Complexes that Mitigate or Mediate Fibrosis: Structural and biophysical characterisation of the ERAD checkpoint and the gal-3-fibrosome

Chronic injury stimuli lead to fibrosis, the excessive deposition of extracellular matrix components to form scar tissue that impairs organ function. These stimuli can arise within the cell or extracellularly. Cellular stimuli can arise through protein misfolding within the endoplasmic reticulum (ER), predisposing to ER stress. Extracellular stimuli interact with membrane proteins at the cell surface to induce inflammatory and pro-fibrotic signalling. This thesis investigates the formation of two complexes that mitigate or mediate such processes. The ER-associated degradation (ERAD) checkpoint (PDI:EDEM heterodimer) clears misfolded proteins from the ER. The gal-3-fibrosome, a galectin-3-nucleated macromolecular assembly at the cell surface, may directly couple inflammation with fibrosis. I have determined the first structure of the PDI:EDEM heterodimer using cryoelectron microscopy (Cryo-EM), to 2.7 ˚Aresolution. The structure uncovers key functional features, including details of the active site, flexibility in peripheral domains, and inter-molecular redox chemistry. In further structural, biochemical, and biophysical studies I focused on degradation of classic ERAD substrates, misfolded variants of alpha-1 antitrypsin. I studied gal-3-fibrosome formation by Fluorophore Localisation Imaging with Photobleaching (FLImP), a novel super-resolution (low nanometre) microscopy method. FLImP and other data from studies in and ex cellula and in cell-free conditions strongly support radial pentamerisation of galectin-3, and formation of higher-order lattices, incorporating pro-inflammatory and pro-fibrotic binding partners at the cell membrane. With high throughput encapsulation reagent screens I optimised the purification of gal-3-fibrosome membrane protein sub-complexes, with subsequent in vitro reconstitution of galectin-3:membrane protein complexes. Preliminary Cryo-EM studies with the TGFBR2 homodimer and CD98 heterodimer:galectin-3 complex yielded lowresolution 3D reconstructions. My findings advance our understanding of fundamental homeostatic and pathological processes at the molecular and mechanistic levels and will inform novel therapeutic approaches. They have direct relevance to alleviating secretory protein misfolding diseases such as alpha-1 antitrypsin deficiency, and progressive fibrotic lung diseases such as idiopathic pulmonary fibrosis (IPF).