TY - JOUR

T1 - Charting the interactome of PDE3A in human cells using an IBMX based chemical proteomics approach

AU - Corradini, Eleonora

AU - Klaasse, Gruson

AU - Leurs, Ulrike

AU - Heck, Albert J R

AU - Martin, Nathaniel I

AU - Scholten, Arjen

PY - 2015/10

Y1 - 2015/10

N2 - In the cell the second messenger cyclic nucleotides cAMP and cGMP mediate a wide variety of external signals. Both signaling molecules are degraded by the superfamily of phosphodiesterases (PDEs) consisting of more than 50 different isoforms. Several of these PDEs are implicated in disease processes inspiring the quest for and synthesis of selective PDE inhibitors, that unfortunately have led to very mixed successes in clinical trials. This may be partially caused by their pharmacological action. Accumulating data suggests that small differences between different PDE isoforms may already result in specific tissue distributions, cellular localization and different involvement in higher order signal protein complexes. The role of PDEs in these higher order signal protein complexes has only been marginally addressed, as no screening methodology is available to address this in a more comprehensive way. Affinity based chemical proteomics is a relatively new tool to identify specific protein-protein interactions. Here, to study the interactome of PDEs, we synthesized a broad spectrum PDE-capturing resin based on the non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Chemical proteomics characterization of this resin in HeLa cell lysates led to the capture of several different PDEs. Combining the IBMX-resin with in-solution competition with the available more selective PDE inhibitors, cilostamide and papaverine, allowed us to selectively probe the interactome of PDE3A in HeLa cells. Besides known interactors such as the family of 14-3-3 proteins, PDE3A was found to associate with a PP2A complex composed of a regulatory, scaffold and catalytic subunit.

AB - In the cell the second messenger cyclic nucleotides cAMP and cGMP mediate a wide variety of external signals. Both signaling molecules are degraded by the superfamily of phosphodiesterases (PDEs) consisting of more than 50 different isoforms. Several of these PDEs are implicated in disease processes inspiring the quest for and synthesis of selective PDE inhibitors, that unfortunately have led to very mixed successes in clinical trials. This may be partially caused by their pharmacological action. Accumulating data suggests that small differences between different PDE isoforms may already result in specific tissue distributions, cellular localization and different involvement in higher order signal protein complexes. The role of PDEs in these higher order signal protein complexes has only been marginally addressed, as no screening methodology is available to address this in a more comprehensive way. Affinity based chemical proteomics is a relatively new tool to identify specific protein-protein interactions. Here, to study the interactome of PDEs, we synthesized a broad spectrum PDE-capturing resin based on the non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Chemical proteomics characterization of this resin in HeLa cell lysates led to the capture of several different PDEs. Combining the IBMX-resin with in-solution competition with the available more selective PDE inhibitors, cilostamide and papaverine, allowed us to selectively probe the interactome of PDE3A in HeLa cells. Besides known interactors such as the family of 14-3-3 proteins, PDE3A was found to associate with a PP2A complex composed of a regulatory, scaffold and catalytic subunit.

KW - 1-Methyl-3-isobutylxanthine

KW - 14-3-3 Proteins

KW - Cyclic Nucleotide Phosphodiesterases, Type 3

KW - HeLa Cells

KW - Humans

KW - Papaverine

KW - Phosphodiesterase Inhibitors

KW - Protein Binding

KW - Protein Interaction Maps

KW - Protein Phosphatase 2

KW - Proteomics

KW - Quinolones

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1039/c5mb00142k

DO - 10.1039/c5mb00142k

M3 - Journal article

C2 - 26205238

VL - 11

SP - 2786

EP - 2797

JO - Molecular BioSystems

JF - Molecular BioSystems

SN - 1742-206X

IS - 10

ER -