Protein Misfolding and Aggregation

National Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

We have carried out detailed investigations of membrane interactions and amyloid formation of alpha-synuclein that have provided residue-specific information and molecular insights into the mechanism of aggregation. Due to the complexity of the amyloid problem, the tools with which we attack have included molecular biology, steady-state and time-resolved fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, electron microscopy, neutron reflectometry, and mass spectrometry. Through our work, we are developing a chemical understanding in how specific biomolecular interactions and cellular environments modulate protein structure and aggregation propensity. In the last review period, we investigated the behavior of alpha-synuclein, traditionally associated with the etiology of Parkinsons disease (PD), in the context of melanoma. Epidemiological evidence has shown that PD patients develop melanoma much more frequently than the normal population, and biochemical studies have identified alpha-synuclein as a commonality and potential molecular link between the two diseases. While studies have established that melanoma cells/tissue with increased amounts of alpha-synuclein have little to no pigmentation, the details of how alpha-synuclein disrupts melanogenesis remains an open question. Here, we use SK-MEL 28 human melanoma cells to show for the first time that endogenous alpha-synuclein is found within melanosomes, the organelle where melanin biosynthesis occurs. We then use recombinant proteins to explore the interaction between alpha-synuclein and the premelanosomal protein (Pmel17), an important pigmentation gene and abundant melanosomal protein. We hypothesized that there may be interplay between Pmel17 and alpha-synuclein, as they are both amyloid-forming proteins with common biochemical and structural properties. However, unlike the pathological alpha-synuclein aggregates, Pmel17 fibrils serve a functional role in melanosomes by sequestering toxic melanin intermediates and acting as a scaffold for melanin deposition. We found that alpha-synuclein fibrils stimulate the aggregation of a Pmel17 fragment constituting the repeat region (RPT, residues 315444), an essential fragment for fibril formation in vivo which forms amyloid fibrils when expressed in vitro. The cross-seeded fibrils displayed ultrastructural features analogous to alpha-synuclein, which persisted upon propagation through multiple generations. Altogether, our data support that cross-propagation between alpha-synuclein and Pmel17, suggesting a molecular connection between PD and melanoma, similar to other amyloid diseases where cross-propagation is postulated as the molecular mechanism underlying disease co-occurrence.