Investigations into the interactions of the high mobility group box 1 protein and their toxicological relevance



Aucott, Hannah
Investigations into the interactions of the high mobility group box 1 protein and their toxicological relevance. Doctor of Philosophy thesis, University of Liverpool.

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Abstract

Drug Induced Liver Injury (DILI) is associated with high morbidity and mortality rates. It is the leading cause of acute liver failure, accounting for 50% of all cases. Moreover, DILI is the most frequent cause of post marketing drug withdrawal and is often cited as a cause of compound attrition during the drug-development process. The High Mobility Group Box 1 (HMGB1) protein is an important inflammatory mediator which alters the immune system to tissue stress and injury. HMGB1 has been implicated in the pathogenesis of multiple inflammatory diseases including immune-mediated DILI. It has been identified as a potential biomarker of hepatic injury and a target for therapeutic intervention. Research is required to elucidate the pro-inflammatory role of HMGB1. HMGB1 has been reported to interact with a diverse range of endogenous (IL-1, DNA, nucleosomes, CXCL12) and exogenous (LPS) molecules to promote inflammation. However, the mechanisms responsible for these synergistic interactions remain poorly defined and therefore, the overall aim of this work was to characterise the interactions of HMGB1. Specifically, this work has focused on the interaction with IL-1β, which is of particular interest since both molecules often co-exist at the site of inflammation. The interaction between HMGB1 and IL-1β was investigated using combined cellular and Nuclear Magnetic Resonance (NMR) methodologies. LPS-free, isotopically labelled recombinant HMGB1 (full length protein, amino acids 1-215) and IL-1β proteins were expressed and purified from BL21 (DE3) cells. To facilitate these studies, three additional HMGB1 mutants were sub-cloned from the HMGB1 plasmid: Δ30 (1-185), A box (1-85) and B box (89-163). The recombinant proteins were characterised using Mass Spectrometry (MS) and NMR spectroscopy. Synovial fibroblasts were isolated from synovial tissue obtained from rheumatoid arthritis patients undergoing joint replacement surgery. Cells were treated with HMGB1 (full length, Δ30, A box or B box) alone or combination with IL-1β. Cell supernatants were collected after 24hr and IL-6 levels were quantified by ELISA. Untreated fibroblasts or cells treated with any HMGB1 construct, or IL-1β alone had no detectable IL-6 release (

Item Type: Thesis (Doctor of Philosophy)
Additional Information: Date: 2014-01 (completed)
Subjects: ?? RM ??
Divisions: Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 05 Aug 2014 10:00
Last Modified: 16 Dec 2022 04:41
DOI: 10.17638/00016877
Supervisors:
URI: https://livrepository.liverpool.ac.uk/id/eprint/16877