Autophagy and Crohn's disease: translating genetics to function

I aim to investigate the molecular biology of Crohn‘s disease (CD), a chronic debilitating inflammatory bowel condition affecting 100 - 250 per 100,000 people in the UK. Although current understanding suggests that an abnormal response to bacteria living in the gut leads to Crohn‘s inflammation, the mechanisms are not understood - yet understanding this mechanism might permit development of improved treatments or even preventative measures. Recent groundbreaking studies have highlighted two genes involved in autophagy as being significantly associated with CD risk. I will study one of these genes, ATG16L1. Autophagy is a cellular process that degrades unwanted large proteins and other cell structures, and is also implicated in clearance of invasive bacteria. We hypothesise that autophagy is abnormal in CD, leading to defective clearance of bowel pathogens and therefore inflammation. A series of experiments are proposed to test this hypothesis. Using established cell lines from animals and humans, and bowel samples from patients with CD, I will study the effects of the CD-associated variant in ATG16L1 on autophagy, characterize cellular components with which Atg16L1 protein interacts and investigate whether autophagy is generally abnormal in CD even without the currently identified genetic variants.

Within the last year genetic studies have identified robust association between a variant (T300A) in the autophagy gene ATG16L1, and susceptibility to Crohn‘s disease (CD). Working in Professor Rubinsztein‘s laboratory at the CIMR I aim to investigate the mechanism by which the disease-associated variant predisposes to CD, and whether autophagy is dysregulated in CD. The experiments proposed will address three key questions:
1. Does the ATG16L1 T300A variant affect mammalian autophagy?
2. Is autophagy generally perturbed in Crohn‘s disease?
3. What proteins interact with Atg16?
Experiments in cell lines include:
-SiRNA knock-down of native APG16 in mouse embryonic fibroblasts, replacement with wild-type/T300A variant ATG1L1, measurement of autophagy;
-Comparison of autophagy in transformed human lymphoblastoid cells homozygous for ATG16L1 wild-type/T300A mutation
-Identification of novel interactors for human Atg16 in epithelial and macrophage cell lines using immuno-affinity purification and mass spectroscopy
Other experiments:
-Measurement of autophagy in normal/inflamed bowel biopsies from patients with CD/normal controls.
-I will also localise and quantify ATG16L1 expression using immunohistochemistry and rt-PCR.
This project offers training in advanced cell biology in an area of cutting-edge medical science within a world-class research institute, and will be the foundation of my career in academic gastroenterology.