Structural disorder of monomeric α-synuclein persists in mammalian cells.

Intracellular aggregation of the human amyloid protein α-synuclein is causally linked to Parkinson's disease. While the isolated protein is intrinsically disordered, its native structure in mammalian cells is not known. Here we use nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to derive atomic-resolution insights into the structure and dynamics of α-synuclein in different mammalian cell types. We show that the disordered nature of monomeric α-synuclein is stably preserved in non-neuronal and neuronal cells. Under physiological cell conditions, α-synuclein is amino-terminally acetylated and adopts conformations that are more compact than when in buffer, with residues of the aggregation-prone non-amyloid-ß component (NAC) region shielded from exposure to the cytoplasm, which presumably counteracts spontaneous aggregation. These results establish that different types of crowded intracellular environments do not inherently promote α-synuclein oligomerization and, more generally, that intrinsic structural disorder is sustainable in mammalian cells.

Paramagnetic relaxation enhancement to improve sensitivity of fast NMR methods: application to intrinsically disordered proteins.

We report enhanced sensitivity NMR measurements of intrinsically disordered proteins in the presence of paramagnetic relaxation enhancement (PRE) agents such as Ni(2+)-chelated DO2A. In proton-detected (1)H-(15)N SOFAST-HMQC and carbon-detected (H-flip)(13)CO-(15)N experiments, faster longitudinal relaxation enables the usage of even shorter interscan delays. This results in higher NMR signal intensities per units of experimental time, without adverse line broadening effects. At 40 mmol·L(-1) of the PRE agent, we obtain a 1.7- to 1.9-fold larger signal to noise (S/N) for the respective 2D NMR experiments. High solvent accessibility of intrinsically disordered protein (IDP) residues renders this class of proteins particularly amenable to the outlined approach.

Megadalton-sized Dityrosine Aggregates of α-Synuclein Retain High Degrees of Structural Disorder and Internal Dynamics.

Heterogeneous aggregates of the human protein α-synuclein (αSyn) are abundantly found in Lewy body inclusions of Parkinson's disease patients. While structural information on classical αSyn amyloid fibrils is available, little is known about the conformational properties of disease-relevant, non-canonical aggregates. Here, we analyze the structural and dynamic properties of megadalton-sized dityrosine adducts of αSyn that form in the presence of reactive oxygen species and cytochrome c, a proapoptotic peroxidase that is released from mitochondria during sustained oxidative stress. In contrast to canonical cross-ß amyloids, these aggregates retain high degrees of internal dynamics, which enables their characterization by solution-state NMR spectroscopy. We find that intermolecular dityrosine crosslinks restrict αSyn motions only locally whereas large segments of concatenated molecules remain flexible and disordered. Indistinguishable aggregates form in crowded in vitro solutions and in complex environments of mammalian cell lysates, where relative amounts of free reactive oxygen species, rather than cytochrome c, are rate limiting. We further establish that dityrosine adducts inhibit classical amyloid formation by maintaining αSyn in its monomeric form and that they are non-cytotoxic despite retaining basic membrane-binding properties. Our results suggest that oxidative αSyn aggregation scavenges cytochrome c's activity into the formation of amorphous, high molecular-weight structures that may contribute to the structural diversity of Lewy body deposits.

Dynamic m6A methylation facilitates mRNA triaging to stress granules.

Reversible post-transcriptional modifications on messenger RNA emerge as prevalent phenomena in RNA metabolism. The most abundant among them is N6-methyladenosine (m6A) which is pivotal for RNA metabolism and function; its role in stress response remains elusive. We have discovered that in response to oxidative stress, transcripts are additionally m6A modified in their 5' vicinity. Distinct from that of the translationally active mRNAs, this methylation pattern provides a selective mechanism for triaging mRNAs from the translatable pool to stress-induced stress granules. These stress-induced newly methylated sites are selectively recognized by the YTH domain family 3 (YTHDF3) "reader" protein, thereby revealing a new role for YTHDF3 in shaping the selectivity of stress response. Our findings describe a previously unappreciated function for RNA m6A modification in oxidative-stress response and expand the breadth of physiological roles of m6A.

Intracellular repair of oxidation-damaged α-synuclein fails to target C-terminal modification sites.

Cellular oxidative stress serves as a common denominator in many neurodegenerative disorders, including Parkinson's disease. Here we use in-cell NMR spectroscopy to study the fate of the oxidation-damaged Parkinson's disease protein alpha-synuclein (α-Syn) in non-neuronal and neuronal mammalian cells. Specifically, we deliver methionine-oxidized, isotope-enriched α-Syn into cultured cells and follow intracellular protein repair by endogenous enzymes at atomic resolution. We show that N-terminal α-Syn methionines Met1 and Met5 are processed in a stepwise manner, with Met5 being exclusively repaired before Met1. By contrast, C-terminal methionines Met116 and Met127 remain oxidized and are not targeted by cellular enzymes. In turn, persisting oxidative damage in the C-terminus of α-Syn diminishes phosphorylation of Tyr125 by Fyn kinase, which ablates the necessary priming event for Ser129 modification by CK1. These results establish that oxidative stress can lead to the accumulation of chemically and functionally altered α-Syn in cells.

Efficient modification of alpha-synuclein serine 129 by protein kinase CK1 requires phosphorylation of tyrosine 125 as a priming event.

S129-phosphorylated alpha-synuclein (α-syn) is abundantly found in Lewy-body inclusions of Parkinson's disease patients. Residues neighboring S129 include the α-syn tyrosine phosphorylation sites Y125, Y133, and Y136. Here, we use time-resolved NMR spectroscopy to delineate atomic resolution insights into the modification behaviors of different serine and tyrosine kinases targeting these sites and show that Y125 phosphorylation constitutes a necessary priming event for the efficient modification of S129 by CK1, both in reconstituted kinase reactions and mammalian cell lysates. These results suggest that α-syn Y125 phosphorylation augments S129 modification under physiological in vivo conditions.