Effect of OPTN and OPTN phospho mutants on the innate immune response to Toll-like receptor and NOD Like receptor stimulation

Abstract

Inflammatory diseases represent a large section of conditions affecting the oral cavity. Many chronic diseases may last for life, with their causal factors deemed multifactorial or unknown. Optineurin (OPTN) is an adaptor protein with multiple roles in cellular function. One of its primary roles is related to the promotion of proinflammatory cytokines and chemokines expression and secretion. Gene mutations within OPTN have been identified to contribute to multiple inflammatory or autoimmune conditions. The main aim of this study is to investigate a link between OPTN and NOD2 activation with MDP in the form of a phosphorylation site in OPTN structure. Also, to test the ability of this phosphorylation site to impact NOD2 activation of the NFkB pathway through phosphoproteomics modification in OPTN structure. Finally, measure the significance of this link to inflammation and bacterial response in the form of cytokines release. During this study, I identified a novel phosphorylation site in OPTN that impacts the NFkB pathway by activating the bacterial receptor NOD2. THP-1, the human monocytic cell line, was genetically modified to express human OPTN tagged with EGFP on its N-terminus (THP- 1 OPTN). The THP-1 OPTN cell line was utilised to study protein localisation intracellularly, immunoprecipitation and phosphoproteomics. I identified a novel Serine 526 (Ser526) phosphorylation site induced after NOD2 stimulation with muramyl dipeptide (MDP). To determine the functional consequence of Ser526, phosphorylation site-directed mutagenesis was used to introduce an amino acid substitution at position 525 in the EGFP￾OPTN, resulting in phosphomimetic amino acids. Phospho-dead and phospho-active OPTN constructs were generated by switching Ser526 to Ala526 (S526A) and Glu526 (S526E), 5 respectively. In addition, stable THP-1 cell lines were produced (THP-1 S526A and THP-1 S526E), and the impact of these mutations on the immune response downstream of NOD2 and Toll￾like receptors (TLR) was investigated. THP-1 S526E cells demonstrated a significant elevation in the induction and secretion of proinflammatory cytokines upon NOD2 stimulation. THP-1 S526A ability to secrete cytokines was significantly impeded upon stimulation. The phosphorylation at Ser526 seemed to augment TLR signalling resulting in a significant boost in the induction and release of cytokines. These studies have identified a novel role for OPTN in the immune response downstream of NOD2. The phosphorylation of Ser526 upon NOD2 activation acts as an immune booster which can cooperate with other receptors, such as TLRs, upon bacterial challenge. Mutations in OPTN and NOD2 are associated with a host of human diseases, and the relevance of this pathway to these conditions warrants further investigation.