Abstract
Death-associated protein kinase 1 (DAPK1) is a pleiotropic hub of a number of networked distributed intracellular processes. Among them, DAPK1 is known to interact with the excitotoxicity driver NMDA receptor (NMDAR), and in sudden pathophysiological conditions of the brain, e.g., stroke, several lines of evidence link DAPK1 with the transduction of glutamate-induced events that determine neuronal fate. In turn, DAPK1 expression and activity are known to be affected by the redox status of the cell. To delineate specific and differential neuronal DAPK1 interactors in stroke-like conditions in vitro, we exposed primary cultures of rat cortical neurons to oxygen/glucose deprivation (OGD), a condition that increases reactive oxygen species (ROS) and lipid peroxides. OGD or control samples were co-immunoprecipitated separately, trypsin-digested, and proteins in the interactome identified by high-resolution LC-MS/MS. Data were processed and curated using bioinformatics tools. OGD increased total DAPK1 protein levels, cleavage into shorter isoforms, and dephosphorylation to render the active DAPK1 form. The DAPK1 interactome comprises some 600 proteins, mostly involving binding, catalytic and structural molecular functions. OGD up-regulated 190 and down-regulated 192 candidate DAPK1-interacting proteins. Some differentially up-regulated interactors related to NMDAR were validated by WB. In addition, a novel differential DAPK1 partner, LRRFIP1, was further confirmed by reverse Co-IP. Furthermore, LRRFIP1 levels were increased by pro-oxidant conditions such as ODG or the ferroptosis inducer erastin. The present study identifies novel partners of DAPK1, such as LRRFIP1, which are suitable as targets for neuroprotection.
Highlights
Modern biological research reveals that, in order to regulate essential cellular functions in a given condition, the cell sets its proteome into highly complex assemblies of multiproteins
Neurons exposed to oxygen/glucose deprivation (OGD) showed increased Death-associated protein kinase 1 (DAPK1) levels (Figure 1B), as assessed both by WB and ICC (Figure 1E), a 70% reduction of phosphorylated DAPK1 at Ser308 (Figure 1F), and a 22% reduction of the full length 160 kDa DAPK1 form that matched the increase in the truncated 100 kDa form (Figure 1D)
As one of the aims of the study was to find novel DAPK1 protein interactors differentially increased in the neuronal DAPK1 interactome after OGD, we focused on leucine-rich repeat of flightless I-interacting protein 1 (LRRFIP1), a.k.a
Summary
Modern biological research reveals that, in order to regulate essential cellular functions in a given condition, the cell sets its proteome into highly complex assemblies of multiproteins. In this regard, death-associated protein kinase 1 (DAPK1) is an emerging hub kinase involved in a number of cellular functions, both in physiological and pathophysiological conditions. While it has been associated to excitotoxic neuronal damage, its role in ischemia is still poorly studied. It has been recently reported that ischemia downregulates miR-98-5p, and that experimentally upregulated miR-98-5p in stroked mice inhibits ROS production, reduces infarction and suppresses DAPK1 signaling [3]
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