Determination of the NADH dehydrogenase subunit 2 binding domain for Src40-49 and its attenuation of inflammatory pain

Abstract

Event Abstract Back to Event Determination of the NADH dehydrogenase subunit 2 binding domain for Src40-49 and its attenuation of inflammatory pain Rongwen Li1, 2*, Andrew Barszczyk1 and Michael Tymianski1, 2 1 Toronto Western Hospital Research Institute, Canada 2 NoNO Inc, Canada NADH dehydrogenase subunit 2 (ND2) is one of several proteins that interacts with the unique domain of the Src tyrosine kinase. Acting as an adapter protein, ND2 anchors Src to the N-methyl-d-aspartate (NMDA) receptor complex in post synaptic densities (PSDs), allowing it to regulate NMDA receptor activity. While the peptide sequence comprising the ND2 interaction domain of Src has been characterized (Src40-49) and has been used as a cell-permeant peptide to treat pain (Src40-49Tat), the Src interaction domain of ND2 has not yet been determined, though it is known to reside in an ND2 segment known as ND2.1. To identify the Src interacting domain of ND2, four Glutathione-S-transferase (GST) fusion proteins containing 4 overlapping sequence fragments of ND2.1 were constructed (ND2.1.1 through ND2.1.4). ND2.1.4–GST was observed to bind directly to the biotinylated domains of Src 40-49. Dot blot and competitive ELISA assays demonstrated that the interaction of Src 40-49Tat with ND2 or ND2.1 is blocked by ND2.1.4–GST. Next, eight peptides containing different segments of ND2.1.4 were synthesized and used in binding assays for Src 40-49Tat. One segment of ND2 (ND2.1.4-7) demonstrated a strong dose-response relationship on binding to Src 40-49-Tat. The interaction of ND2 with Src 40-49 was significantly reduced by pre-incubation with the ND2.1.4-7 peptide in competitive ELISA assays and dot blot assays. Additionally, endogenous colocalization of ND2 with Src was significantly reduced in primary cultures of hippocampal neurons treated with ND2.1.4-7. It was also determined that ND2.1.4-7, when affixed to the cell-permeating HIV1 Tat domain, alleviated the reduction in paw withdrawal threshold induced by the Complete Freund’s Adjuvant model of inflammatory pain. This suggests that inhibiting the interaction between Src and ND2 with the Src-interacting domain of ND2 can alleviate the NMDAR-mediated central sensitization that contributes to inflammatory and neuropathic pain. Conference: B.R.A.I.N. platform in Physiology poster day 2009, Toronto, ON, Canada, 16 Dec - 16 Dec, 2009. Presentation Type: Poster Presentation Topic: Poster presentations Citation: Li R, Barszczyk A and Tymianski M (2009). Determination of the NADH dehydrogenase subunit 2 binding domain for Src40-49 and its attenuation of inflammatory pain. Front. Neurosci. Conference Abstract: B.R.A.I.N. platform in Physiology poster day 2009. doi: 10.3389/conf.neuro.03.2009.17.029 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 17 Dec 2009; Published Online: 17 Dec 2009. * Correspondence: Rongwen Li, Toronto Western Hospital Research Institute, Toronto, Canada, rongwen_li@hotmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Rongwen Li Andrew Barszczyk Michael Tymianski Google Rongwen Li Andrew Barszczyk Michael Tymianski Google Scholar Rongwen Li Andrew Barszczyk Michael Tymianski PubMed Rongwen Li Andrew Barszczyk Michael Tymianski Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.