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-β 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 aggregate diversity in Lewy body deposits

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

Abstract 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 mmolÁL -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

CNBP controls transcription by unfolding DNA G-quadruplex structures

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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

Site-Specific Mapping and Time-Resolved Monitoring of Lysine Methylation by High-Resolution NMR Spectroscopy

Methylation and acetylation of protein lysine residues constitute abundant post-translational modifications (PTMs) that regulate a plethora of biological processes. In eukaryotic proteins, lysines are often mono-, di-, or trimethylated, which may signal different biological outcomes. Deconvoluting these different PTM types and PTM states is not easily accomplished with existing analytical tools. Here, we demonstrate the unique ability of NMR spectroscopy to discriminate between lysine acetylation and mono-, di-, or trimethylation in a site-specific and quantitative manner. This enables mapping and monitoring of lysine acetylation and methylation reactions in a nondisruptive and continuous fashion. Time-resolved NMR measurements of different methylation events in complex environments including cell extracts contribute to our understanding of how these PTMs are established <i>in vitro</i> and <i>in vivo</i>

Site-Specific Mapping and Time-Resolved Monitoring of Lysine Methylation by High-Resolution NMR Spectroscopy

Methylation and acetylation of protein lysine residues constitute abundant post-translational modifications (PTMs) that regulate a plethora of biological processes. In eukaryotic proteins, lysines are often mono-, di-, or trimethylated, which may signal different biological outcomes. Deconvoluting these different PTM types and PTM states is not easily accomplished with existing analytical tools. Here, we demonstrate the unique ability of NMR spectroscopy to discriminate between lysine acetylation and mono-, di-, or trimethylation in a site-specific and quantitative manner. This enables mapping and monitoring of lysine acetylation and methylation reactions in a nondisruptive and continuous fashion. Time-resolved NMR measurements of different methylation events in complex environments including cell extracts contribute to our understanding of how these PTMs are established <i>in vitro</i> and <i>in vivo</i>

Physicochemical Properties of Cells and Their Effects on Intrinsically Disordered Proteins (IDPs)

It has long been axiomatic that a protein’s structure determines its function. Intrinsically disordered proteins (IDPs) and disordered protein regions (IDRs) defy this structure–function paradigm. They do not exhibit stable secondary and/or tertiary structures and exist as dynamic ensembles of interconverting conformers with preferred, nonrandom orientations.(1-4) The concept of IDPs and IDRs as functional biological units was initially met with skepticism. For a long time, disorder, intuitively implying chaos, had no place in our perception of orchestrated molecular events controlling cell biology. Over the past years, however, this notion has changed. Aided by findings that structural disorder constitutes an ubiquitous and abundant biological phenomenon in organisms of all phyla,(5-7) and that it is often synonymous with function,(8-11) disorder has become an integral part of modern protein biochemistry. Disorder thrives in eukaryotic signaling pathways(12) and functions as a prominent player in many regulatory processes.(13-15) Disordered proteins and protein regions determine the underlying causes of many neurodegenerative disorders and constitute the main components of amyloid fibrils.(16) They further contribute to many forms of cancer, diabetes and to cardiovascular and metabolic diseases.(17, 18) Research into disordered proteins produced significant findings and established important new concepts. On the structural side, novel experimental and computational approaches identified and described disordered protein ensembles(3, 19, 20) and led to terms such as secondary structure propensities, residual structural features, and transient long-range contacts.(1, 21) The discovery of coupled folding-and-binding reactions defined the paradigm of disorder-to-order transitions(22) and high-resolution insights into the architectures of amyloid fibrils were obtained.(23, 24) On the biological side, we learned about the unexpected intracellular stability of disordered proteins, their roles in integrating post-translational protein modifications in cell signaling and about their functions in regulatory processes ranging from transcription to cell fate decisions.(15, 25, 26) One open question remaining to be addressed is how these in vitro structural insights relate to biological in vivo effects. How do complex intracellular environments modulate the in vivo properties of disordered proteins and what are the implications for their biological functions (Figure 1)?(27-29