Catégorie d'actualités: significant publications

Bispidines and manganese: a winning couple

As an essential metal ion and an efficient relaxation agent, Mn2+ holds great promise as a substitute for Gd3+ in MRI contrast agent applications, if its stable and inert complexation can be achieved. To achieve this goal, the “Metal complexes and MRI” team of CBM and their collaborators from the University of Heidelberg, Germany, created a Mn2+ selective chelator by introducing four pyridine and one carboxylate donors on a bispidine skeleton. Thanks to a highly rigid and preorganized structure and perfect size-match for Mn2+, the new ligand L provides not only remarkably high thermodynamic stability, but also excellent selectivity over the major biological competitor Zn2+, as well as kinetic inertness. The unusual eight-coordinate structure of the Mn2+ complex, in contrast to the six-coordinate structure of the Zn2+ analogue, underlines that the coordination cavity is perfectly adapted for Mn2+, while it is too large for Zn2+. The MRI efficiency of this MnL complex is about 30% higher than that of typical Mn2+ systems. In vivo MRI experiments realized in control mice at a very low dose (0.02 mmol/kg) indicate good signal enhancement and fast renal clearance. Taken together, MnL is the first chelate that combines such excellent stability, selectivity, inertness and relaxation properties, all of primary importance for MRI use.

D. Ndiaye, P. Cieslik, H. Wadepohl, A. Pallier, S. Même, P. Comba, and É. Tóth, Mn2+ bispidine complex combining exceptional stability, inertness and MRI efficiency, J. Am. Chem. Soc. 2022, doi : 10.1021/jacs.2c10108
JACS spotlight sur cet article : https://pubs.acs.org/doi/pdf/10.1021/jacs.2c12719

A major advance in the understanding of DNA damage repair

The "DNA repair: structure, function and dynamics" team has just revealed, in the prestigious journal Nucleic Acid Research, how archaeal DNA glycosylases are able to recognize and repair, at the molecular level, certain lesions in their DNA.

To know more :
Structural and functional determinants of the archaeal 8-oxoguanine-DNA glycosylase AGOG for DNA damage recognition and processing
Coste Franck, Goffinont Stéphane, Cros Julien, Gaudon Virginie, Guérin Martine, Garnier Norbert, Confalonieri Fabrice, Flament Didier, Suskiewicz Marcin Josef, Castaing Bertrand https://doi.org/10.1093/nar/gkac932

Identification of a ‘double‘ protein post-translational modification

Proteins are the main ‘molecular machines’ of the cell. To efficiently perform their tasks, they have to be dynamically switched on and off, recruited to specific cellular locations, and degraded in a timely manner. One of the main mechanisms that regulate these processes is temporary covalent attachment, to a protein, of extra regulatory elements known as protein post-translational modifications. The modification reaction is catalysed by specific enzymes and can lead to changes in protein activity, localisation, or half-life. Two of the common protein modifications are ubiquitin and ADP-ribose, each of which can be linked directly to a protein substrate.

In the study published in Science Advances, an international team of researchers, including Vincent Aucagne, Marcin Suskiewicz, and Hervé Meudal from the CBM in Orléans, led by Ivan Ahel and Dragana Ahel groups at the University of Oxford, have demonstrated that these two individual modifications can be joined together, producing a ‘double’ protein modification. The enzymes responsible for this process are DELTEX E3 ligases, which can efficiently attach ubiquitin to protein-linked ADP-ribose. A key contribution of Orléans scientists to the project was the analysis of the ubiquitin-ADP-ribose linkage performed using mass spectrometry (MS) and nuclear magnetic resonance (NMR) equipment of the new MOV2ING platform in Orléans.

The study shows that different protein modifications can be joined together to either combine two regulatory signals or produce a third, distinct signal, with a specific function. This shows previously unappreciated level of complexity in protein regulation.

While the role of ubiquitin-ADP-ribose in cells remains unclear, DELTEX enzymes have previously been linked to both development and antiviral response. The authors showed that the SARS-CoV-2 virus possesses enzymes that can remove the new modification, possibly allowing the virus to inhibit the host immune response.

References :
Kang Zhu, Marcin J. Suskiewicz, Hloušek-Kasun, Hervé Meudal, Andreja Mikoč, Vincent Aucagne, Dragana Ahel and Ivan Ahel
DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on protein substrates
Science Advances, 5 Oct 2022, Vol 8, Issue 40 DOI: 10.1126/sciadv.add4253

A versatile new approach to seek constitutive or conditional helicase substrates at global scale

Helicases are ubiquitous ‘molecular motor’ enzymes that disrupt nucleic acid (NA) helices and NA-protein interactions. Despite the key roles of helicases in many cellular processes and diseases, their target repertoires and the determinants of their functional specialization are often unknown. Scientists from the ‘RNA remodeling’ group of CBM have developed a new screening scheme, Helicase-SELEX, to elucidate helicase substrate requirements and find natural or synthetic helicase substrates in large NA sequence libraries. Using the transcription termination Rho helicase as prototype, the CBM scientists have discovered ~3300 functional substrate sequences in Escherichia coli, thereby providing the first detailed map of Rho utilization (Rut) sites at genome scale. Further, they have shown that inclusion of a Rho cofactor (NusG) in the selection scheme can modulate the H-SELEX outcome and help probe specificity determinants at global scale. Finally, they have used H-SELEX to evolve synthetic Rut sequences operating as riboswitches able to elicit Rho activity in vitro and in vivo only in presence of an orthogonal cofactor (serotonin). Thus, Helicase-SELEX is a versatile new approach to characterize or exploit helicases for fundamental or biotechnology purposes.

