Typologie d'actualités: CBM

Structural insights into the SUMOylation reaction

SUMOylation and ubiquitylation are related protein modifications where small proteins (SUMO or ubiquitin) become covalently attached to protein substrates to regulate their function. Both these protein modifications are essential for viability and are strongly implicated in human disease, but SUMOylation remains less studied than ubiquitylation. A key step in both SUMOylation and ubiquitylation reactions is the formation of a reactive thioester molecule in which SUMO or ubiquitin becomes linked to a cysteine residue on proteins called E2. It is from there that SUMO/ubiquitin is transferred onto the final protein substrate. In the study just published in Journal of Biological Chemistry, the researchers from the CBM used site-directed mutagenesis to create a version of the human E2-SUMO thioester that – unlike the native reactive thioester – is chemically stable and can be studied with structural biology methods. The crystal structure of this molecule revealed potential regulatory mechanisms for the SUMOylation process. The mutagenesis approach was inspired by a method developed for the yeast SUMOylation pathway by the group of Chris Lima.

The article, authored by the CBM engineer Stéphane Goffinont and other members of the team “Protein Post-Translational Modifications and DNA Repair: Structure, Function, and Dynamics”, is the first publication from the project “SUMOwriteNread”. The project is led by the CBM researcher Marcin J. Suskiewicz and funded by the Horizon Europe programme of the European Union (European Research Council Starting Grant no 101078837).

Stéphane Goffinont, Franck Coste, Pierre Prieu-Serandon, Lucija Mance, Virginie Gaudon, Norbert Garnier, Bertrand Castaing and Marcin Józef Suskiewicz
Structural insights into the regulation of the human E2∼SUMO conjugate through analysis of its stable mimetic.
Journal of Biological Chemistry, Volume 299, Issue 7, 2023, 104870 - https://www.sciencedirect.com/science/article/pii/S0021925823018987

Valentin BEAUVAIS thesis defense

Valentin BEAUVAIS, PhD student in the "DAIH Team: Dysregulation of Authophay in Inflammation Due to HIV", will defend his thesis on Wednesday July 5, 2023 at 2:00 p.m. at the Charles Auditorium, CNRS Campus in Orléans.

Download the thesis defense notice (the link will be activated soon)

Abstract:

The transcription of messenger RNA (mRNA) is a complex process implicating a large diversity of players at very specific steps. The production of an mRNA also requires its maturation into a ribonucleoproteic particle (mRNP) through the binding of proteins that will package, protect and allow the mRNP to be exported to the cytoplasm where it will be translated into a protein. This crucial step is monitored by a quality control system (QC) that detects the rare aberrant mRNPs and induces their degradation by one of the exosome-dependent exonuclease, Rrp6. The error rate during mRNA maturation is very low and complicates the study of the QC. The induction of the bacterial factor Rho leads to the formation of aberrant mRNPs while avoiding the deletion of any protein potentially implicated in the QC system. The present study focuses on the THO complex, a keystone for the maturation of mRNA as it binds to chromatin early during transcription and then serves as a recruitment platform for other packaging proteins. The use of genome-wide analysis techniques (ChIP-seq) revealed the implication of Tho2 for the detection of aberrant transcripts and their degradation by Rrp6, independently from the THO complex. Furthermore, a similar approach (RNA-seq) on Rrp6-depleted yeast highlighted once again the importance of ncRNA degradation for the regulation of coding gene expression. Finally, the expansion of Rho utilization from yeast to human could bring to light the implication of nuclear speckles in the quality control of mRNPs in humans.

Keywords :

mRNP biogenesis, HTS techniques, Rho, THO complex, Rrp6, Nuclear speckles

Edison ZHAMUNGUI thesis defense

Edison ZHAMUNGUI, PhD student in the "Functional mass spectrometry of molecular assemblies" team, will defend his thesis on Wednesday July 5, 2023 at 10:30 a.m. at the Charles Auditorium, CNRS Campus in Orléans.

Download the thesis defense notice.

Abstract:

Although membrane proteins (MPs) represent 2/3rds of potential therapeutic targets, so far only 10% of these proteins have been targeted by a marketed drug. This is due to a lack of structural information which derives from the challenges associated with producing MPs and the need for solubilizing agents. Major biological functions rely on critical proteins interacting with other biomolecules such as nucleic acids, peptides, or other proteins, thus forming larger complexes. The size of these complexes (i.e., 100 000 Da or higher) as well as their high heterogeneity impose limitations on their structural characterization.

In the past decade, the characterization of noncovalent complexes by native mass spectrometry has emerged as a valuable ally of traditional and emerging Structural Biology techniques such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, or cryo-EM. Here we show how a novel method called Native Liquid MALDI (NALIM) can be used to answer questions about the structural biology of membrane proteins and large soluble complexes.

NALIM-TOF MS relies on the known strengths of MALDI-TOF MS such as a high tolerance to contaminants, a low sample consumption and in principle, a limitless mass range of analysis. With carefully controlled non-denaturing conditions and instrumental parameters optimizations, NALIM enabled rapid access to characterize various membrane transporter proteins, including an ABC transporter and an ion channel. The stabilization of the dimer of Bacillus subtilis multidrug resistance (BmrA) protein by ligand binding was demonstrated and the binding site of the Tx7335 actitoxin in the potassium ion channel KcsA shown to differ from the site of pore blockers. To get closer to the in vivo conditions of the native membrane's lipid bilayer, NALIM was applied to the characterization of membrane proteins in proteoliposomes and membrane vesicles.

