Biochemical approach to understand Huntington's disease and poly-glutamine-related pathologies

Huntington's Disease (HD) is a hereditary neurodegenerative disorder that is characterized by involuntary movements, general motor impairment, psychiatric disorders and dementia. The molecular basis of HD has been the subject of studies since the early 1990s, when the causal gene and its tri-nucleotide expansion were discovered. In the resulting gene transcript, the huntingtin protein (htt), the mutation leads to poly glutamine (poly Q) expansions at the N-terminus, and poly Q tracts with more than 35 repetitions are pathogenic.

Even though htt has been the focus of many studies, its high-resolution characterization has been impaired because htt is aggregation prone. Furthermore, the N-terminal part harboring the mutation does not adopt a globular structure, prohibiting X-ray crystallization studies and the poly Q repetition is poorly resolved in NMR spectra, precluding data analysis.

To overcome the current limitations of poly Q analysis, we combined nonsense suppression and cell-free expression to site-specifically label a single glutamine in htt. We then performed NMR experiments to obtain well-resolved spectra that allowed us to precisely measure backbone and side-chain chemical shifts and enabled the derivation of htt secondary structure elements. Our analysis of htt indicates that the 17 N-terminal residues have a strong propensity for α-helical structure that increases when approaching the poly Q tract. This propensity then decreases along the poly Q tract and the following residues, including two poly P tracts, show a greater tendency towards extended structure, which is most likely due to the enrichment in poly-proline-II conformations.

With this methodology we can now obtain other NMR observables such as residual dipolar couplings, relaxation rates and paramagnetic relaxation enhancements that can be used to decipher local conformational propensities, structural cooperativity and intra-molecular interactions in poly Q regions. This paves the way to understand the structural bases of the pathological htt species and the putative presence of a subpopulation of toxic conformers that remained speculative up to this point.

A. Urbanek, A. Morato, F. Allemand, E. Delaforge, A. Fournet, M. Popovic, S. Delbecq, N. Sibille, P. Bernado, Angew. Chem. Int. Ed. Engl. 2018, DOI 10.1002/anie.201711530.

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Teaching bacteria to recognize new molecules

Microbes can be engineered for different applications in our daily life, such as microbiome therapeutics, diagnosis of disease, or environmental monitoring. However, these applications are all based on the abilities of microbes to recognize and respond to novel signals such as disease biomarkers or environmental pollutants.

Here the members of the synthetic biology group at the CBS have developed a general framework to build synthetic receptors for bacteria to respond to novel ligands. The synthetic receptors are composed of: 1) The VHH camelid antibody which has shown the potential to recognize arbitrary targets; and 2) bacterial transcription factors which are responsible for controlling bacteria behavior. Once the synthetic receptors are activated by ligand-induced receptor dimerization, they can control bacteria response by changing gene expression. We provide a method to optimize receptor behavior by finely tuning protein expression levels and optimizing inter-domains linker regions. Finally, we show that these receptors can be connected to downstream synthetic gene circuits for further signal processing. The general nature of this platform and the versatility of antibody-based detection should support the deployment of these receptors into various hosts to detect ligands for which no receptor is found in nature.

For more details, please see our latest publication in ACS synthetic biology:
"A Modular Receptor Platform To Expand the Sensing Repertoire of Bacteria".

25th anniversary of the CBS

The CBS organizes a special event to celebrate its twenty-fifth anniversary on May 24 and 25, 2018. The research that has been conducted there for 25 years will be presented through presentations by internationally renowned scientists, collaborators and former members of the CBS, as well as by flash intervention of PhD students and post-doctorants. During the first half-day a tribute will also be given to the memory of Michel Kochoyan, former Director of the CBS.

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Une nouvelle méthode ouvre la voie à l’étude des protéines à séquence répétée

Certaines pathologies – c'est notamment le cas de la maladie de Huntington - sont causées par des protéines présentant un nombre anormalement élevé de répétitions d'un même acide aminéacide aminé. La structure protéique adoptée au niveau de ces répétitions est désordonnée et non accessible aux outils d'analyse actuels. Mais à Montpellier, l'équipe de Pau Bernadó vient de mettre au point une nouvelle méthodologie permettant de s'affranchir de cette limite. Ce travail ouvre la voie à une meilleure compréhension de la maladie de Huntington, mais également à l'étude d'autres maladies héréditaires associées à ce type de phénomène.

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A new mecanism for transcription initiation in M. Tuberculosis

In bacteria, the sigma subunit of RNA polymerase controls promoter recognition and initiation of RNA synthesis. Using Förster energy transfer between single molecules (smFRET), we have demonstrated that Mycobacterium tuberculosis, the causative agent of tuberculosis, uses a yet unknown transcriptional activation mechanism in which the RNA polymerase retains the sigma subunit in inactive « closed » conformation unless it is stabilized in the "open" active conformation by the activator protein RbpA.

This collaborative work between the groups of Emmanuel Margeat (CBS) and Konstantin Brodolin (IRIM, Montpellier) was published on May 23, 2018 in Science Advances