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27 mai 2019: 1 événement

  • Séminaires Invités

    Lundi 27 mai 14:00-15:30 - Uwe Schlattner

    Imaging AMPK activation and cellular energy state with FRET sensors

    Résumé : AMP-activated protein kinase (AMPK) represents a key cell signaling hub that senses and regulates energy state. AMPK activation by increasing AMP and ADP concentrations involves a conformational switch within the heterotrimeric complex. This is exploited here for the construction of a synthetic sensor of cellular energetics and allosteric AMPK activation, AMPfret. Based on engineered AMPK fused to fluorescent proteins, the sensor allows direct, real-time readout of the AMPK conformational state by fluorescence resonance energy transfer (FRET). AMPfret faithfully and dynamically reports the binding of AMP and ADP to the AMPK γ-CBS sites, competed by Mg2+ free ATP. The FRET signal correlates with activation of AMPK by allosteric mechanisms and protection from dephosphorylation, attributed here to specific CBS sites, but does not require AMPK activation loop phosphorylation. Moreover, AMPfret is capable to report binding of pharmacological activators to both allosteric sites, CBS and α/β-ADaM, thus enabling activator screening. Cellular assays demonstrate that AMPfret is applicable in vivo for spatiotemporal analysis of energy state and allosteric AMPK activation.

    contact : Marco Canepari & Aurélie Dupont

    Lieu : LIPhy, conference room - 140 Avenue de la Physique 38402 Saint Martin d’Hères

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