Biology subjects

Theillet, F.-X.

Publications and source records attributed to Theillet, F.-X..

5 recordsLinked to original sources

bioRxiv2024

In-cell residue-resolved NMR of micromolar α-synuclein and tau at 310K

Aggregates of non-globular proteins are associated to several degenerative disorders, e.g. -synuclein and tau involved in Parkinsons and Alzheimers diseases. Do these proteins suffer progressive changes in conformations and interactions in pathologic situations? In-cell NMR provides atomic-scale information in live cells but, until now, only at ~283 K in the case of unfolded proteins. Here, we report new labeling and acquisition methods enabling in-cell NMR at 310 K to study these proteins at micromolar concentrations, i.e. native cellular abundances. We used stable human cell lines expressing -synuclein or tau upon induction in a culture medium supplemented with 13C-labeled amino acids, or precursors thereof. Acquiring 13C-13CO spectra permitted an early residue-resolved analysis of -synuclein and tau at 310 K and <10 M in HEK cells at 700 MHz. We detected disordered conformations and identical patterns of cellular interactions for -synuclein wild-type and two mutants (F4A, A30P). Only the disordered N-terminus of tau was observable, even upon microtubule dismantling by colchicin. Our approach offers an excellent scalability -in signal and resolution-up to 1.2 GHz. 13C-labeling and 13C-detected NMR in live human cells are thus viable techniques for in-cell structural biology.

biochemistry↗

bioRxiv2024

α-Synuclein acts as a cholesteryl-ester sensor on lipid droplets regulating organelle size and abundance.

While aggregated alpha-Synuclein (Syn) is commonly associated with Parkinsons disease, its physiological function as a membrane-binding protein is poorly understood. Here, we show that endogenous Syn binds lipid droplets (LDs) in multiple human cell lines and in stem cell-derived dopaminergic neurons. LD-binding encompasses Syn residues 1-100, which masks their detection by immunofluorescence microscopy, probably explaining the scarcity of similar observations in earlier studies. Syn-LD interactions are highly temperature-sensitive and selective for cholesteryl-ester-rich LDs. They promote the formation of Syn multimers that dissociate from LDs at non-permissive temperatures. Syn remains LD-bound throughout starvation-induced lipolysis, whereas siRNA-knockdown diminishes LD abundance and compromises cell viability upon nutrient depletion, without affecting LD biosynthesis. Reciprocally, excess Syn stimulates LD accumulation in dependence of lipid availability, restricts organelle size and ensures intracellular LD organization, which strictly depends on functional membrane-binding. Supporting a general role of Syn in cellular lipid and cholesterol metabolism, our results point to additional loss-of-function similarities between Parkinsons, Alzheimers and Gauchers disease.

cell biology↗

bioRxiv2024

KIF2C-induced nuclear condensation concentrates PLK1 and phosphorylated BRCA2 at the kinetochore microtubules in mitosis

During mitosis, the human microtubule depolymerase KIF2C increases the turnover of kinetochore-microtubule attachments. This facilitates the correction of attachment errors. Moreover, BRCA2 phosphorylated at Thr207 by PLK1 (BRCA2-pT207) assembles a complex including PLK1, PP2A and BUBR1 that contributes to the stability of the kinetochore-microtubule attachments. PLK1, together with Aurora B, critically regulate the accurate segregation of chromosomes. Here we demonstrate that KIF2C contains an N-terminal domain that binds directly to several phosphorylated peptides, including BRCA2-pT207. Using an optogenetic platform, we reveal that KIF2C assembles into membrane-less compartments or biomolecular condensates that are located next to microtubules. We provide evidence that condensate assembly depends on the presence of the newly defined N-terminal phospho-binding domain of KIF2C and on the kinase activities of Aurora B and PLK1. Moreover, KIF2C condensates concentrate active PLK1 and colocalize with BRCA2-pT207. We propose that, because of its phospho-dependent binding and oligomerization capacities, KIF2C forms biomolecular condensates that partition PLK1 and locally amplify its kinase activity during mitosis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/589357v1_ufig1.gif" ALT="Figure 1"> View larger version (80K): org.highwire.dtl.DTLVardef@3ff5bforg.highwire.dtl.DTLVardef@11efbdcorg.highwire.dtl.DTLVardef@1295118org.highwire.dtl.DTLVardef@18b3dd3_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Structural characterization of stem cell factors Oct4, Sox2, Nanog and Esrrb disordered domains, and a method to identify their phospho-dependent binding partners.

The combined expression of a handful of pluripotency transcription factors (PluriTFs) in somatic cells can generate induced pluripotent stem cells (iPSCs). Here, we report the structural characterization of disordered regions contained in four important PluriTFs, namely Oct4, Sox2, Nanog and Esrrb. Moreover, many post-translational modifications (PTMs) have been detected on PluriTFs, whose roles are not yet characterized. To help in their study, we also present a method i) to produce well-characterized phosphorylation states of PluriTFs, using NMR analysis, and ii) to use them for pull-downs in stem cell extracts analyzed by quantitative proteomics to identify of Sox2 binders.

biochemistry↗

bioRxiv2020

Megadalton-sized dityrosine aggregates of α-synuclein retain high degrees of structural disorder and internal dynamics.

Heterogeneous aggregates of the human protein -synuclein (Syn) are abundantly found in Lewy body inclusions of Parkinsons disease patients. While structural information on classical Syn 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 Syn 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-{beta} 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 Syn 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 Syn in its monomeric form and that they are non-cytotoxic despite retaining basic membrane-binding properties. Our results suggest that oxidative Syn aggregation scavenges cytochrome cs activity into the formation of amorphous, high molecular-weight structures that may contribute to aggregate diversity in Lewy body deposits.

biophysics↗