of the previously used RPT with a C-terminal hexahistidine tag. We characterized the aggregation of nRPT, which formed amyloid fibrils under acidic conditions (pH 6) but not at neutral pH. Characterizing the morphologies of nRPT aggregates using transmission electron microscopy revealed a pH-dependent maturation from short, curved structures at pH 4 to paired, rod-like fibrils at pH 6. This was accompanied by a secondary structural transition from mixed random coil/beta-sheet at pH 4 to canonical beta-sheet at pH 6. We also show that pre-formed nRPT fibrils undergo disaggregation upon dilution into pH 7 buffer. More broadly, this strategy can be utilized to generate native amyloidogenic domains from larger proteins by utilizing intrinsic N-terminal glycine or serine residues.