Skip to Content
Merck
  • Comparative Proteomics Unveils LRRFIP1 as a New Player in the DAPK1 Interactome of Neurons Exposed to Oxygen and Glucose Deprivation.

Comparative Proteomics Unveils LRRFIP1 as a New Player in the DAPK1 Interactome of Neurons Exposed to Oxygen and Glucose Deprivation.

Antioxidants (Basel, Switzerland) (2020-12-04)
Núria DeGregorio-Rocasolano, Verónica Guirao, Jovita Ponce, Marc Melià-Sorolla, Alicia Aliena-Valero, Alexia García-Serran, Juan B Salom, Antoni Dávalos, Octavi Martí-Sistac, Teresa Gasull
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-Actin antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Anti-DAP Kinase 1 antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Donkey Anti-Mouse IgG Antibody, Cy3 conjugate, Species Adsorbed, Chemicon®, from donkey
Sigma-Aldrich
Anti-NMDAR2B Antibody, NT, ascites fluid, clone 1A8.9B3, Chemicon®
Sigma-Aldrich
Monoclonal Anti-Death Associated Protein Kinase antibody produced in mouse, clone DAPK-55, ascites fluid
Sigma-Aldrich
Anti-α-Tubulin antibody, Mouse monoclonal, clone B-5-1-2, purified from hybridoma cell culture
Sigma-Aldrich
Anti-phospho-DAP-Kinase (pSer308) antibody, Mouse monoclonal, clone DKPS308, purified from hybridoma cell culture