Cellular and structural studies of the role of CD98:integrin complexes in fibrosis.

(i)我们将通过免疫组织化学研究由A1AT（肝细胞）错误折叠突变体或SP-C（肺细胞）错误折叠突变体引起的纤维化细胞表面复合物的形成。为了探究细胞对糖蛋白错误折叠反应的依赖性，我们将使用WT细胞和编码内质网糖蛋白折叠检查点酶UGGT基因被删除的细胞；（ii）共免疫沉淀复合物的质谱法将用于鉴定其他相互作用的膜蛋白（图1C），包括鉴定-整合素与1-整合素配对；（iii）在不存在LPS（模拟纤维化“加剧”刺激）的情况下，纯化纤维化细胞表面复合物，并通过质谱法和生化方法对其组成和化学计量进行表征。这些复合物将由重组表达的组分重组并纯化；它们的结构将通过x射线晶体学（与安德鲁·奎格利博士，哈维尔）和低温电子显微镜（在莱斯特的米德兰兹低温电子显微镜设施）进行研究。预期结果：将阐明促纤维化复合物结构/功能的分子细节，并评估其对内质网糖蛋白折叠机制的依赖性。

(i) We shall investigate by immuno-histochemistry the formation of profibrotic cell surface complexes caused by misfolded mutants of A1AT (liver cells) or misfolded mutants of SP-C (lung cells). To probe the dependency of the cell's response to glycoprotein misfolding, we shall use WT cells and cells in which the gene encoding the ER glycoprotein folding checkpoint enzyme UGGT has been deleted;(ii) Mass spectrometry of co-immunoprecipitated complexes will be used to identify other interacting membrane proteins (Figure 1C) including the identity of the -integrin pairing with 1-integrin here;(iii) profibrotic cell surface complexes will be purified, in the absence and presence of LPS (modelling profibrotic 'exacerbation' stimulus) and their composition and stoichiometry characterised by mass-spectrometry and biochemical methods. The complexes will be reconstituted from recombinantly expressed components and purified; their structures will be studied by X-ray crystallography (with Dr Andrew Quigley, Harwell) and Cryo-EM (at the Midlands Cryo-EM Facility in Leicester).Expected outcomes: the molecular detail of the profibrotic complexes structure/function will be elucidated and their dependency on the ER glycoprotein folding machinery assessed.