A Prolyl-Isomerase Mediates Dopamine-Dependent Plasticity and Cocaine Motor Sensitization

SummarySynaptic plasticity induced by cocaine and other drugs underlies addiction. Here we elucidate molecular events at synapses that cause this plasticity and the resulting behavioral response to cocaine in mice. In response to D1-dopamine-receptor signaling that is induced by drug administration, the glutamate-receptor protein metabotropic glutamate receptor 5 (mGluR5) is phosphorylated by microtubule-associated protein kinase (MAPK), which we show potentiates Pin1-mediated prolyl-isomerization of mGluR5 in instances where the product of an activity-dependent gene, Homer1a, is present to enable Pin1-mGluR5 interaction. These biochemical events potentiate N-methyl-D-aspartate receptor (NMDAR)-mediated currents that underlie synaptic plasticity and cocaine-evoked motor sensitization as tested in mice with relevant mutations. The findings elucidate how a coincidence of signals from the nucleus and the synapse can render mGluR5 accessible to activation with consequences for drug-induced dopamine responses and point to depotentiation at corticostriatal synapses as a possible therapeutic target for treating addiction

Stereospecific Phosphorylation by the Central Mitotic Kinase Cdk1-Cyclin B

The <i>cis</i> vs <i>trans</i> conformation, or shape, of phosphoserine-proline (pSer-Pro), a prevalent motif in cell cycle proteins, may play a significant role in regulating mitosis. We demonstrate that Cdk1-cyclin B, the central mitotic kinase, is specific for the <i>trans</i> conformation, not <i>cis</i>, of synthetic, locked Ser-Pro 11-residue peptide substrates, using LC-MSMS detection and sequencing of phosphorylated products. This substrate stereospecificity may contribute an additional level of mitotic regulation

Cyclohexyl ketone inhibitors of Pin1 dock in a trans-diaxial cyclohexane conformation.

Cyclohexyl ketone substrate analogue inhibitors (Ac-pSer-Ψ[C = OCH]-Pip-tryptamine) of Pin1, the cell cycle regulatory peptidyl-prolyl isomerase (PPIase), were designed and synthesized as potential electrophilic acceptors for the Pin1 active site Cys113 nucleophile to test a proposed nucleophilic addition-isomerization mechanism. Because they were weak inhibitors, models of all three stereoisomers were docked into the active site of Pin1. Each isomer consistently minimized to a trans-diaxial cyclohexane conformation. From this, we hypothesize that Pin1 stretches substrates into a trans-pyrrolidine conformation to lower the barrier to isomerization. Our reduced amide inhibitor of Pin1 adopted a similar trans-pyrrolidine conformation in the crystal structure. The molecular model of 1, which mimics the l-Ser-l-Pro stereochemistry, in the Pin1 active site showed a distance of 4.4 Å, and an angle of 31° between Cys113-S and the ketone carbon. The computational models suggest that the mechanism of Pin1 PPIase is not likely to proceed through nucleophilic addition

Pin1 is proposed to stretch the prolyl ring by binding phosphate and C-terminal residues tightly, creating a <i>trans</i>-pyrrolidine conformation of the substrate and forcing pyramidalization of the prolyl nitrogen in the twisted-amide mechanism.

<p>Distance measurements are from calculated structures of Ac–Pro–OH in the ground state and the <i>trans</i>-pyrrolidine transition state.</p

X-ray crystal structures of intermediates (1<i>S</i>,3<i>R</i>,4<i>R</i>)-11 and <i>rac</i>-11 are shown above as displacement ellipsoid drawings (50%).

<p>The positional disorder of the benzyl group in <b><i>rac</i></b><b>-11</b> is shown as lighter lines. Hydrogen atoms are omitted for clarity. Structural depiction of the stereochemistries of <b>(1</b><b><i>S</i></b><b>,3</b><b><i>R</i></b><b>,4</b><b><i>R</i></b><b>)-11</b> and <b><i>rac</i></b><b>-11</b> are shown below each crystal structure.</p

Models of cyclohexyl ketone inhibitors were docked with dynamic minimization.

<p>(A) <b>(1</b><b><i>S,</i></b><b>3</b><b><i>R,</i></b><b>4</b><b><i>R</i></b><b>)-1</b> in orange, (B) <b>(1</b><b><i>R,</i></b><b>3</b><b><i>R,</i></b><b>4</b><b><i>R</i></b><b>)-2</b> in blue, (C) <b>(1</b><b><i>S,</i></b><b>3</b><b><i>S,</i></b><b>4</b><b><i>S</i></b><b>)-2</b> in green, and (D) superposition of all atoms of <b>1</b> and <b><i>rac</i></b><b>-2</b>. Models were based on PDB 2Q5A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044226#pone.0044226-Zhang2" target="_blank">[32]</a>, and minimized using Sybyl 8.1.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044226#pone.0044226-1" target="_blank">[42]</a>. Images were prepared using MacPyMol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044226#pone.0044226-DeLano1" target="_blank">[44]</a>.</p

Ketone inhibitors were designed to mimic the tetrahedral intermediate of proposed mechanism B.

<p>(A) Proposed Pin1 hydrogen-bond assisted twisted amide mechanism <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044226#pone.0044226-Schroeder1" target="_blank">[25]</a>, (B) Pin1 Cys113 nucleophilic-addition mechanism tetrahedral intermediate proposed by Ranganathan et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044226#pone.0044226-Ranganathan1" target="_blank">[26]</a>. (C) Electrophilic ketone inhibitor designed to mimic the proposed tetrahedral intermediate upon Cys113-S nucleophilic addition.</p