Intervention of Death-Associated Protein Kinase 1–p53 Interaction Exerts the Therapeutic Effects Against Stroke

Abstract

D eath-associated protein kinase 1 (DAPK1) is a Ca 2+ / calmodulin-dependent kinase 1 and plays roles in several modes of cell death in ischemic stroke. 2,3 To define a promising target that allows a delayed intervention being clinically effective against stroke, we searched for cell death signals downstream of DAPK1. We identified a direct binding of DAPK1 to a DNA-binding motif (DM) of the tumor suppressor protein p53 (p53DM), which is known as a transcriptional regulator of genes that control cell necrosis and apoptosis. [4][5][6][7] We thus synthesized a membrane-permeable p53DM peptide consisting of amino acid 271 to 282 (Tat-p53DM) that specifically blocked DAPK1-p53 interaction.3 Activation of DAPK1 phosphorylates p53 at serine-23 (pS 23 ) in the cultured cortical neurons. 3 In the nucleus, the pS 23 induces expression of the proapoptotic genes such as Bax, whereas in the mitochondrial matrix, the pS 23 interacts with a necrotic factor of cyclophilin D. Application of Tat-p53DM effectively blocks these dual pathways of the pS 23 actions in the cortical cultures in vitro, 3 suggesting that Tat-p53DM could be clinically effective against stroke. To test this possibility, here we validated the therapeutic effects of Tat-p53DM in vivo in a mouse model with stroke. Methods Focal Cerebral IschemiaFocal cerebral ischemia was induced by intraluminal middle cerebral artery occlusion (MCAO), as we described previously. 2 Briefly, a 7/0 surgical nylon monofilament with rounded tip was introduced into the left internal carotid through the external carotid stump and advanced 10 to 13 mm past the carotid bifurcation. The filament was left in place for 60 minutes and then withdrawn. Sham-operated animals were treated identically, except that the middle cerebral artery was not occluded after the neck incision. PeptidesTat-p53DM (Tat-RVCACPGRDRRT) or Tat-s-p53DM (Tat-CCPGECVRTRRR) peptides with 99% purity were synthesized by Bioyeargene Biosciences (Wuhan, China). The peptides were numbered, and the experimenters were unaware of which one was applied in all experiments. Peptides Dose-Effect ExperimentsSixty mice underwent a 60-minute MCAO and were injected (intravenously) with vehicle (control) or with Tat-p53DM or Tat-s-p53DM at a single dose of 0.1, 0.5, 1, or 2 mg/kg body weight. And 1 mg/kg was identified as the optimal dose. Twelve hours later, all mice were euthanized and the cortical extracts were prepared. The proteins were Background and Purpose-Death-associated protein kinase 1 (DAPK1) interacts with the tumor suppressor gene p53 via a direct binding of a death domain of DAPK1 to a DNA-binding motif (DM) of p53 (p53DM) and converges multiple cell death pathways in stroke. The goals of this study are to determine whether disruption of DAPK1-p53 interaction is therapeutically effective against stroke. Methods-We synthesized a membrane-permeable p53DM peptide (Tat-p53DM) and tested the therapeutic effects of Tatp53DM in a mouse model with stroke. Results-We showed that Tat-p53DM blocked DAPK1-p53 interaction in bra...

Neuroprotective peptides fused to arginine-rich cell penetrating peptides: Neuroprotective mechanism likely mediated by peptide endocytic properties

“…The DAPK1 protein has been shown to interact with extra-synaptic NMDA receptors containing the NR2B subunit, a process that is thought to induce and/or exacerbate injurious calcium influx (Tu et al, 2010). In addition, activated DAPK1 is associated with other signalling pathways linked to ischaemic cell death Wang et al, 2014). TAT-NR2Bct competitively inhibits activated DAPK1 binding to the NR2B subunit protein and thereby blocks subsequent downstream damaging cellular events caused by NMDA receptor over-activation.…”

A Novel Mechanism of Spine Damages in Stroke via DAPK1 and Tau

“…Our previous work demonstrated that following stroke, DAPK1 in the postsynaptic domain phosphorylated NMDA receptor NR2B at Serine-1303, eventually leading to neuronal death ( Tu et al 2010 ). Recently, we found that DAPK1 could also phosphorylate p53 at Serine-23 via direct binding of the DAPK1 death domain (DD) to the DNA-binding motif of p53 (p53DM) ( Pei et al 2014 ; Wang et al 2014 ). Though a handful of DAPK1 substrates have been identified, downstream signaling of DAPK1 that specifically mediates early spine damage after ischemic injuries is yet to be characterized.…”

[P2–202]: Dapk1 Signaling Pathways in Stroke: From Mechanisms to Therapies

“…Using yeast two-hybrid analysis and GST affinity binding assay, the authors confirmed that DAPK1DD is bound to a p53 DNA-binding motif consisting of amino acids 241-281 (p53DM ) [48]. The application of a synthesized membrane-permeable p53DM 241-281 peptide (Tat-p53DM) that interrupts DAPK1-p53 interaction blocks these dual pathways of pS23 actions in vitro and in vivo [54]. Thus, the DAPK1-p53 interaction is a signaling point of convergence of necrotic and apoptotic pathways and is a desirable target for the treatment of ischemic insults.…”