Characterisation of the molecular mechanics underpinning idiopathic pulmonary fibrosis (IPF) using quantitative protemics

Abstract

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease. Pathogenesis involves the aberrant accumulation of myofibroblasts and excessive deposition of matrix components. Its aetiology is unknown and treatment options are limited. The histone deacetylase inhibitor FK228 has been shown to have anti-proliferative effects on fibroblasts, and therefore may be a candidate for IPF therapy.

This thesis describes the in-depth proteomic characterisation of fibroblasts from patients with IPF, and their response to FK228. Comparison of protein expression between IPF and non-IPF fibroblasts revealed over 120 aberrantly expressed proteins in this disease, including those involved in transcriptional regulation, which have the potential for use as diagnostic or monitoring biomarkers. Extensive validation is now required for their translation to the clinic. IPF biomarkers are greatly sought after as there are currently none in clinical use. FK228 treatment caused a decrease in IPF fibroblast metabolic activity and proliferation through cell cycle arrest, and significantly affected the expression of hundreds of proteins. The proteomic responses of IPF fibroblasts grown in 2D and 3D culture were shown to differ substantially; there was little difference in the number of proteins quantified between culture systems, but treatment affected the expression of different proteins. This highlights the importance of culture system selection for studying IPF and for testing therapeutics effectively in vitro.

The method for profiling cultured fibroblasts using proteomics was firstly developed in the MRC-5 cell line, to optimise cell lysis, protein isolation and digestion, and mass spectrometry analysis for global protein identification. Intracellular and extracellular proteomic analysis of these cells following optimisation, with and without transforming growth factor beta-1 (TGF-β₁) stimulation is also discussed in this thesis. This dataset provides to the author’s knowledge the most comprehensive characterisation of the fibroblastic response to this profibrotic cytokine to date, with more than 5000 proteins quantified. Data analyses highlighted biological processes that may be dysregulated in fibrotic disease, including cell cycle regulation and cellular organisation.

Finally, the first steps in the development of a method using immunoprecipitation enrichment to study the IPF fibroblast acetylome in response to FK228 are described, for the identification of off-target effects. Fewer than 100 acetylated peptides were identified by mass spectrometry analysis, a considerably lower number than found in previously published reports, highlighting the inefficiency of the technique for global enrichment of acetylated peptides in this study.

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Identifiers

ORCID for Donna Davies: orcid.org/0000-0002-5117-2991

ORCID for Paul Skipp: orcid.org/0000-0002-2995-2959

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