Abstract
Parkinson’s disease (PD) is the second most common neurological disease and belongs to a group of neurodegenerative disorders called synucleinopathies in which pathological aggregates of N-terminally acetylated α-synuclein (NAcα-Syn) accumulate in various regions of the brain. In PD, these NAcα-Syn aggregates have been found to contain covalent dityrosine crosslinks, which can occur either intermolecularly or intramolecularly. Cerebral metal imbalance is also a hallmark of PD, warranting investigations into the effects of brain biometals on NAcα-Syn. NAcα-Syn is an intrinsically disordered protein, and metal-mediated conformational modifications of this structurally dynamic protein have been demonstrated to influence its propensity for dityrosine formation. In this study, a library of tyrosine-to-phenylalanine (Y-to-F) NAcα-Syn constructs were designed in order to elucidate the nature and the precise residues involved in dityrosine crosslinking of Fe-bound NAcα-Syn. The structural capacity of each mutant to form dityrosine crosslinks was assessed using Photo-Induced Cross-Linking of Unmodified Proteins (PICUP), demonstrating that coordination of either FeIII or FeII to NAcα-Syn inhibits dityrosine crosslinking among the C-terminal residues. We further demonstrate that Y39 is the main contributor to dityrosine formation of Fe-bound NAcα-Syn, while Y125 is the main residue involved in dityrosine crosslinks in unmetalated NAcα-Syn. Our results confirm that iron coordination has a global effect on NAcα-Syn structure and reactivity.
Highlights
Synucleinopathies are a group of neurodegenerative disorders in which insoluble aggregates, made up predominantly of the neuronal protein α-synuclein, accumulate in neurons, glial cells, and nerve fibers [1]
Tyrosine substituted NAc αSyn variants were created through site-specific substitution of each tyrosine with phenylalanine in order to investigate their intrinsic differences in protein dynamics following the coordination of iron
To examine the impact of each individual tyrosine on the photochemical behavior of NAc αSyn, Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) studies were first performed in the absence of metal ions on NAc WT, which retains the native sequence of NAc αSyn, Life 2020, 10, x FOR PEER
Summary
Synucleinopathies are a group of neurodegenerative disorders in which insoluble aggregates, made up predominantly of the neuronal protein α-synuclein (αSyn), accumulate in neurons, glial cells, and nerve fibers [1]. The toxicity of the αSyn aggregates manifests through many different biological pathways, such as abnormal mitochondrial activity, membrane damage, and impaired synaptic function among others [2,3]. ΑSyn has long been considered an intrinsically disordered protein (IDP) because it is natively unfolded in its monomeric state [6,7,8]. It is constitutively acetylated at the N-terminus in humans (NAc αSyn), and this post-translational modification (PTM) has dramatic consequences on the metal binding characteristics and self-assembly mechanisms of this structurally dynamic protein [9,10,11,12].
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