The CNRS Institute of Chemistry has reported this new original screening approach on its website

Référence

Delaleau M., Eveno E., Simon I., Schwartz A & Boudvillain M.
A scalable framework for the discovery of functional helicase substrates and helicase-driven regulatory switches
PNAS 2022

https://www.pnas.org/doi/10.1073/pnas.2209608119

In Cellulo and In Vivo Comparison of 3 helper lipids for Lipid Nanoparticle Formulation of mRNA

LNPs are a leading class of mRNA delivery systems. LNPs are made of an ionizable lipid, a polyethyleneglycol (PEG)-lipid conjugate and helper lipids. The success of LNPs is due to proprietary ionizable lipids and appropriate helper lipids.

Using a benchmark lipid (D-Lin-MC3) researchers compared the ability of three helper lipids to transfect dendritic cells in cellulo and in vivo. Studies revealed that the choice of helper lipid does not influence the transfection efficiency of immortalized cells but, LNPs prepared with DOPE (dioleylphosphatidylethanolamine) and β-sitosterol were more efficient for mRNA transfection in murine dendritic cells than LNPs containing DSPC (distearoylphosphatidylcholine).

This higher potency of DOPE and β-sitosterol LNPs for mRNA expression was also evident in vivo but only at low mRNA doses.

References of the article published in Nanomaterials:
Ayoub Medjmedj, Albert Ngalle-Loth, Rudy Clemnçon, Josef Hamacek, Chantal Pichon and Federico Perche
In Cellulo and In Vivo Comparison of Cholesterol, Beta-Sitosterol and Dioleylphosphatidylethanolamine for Lipid Nanoparticle Formulation of mRNA
Nanomaterials 2022, 12(14), 2446; https://doi.org/10.3390/nano12142446

A unique biosensor able to detect as low as 10 nM bioavailable copper based on whole-cell eukaryotic Saccharomyces cerevisiae

Cooper in a micronutrient essential to life, its lack induces neurological and blood disorders. It is extensively used in industry, in particular in the batteries of electric cars, but also as a fertilizer and fungicide. However, it is toxic at high concentrations, and is considered as a critical emerging pollutant. Copper detection in water constitutes a major societal and environmental issue.

Currently, copper concentrations are monitored by sophisticated analytical methods requiring time, expansive equipments and deep expertises. Moreover, these technics quantify total copper present in a sample and not copper interacting with living organisms.

Researchers of CBM developed a new and atypical device able to detect copper in a very sensitive and easy way. Their biosensor is based on whole-cell eukaryotic living Saccharomyces cerevisiae, allowing detection of bioavailable copper. It is a ratiometric biosensor, measuring the ratio between (i) a constitutively expressed fluorescent protein and (ii) another fluorescent protein whose expression is directly correlated with copper concentrations as its expression is in under the control of CUP1 promoter, a well-known promoter in Saccharomyces.

By genetic engineering, researchers created many different variants to optimize the response of our biosensor. Their best biosensor is able to detect as low as 10 nM of copper in a linear range from 10-3 to 10-8 M, much better features compared to other currently reported whole cell copper biosensors. This biosensor was also validated on “real” samples: detected concentrations are totally in agreement with manufacturers’ values.

Reference of the article :
Bojan Zunar, Christine Mosrin, Hélène Bénédetti, Béatrice Vallée
Re-engineering of CUP1 promoter and Cup2/Ace1 transactivator to convert Saccharomyces cerevisiae into a whole-cell eukaryotic biosensor capable of detecting 10 nM of bioavailable copper
Biosensors and Bioelectronics 214 (2022) 114502

The article was reported by the CNRS Institute of Chemistry on its website and in its letter "En direct des labos".

 

A new mechanism of antiobiotic resistance

The bacterial Rho factor is a molecular motor that induces genome-wide transcription termination. Rho is essential in many species, including in Mycobacterium tuberculosis where inactivation of the rho gene leads to rapid death. Nevertheless, the Rho factor of M. tuberculosis [MtbRho] displays idiosyncrasies, including resistance to the antibiotic bicyclomycin [BCM], which remain unexplained. To identify the molecular origin of these idiosyncrasies, we solved the structure of MtbRho by cryo-EM at 3.3 Å. This atomic structure notably reveals a leucine → methionine substitution that creates steric hindrance in the binding pockets of BCM, close to the ATPase sites, thereby conferring resistance to BCM at the expanse of molecular motor efficiency. Our work contributes to explain the unusual properties of MtbRho and provides groundwork for the development of new antibiotics.

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