Engineering of a red-light-activated human cAMP/cGMP-specific phosphodiesterase

Proc Natl Acad Sci U S A. 2014 Jun 17;111(24):8803-8. doi: 10.1073/pnas.1321600111. Epub 2014 Jun 2.

Abstract

Sensory photoreceptors elicit vital physiological adaptations in response to incident light. As light-regulated actuators, photoreceptors underpin optogenetics, which denotes the noninvasive, reversible, and spatiotemporally precise perturbation by light of living cells and organisms. Of particular versatility, naturally occurring photoactivated adenylate cyclases promote the synthesis of the second messenger cAMP under blue light. Here, we have engineered a light-activated phosphodiesterase (LAPD) with complementary light sensitivity and catalytic activity by recombining the photosensor module of Deinococcus radiodurans bacterial phytochrome with the effector module of Homo sapiens phosphodiesterase 2A. Upon red-light absorption, LAPD up-regulates hydrolysis of cAMP and cGMP by up to sixfold, whereas far-red light can be used to down-regulate activity. LAPD also mediates light-activated cAMP and cGMP hydrolysis in eukaryotic cell cultures and in zebrafish embryos; crucially, the biliverdin chromophore of LAPD is available endogenously and does not need to be provided exogenously. LAPD thus establishes a new optogenetic modality that permits light control over diverse cAMP/cGMP-mediated physiological processes. Because red light penetrates tissue more deeply than light of shorter wavelengths, LAPD appears particularly attractive for studies in living organisms.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allosteric Site
  • Animals
  • CHO Cells
  • Cell Line
  • Cricetinae
  • Cricetulus
  • Cyclic AMP / chemistry*
  • Cyclic GMP / chemistry*
  • Cyclic Nucleotide Phosphodiesterases, Type 2 / genetics*
  • Escherichia coli / metabolism
  • Genes, Reporter
  • Humans
  • Hydrolysis
  • Kinetics
  • Light
  • Models, Molecular
  • Phosphoric Diester Hydrolases / chemistry*
  • Protein Engineering*
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / chemistry
  • Synechocystis / metabolism
  • Temperature
  • Zebrafish

Substances

  • Recombinant Fusion Proteins
  • Cyclic AMP
  • Phosphoric Diester Hydrolases
  • Cyclic Nucleotide Phosphodiesterases, Type 2
  • PDE2A protein, human
  • Cyclic GMP