Francois-Xavier Theillet

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-β 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.

Bacterial in-cell NMR of human α-synuclein: a disordered monomer by nature?

The notion that human α-synuclein is an intrinsically disordered monomeric protein was recently challenged by a postulated α-helical tetramer as the physiologically relevant protein structure. The fact that this alleged conformation had evaded detection for so many years was primarily attributed to a widely used denaturation protocol to purify recombinant α-synuclein. In the present paper, we provide in-cell NMR evidence obtained directly in intact Escherichia coli cells that challenges a tetrameric conformation under native in vivo conditions. Although our data cannot rule out the existence of other intracellular protein states, especially in cells of higher organisms, they indicate clearly that inside E. coli α-synuclein is mostly monomeric and disordered.

Cell signaling, post-translational protein modifications and NMR spectroscopy

Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukaryotic post-translational protein modifications (PTMs) denote reversible, covalent additions of small chemical entities such as phosphate-, acyl-, alkyl- and glycosyl-groups onto selected subsets of modifiable amino acids. In turn, these modifications induce highly specific changes in the chemical environments of individual protein residues, which are readily detected by high-resolution NMR spectroscopy. In the following, we provide a concise compendium of NMR characteristics of the main types of eukaryotic PTMs: serine, threonine, tyrosine and histidine phosphorylation, lysine acetylation, lysine and arginine methylation, and serine, threonine O-glycosylation. We further delineate the previously uncharacterized NMR properties of lysine propionylation, butyrylation, succinylation, malonylation and crotonylation, which, altogether, define an initial reference frame for comprehensive PTM studies by high-resolution NMR spectroscopy.

Thermodynamics of protein destabilization in live cells

Significance A key question in structural biology is how protein properties mapped out under simplified conditions in vitro transfer to the complex environment in live cells. The answer, it appears, varies. Defying predictions from steric crowding effects, experimental data have shown that cells in some cases stabilize and in other cases destabilize the native protein structures. In this study, we reconcile these seemingly conflicting results by showing that the in-cell effect on protein thermodynamics is sequence specific: The outcome depends both on the individual target protein and on its detailed host-cell environment. Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein’s interplay with the functionally optimized “interaction landscape” of the cellular interior.

The alphabet of intrinsic disorder

A significant fraction of every proteome is occupied by biologically active proteins that do not form unique three-dimensional structures. These intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) have essential biological functions and are characterized by extensive structural plasticity. Such structural and functional behavior is encoded in the amino acid sequences of IDPs/IDPRs, which are enriched in disorder-promoting residues and depleted in order-promoting residues. In fact, amino acid residues can be arranged according to their disorder-promoting tendency to form an alphabet of intrinsic disorder that defines the structural complexity and diversity of IDPs/IDPRs. This review is the first in a series of publications dedicated to the roles that different amino acid residues play in defining the phenomenon of protein intrinsic disorder. We start with proline because data suggests that of the 20 common amino acid residues, this one is the most disorder-promoting.

Phosphorylation of histone H3 Ser10 establishes a hierarchy for subsequent intramolecular modification events

Phosphorylation of Ser10 of histone H3 regulates chromosome condensation and transcriptional activity. Using time-resolved, high-resolution NMR spectroscopy, we demonstrate that histone H3 Ser10 phosphorylation inhibits checkpoint kinase 1 (Chk1)– and protein kinase C (PKC)–mediated modification of Thr11 and Thr6, the respective primary substrate sites of these kinases. On unmodified H3, both enzymes also target Ser10 and thereby establish autoinhibitory feedback states on individual H3 tails. Whereas phosphorylated Ser10 does not affect acetylation of Lys14 by Gcn5, phosphorylated Thr11 impedes acetylation. Our observations reveal mechanistic hierarchies of H3 phosphorylation and acetylation events and provide a framework for intramolecular modification cross-talk within the N terminus of histone H3.

NMR profiling of histone deacetylase and acetyl-transferase activities in real time.

Histone deacetylases (HDACs) and histone acetyl-transferases (HATs) are universal regulators of eukaryotic transcriptional activity and emerging therapeutic targets for human diseases. Here we describe the generation of isotope-labeled deacetylation and acetylation reporters for simultaneous NMR readouts of multiple deacetylation and acetylation reactions at different histone H4 sites. The site preferences of two prototypic histone deacetylases (Sir2.1 and HDAC8) and two acetyl-transferases (HAT1 and p300/CBP) were studied in intramolecular competition assays. We identify a previously ill-defined acetylation site, lysine 20 of histone H4, as a preferred target of three of theses enzymes. In situ analyses of endogenous deacetylation reactions at H4 sites in HeLa nuclear extracts point to abundant HDAC activities in human cellular environments.

Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts

We outline NMR protocols for site-specific mapping and time-resolved monitoring of protein phosphorylation reactions using purified kinases and mammalian cell extracts. These approaches are particularly amenable to intrinsically disordered proteins and unfolded, regulatory protein domains. We present examples for the 15N isotope-labeled N-terminal transactivation domain of human p53, which is either sequentially reacted with recombinant enzymes or directly added to mammalian cell extracts and phosphorylated by endogenous kinases. Phosphorylation reactions with purified enzymes are set up in minutes, whereas NMR samples in cell extracts are prepared within 1 h. Time-resolved NMR measurements are performed over minutes to hours depending on the activities of the probed kinases. Phosphorylation is quantitatively monitored with consecutive 2D 1H-15N band-selective optimized-flip-angle short-transient (SOFAST)-heteronuclear multiple-quantum (HMQC) NMR experiments, which provide atomic-resolution insights into the phosphorylation levels of individual substrate residues and time-dependent changes thereof, thereby offering unique advantages over western blotting and mass spectrometry.

In-Cell Protein Structures from 2D NMR Experiments

In-cell NMR spectroscopy provides atomic resolution insights into the structural properties of proteins in cells, but it is rarely used to solve entire protein structures de novo. Here, we introduce a paramagnetic lanthanide-tag to simultaneously measure protein pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) to be used as input for structure calculation routines within the Rosetta program. We employ this approach to determine the structure of the protein G B1 domain (GB1) in intact Xenopus laevis oocytes from a single set of 2D in-cell NMR experiments. Specifically, we derive well-defined GB1 ensembles from low concentration in-cell NMR samples (∼50 μM) measured at moderate magnetic field strengths (600 MHz), thus offering an easily accessible alternative for determining intracellular protein structures.

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 Parkinsons disease. Here we use in-cell NMR spectroscopy to study the fate of the oxidation-damaged Parkinsons 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.