The development of the NALIM method involved exploring different proteins to find a suitable calibrant for high mass range analysis. A preparation of alpha1-antitrypsin (α1AT) that forms a molecular ladder over a wide mass range was validated as a calibrant. Furthermore, instrumental parameters were tuned to improve the resolution and sensitivity in the high mass range, making it possible to obtain information on the stoichiometry and stability of large soluble complexes. For instance, NALIM revealed the stoichiometry and specificity of ZBTB8A oligomers and the stabilizing effect of NusG on the Rho hexamer.

In summary, NALIM was shown to be a fast and straightforward method for the structural characterization of membrane protein complexes and large soluble oligomers in their native state. The information obtained through NALIM can be of significant value in the fields of molecular biology and pharmacology. Moreover, NALIM promises to be an alternative for new strategies of drug discovery.

 

Conference by Prof. Ken LAU and Dr Andrea ROLONG – June 5, 2023

This international conference is organized within the framework of the ARD CVL Biomedicaments Program.

Abstract: Tissue complexity emerges from interactions of components across various biological systems, such as exogenous factors from the microbiota and different types of host cells, and the body's immune cells to the presence of tumors. These interactions occur across genetic, molecular, and spatial domains. Although single-cell and spatial -omics approaches are already capable of profiling various components at an atlas scale, there is still a significant gap in effectively transforming these methods from correlative studies to hypothesis-driven studies. Here, we present two stories on how-omic level data and computational analyzes can be integrated with experimental models (human, mouse, and organoid) for mechanistic studies:

- in understanding rare epithelial cell populations in modulating inflammation in the gut,

- in modeling a pre-cancer-to-cancer transition in the colon. We present emerging techniques, analyses, and the key roles they play in understanding the complex interactions that dictate tissue function in homeostasis and disease.

Conference website

Conference registration

Truly life materials?

Physiological characterization of life in Engineered Living Materials by confocal microscopy at single cell resolution.

The CNRS Institute of Chemistry reported this remarkable research on its site. See the article

Engineered Living Materials (ELMs) combine living cells with non-living scaffolds to get life-like characteristics, such as biosensing, growth, and self-repair. Some ELMs are 3D-printed, and called bio-ink. For ELMs to be functional, cells in ELMs has to remain alive and active. However, currently, microorganism physiology in ELMs is still elusive and restrict their use.

Researchers of the team "Cell signalling and neurofibromatosis" reconstituted such bioprinted ELMs by associating the yeast Saccharomyces cerevisiae with the hydrogel Pluronic F-127. Theydeveloped genetically engineered yeast by integrating fluorescent gene whose expression is correlated to a physiological parameter: ATP concentration (metabolism), intracellular pH (growth phase), morphology … These engineered and ratiometric biosensors are effective and allow to assess yeast physiological status in ELM directly in situ by confocal microscopy at single cell scale level. They constitute a valuable tool easy to adapt to any other system by associating them to other materials to evaluate their biocompatibility.

Furthermore, the researchers tested their recently developed copper biosensor embedded into this hydrogel F-127, and showed it is fully functional into this ELM. Yeast biosensor association with hydrogel provides several very interesting advantages such as protecting yeast from contaminations and supplying them with nutrients.

This work allows to establish the proof of concept that F127 associated with engineered yeast S. cerevisiae is a promising ELM in order to develop easy to use whole-cell biosensors able to detect copper directly on samples collected in the environment.

Bojan Žunar B., Ito T., Mosrin C., Sugahara Y., Bénédetti H., Guégan R. and Vallée B.
Confocal imaging of biomarkers at a single-cell resolution: quantifying 'living' in 3D-printable engineered living material based on Pluronic F-127 and yeast Saccharomyces cerevisiae.
Biomater Res 26, 85 (2022). https://doi.org/10.1186/s40824-022-00337-8

Milk thistle, a plant extract with promising –green- medicinal properties against psoriasis

Considering the relative low efficacy and high toxicity of current drug treatments against psoriasis, new therapeutic strategies are needed.

Scientists from CBM have searched for natural products unable to modulate the TGFb/miRNA-21-5p pathway in keratinocyte cells. This axis of regulation was chosen not only because it plays a pivotal role in epidermal haemostasis but also because its dysregulation is systematically associated with skin disorders including psoriasis.

To identify such bioactive compounds, a library of medicinal plant extracts was screened using the miR-ON RILES screening system placed under the control of the miRNA-21-5p in keratinocytes treated with TGFb. Silymarin, a mixture of flavonolignans extracted from Silybum marianum (L.) Gaertn., was identified as the most potent regulator of miRNA-21-5p expression. RNA-sequencing analysis revealed three unexpected transcriptomic signatures associated with keratinocyte differentiation, cell cycle, and lipid metabolism.

Mechanistically, Silymarin blocks cell cycle progression, inhibits keratinocyte differentiation through repression of Notch3 expression, stimulates lipid synthesis via activation of PPARg signaling and inhibits inflammatory responses by suppressing the transcriptional activity of NF-kB. Notably, the topical application of silymarin alleviates the development of psoriasiform lesions in mice by abrogating the altered expression levels of markers involved in inflammation, proliferation, differentiation, and lipid metabolism without inducing toxicity.

Therefore this plant extract might represent a promising "green" alternative to current pharmacological treatments for the management of this pathology.


Elodie Henriet,Florence Abdallah, Yoan Laurent, Cyril Guimpied, Emily Clement, Michel Simon, Chantal Pichon and Patrick BarilTargeting TGF-β1/miR-21 Pathway in Keratinocytes Reveals Protective Effects of Silymarin on Imiquimod-Induced Psoriasis Mouse ModelVolume 3, ISSUE 3, 100175, May 2023 - DOI:https://doi.org/10.1016/j.xjidi.2022.100175