AMPK,
AMPK, "AMP-activated protein kinase"
Science and technology
Article
An international team of scientists, led by Uwe Schlattner at the University Grenoble Alpes & Inserm, and Imre Berger at the University of Bristol UK has now succeeded to engineer AMPfret, the first nano-sensor capable of measuring energy states in cells in vivo.

The energy flow through cells is at the core of all physiological processes in health and disease. Energy in cells is stored in the form of molecules called adenylates. Transformation of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and finally adenosine monophosphate (AMP) fuels metabolism and mechanical work. While the levels of ATP do not fluctuate a lot under healthy conditions, those of ADP and AMP do so much more. Thus, only ATP/ADP and ATP/AMP ratios appear to faithfully represent the true energy state of a cell.  

Because of the key role of energy flux for all life processes, a tool that monitors such cellular energy states directly within cells would be highly desirable. Much effort has been spent to develop such sensors, but success has been limited. To track cellular energy levels, a sensor would need to gauge ATP, ADP and AMP concentrations at physiological levels, this however remained unfeasible to date.

An international team of scientists, led by Uwe Schlattner at the University Grenoble Alpes & Inserm, and Imre Berger at the University of Bristol UK has now succeeded to engineer AMPfret, the first nano-sensor capable of measuring energy states in cells in vivo. For their design they exploited the unique properties of a protein complex, the AMP-activated protein kinase (AMPK).

"When we first succeeded to produce functional AMPK some time ago, we soon discovered that AMPK shape is considerably altered when it binds AMP instead of ATP (1)" recalls Uwe Schlattner, director of the Laboratory of Fundamental and Applied Bioenergetics (LBFA) in Grenoble. "With our 15 years of experience working on AMPK, we asked if we could translate this shape-shifting capacity into a detectable signal. We decided to use an effect called Förster Resonance Energy Transfer (FRET) which occurs between two fluorophores dependent on their distance." This raised the question: how to place and orient the fluorophores on the AMPK structure to obtain a maximal signal?

To tackle this challenge, the Schlattner and Berger teams joined forces. By outfitting AMPK at the appropriate places with a specific pair of fluorophores, they generated a sensitive nano-device emitting light signals they could measure, now published in Nature Communications (2). "The fluorophores have to be aligned and closely spaced to produce FRET" says Imre Berger, director of Bristol’s Synthetic Biology Center (BrisSynBio). "We iteratively truncated, modified and fluorophore-tagged the subunits of AMPK to get the geometry right."

"We were thrilled to see our sensor works" concluded Martin Pelosse, first author of the study. "AMPfret enabled us to detect, for the first time, energy states in vivo in cells by using a genetically encoded nano-device. Moreover, by analysing AMPfret in detail in vitro, we could decipher long elusive molecular mechanisms of the AMPK enzyme itself as well.

AMP-activated protein kinase (AMPK)
AMP-activated protein kinase (AMPK), a protein complex that changes shape upon AMP binding, has been reengineered into a fluorescent nano-sensor faithfully detecting physiological AMP levels in vitro and in vivo

Notes:

(1) Riek, U., Scholz, R., Konarev, P. Rufer, A., Suter, M., Nazabal, A., Ringler, P., Chami, M., Müller, S.A., Neumann, D., Forstner, M. Hennig, M., Zenobi, R., Engel, A. Svergun, D., Schlattner, U., and Wallimann, T. (2008) Structural properties of AMP-activated protein kinase (AMPK) : dimerization, molecular shape, and changes upon ligand binding. J. Biol. Chem. 283, 18331-18343.

(2) Pelosse, M., Cottet-Rousselle, C., Bidan, C., Dupont, A., Gupta, K., Berger, I., and Schlattner, U. (2019) Synthetic energy sensor AMPfret deciphers adenylate-dependent AMPK activation mechanism. Nat. Commun.

Publié le March 4, 2019
Mis à jour le March 5, 2019

Vous aimerez peut-être aussi