Endoplasmic Reticulum Resident Chaperones and their Implications in Health and Disease

Abstract

The endoplasmic reticulum (ER) is a key organelle in the eukaryotic cell and is responsible for protein folding and quality control, mediated by protein chaperones. It is also home to the oxidoreductase system which is required for disulphide bond formation and is the main theme of this thesis. Two of the vital proteins in the ER-localised oxidoreductase system are protein disulphide isomerase (PDI) and endoplasmic reticulum oxidoreductase 1 α (Ero1α). These proteins work alongside other specialist PDI family members such as anterior gradient 2 (AGR2). This thesis examines the role of both PDI and AGR2 in protein quality control, together with important clients such as collagen, a major component of the extracellular matrix (ECM) and a secreted product of fibroblast cells present in the skin. The role of AGR2 in oesophageal cancer was explored, with data dependant acquisition (DDA) mass spectrometry (MS) used to identify novel interacting partners of AGR2. AGR2 was found to be over expressed in certain oesophageal cell lines and key interacting partners such as MUC5AC and SQSTM1 were identified. Research was undertaken in this thesis to compare the effectiveness and application of the reducing agent dithiobutylamine (DTBA) to biological systems and how this reducing agent affects the cellular proteome and the redox state of PDI. Live cell imaging was used to see how the structure of the ER was affected by reductive stress. Data independent acquisition (DIA) MS was used to identify new molecular players in the cellular response to reductive stress, in the response to PDI inhibitors, in the response to growth factors (PDGF) and in the response to skin bioactive (e.g. niacinamide). Taken together, these analyses lay the foundations for understanding new mechanistic control points in the quality control of protein secretion and homeostasis.