Role of The ATG16L1 WD domain in lipid transport and membrane integrity

Griffith, Luke (2023) Role of The ATG16L1 WD domain in lipid transport and membrane integrity. Doctoral thesis, University of East Anglia.

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Influenza A (IAV) and SARs-CoV-2 are important respiratory viruses causing widespread infections and mortality. The severity of both viruses is dependent on the host immune response. Recent work has implicated specific single nucleotide polymorphisms (SNPs) in the interferon induced transmembrane protein 3 (IFITM3) gene with increased severity of IAV and SARs-CoV-2 in humans and mice. IFITM3 has been shown to restrict Influenza infection through modulation of endosomal membrane cholesterol concentration, inhibiting key viral fusion processes. Recently, our laboratory has shown that ATG16L1, a protein normally associated with autophagy, is protective against Influenza infection in mice. Transgenic mice expressing a truncated ATG16L1 protein lacking the WD domain (ΔWD) show increased Influenza pathology and virus fusion efficiency.

Non-canonical roles of the ATG16L1 WD domain may include damaged plasma membrane repair through lysosomal exocytosis of cholesterol. I hypothesise that changes in membrane cholesterol composition may increase virus fusion. ATG16L1 WD truncation is thought to occur in the human SNP rs2241880 (T300A), which creates a caspase 3 cleavage site upstream of the WD domain.

To examine if SNPs in IFITM3 and ATG16L1 modulate respiratory virus infection, I investigated if nasopharyngeal swabs from patients could be used to isolate DNA for host SNP analysis at point of care. This would provide clinicians with rapid predictive insight into patient prognosis following viral diagnosis. Nucleic acids from a Norfolk cohort of volunteers previously diagnosed with Covid-19 were extracted using both NHS diagnostic platforms and lab methods for SNP analysis.

The Norfolk cohort had SNP allelic frequencies of IFITM3 rs12252, rs34481144 and ATG16L1 T300A consistent with European populations. DNA isolated from clinical swabs can be used for SNP genotyping during the diagnostic process.

Through in vitro experimentation, I have shown that deletion of the ATG16L1 WD domain modulates intracellular cholesterol transport to the plasma membrane. The intracellular distribution of cholesterol, phosphatidylserine (PS), and IFITM3 was characterised using immunostaining with fluorescent probes and subcellular fractionation, followed by biochemical analysis of cholesterol and protein distribution. In ΔWD MEFs there was enhanced intracellular accumulation of cholesterol, together with PS and IFITM3, in Rab7 positive late endosomes. There was lower cholesterol in the plasma membranes of ΔWD brains and ΔWD MEFs, which also displayed reduced PS in the plasma membrane. Treatment with T0901317 drug, which induces efflux of cholesterol from endosomes, reversed cholesterol and PS accumulation in late endosomes of ΔWD MEFs. However, there was no effect on IFITM3 localisation. T0901317 treatment normalised plasma membrane cholesterol concentrations of ΔWD MEFs and brains, and restored PS in the plasma membrane of ΔWD MEFs. This research shows a novel role for ATG16L1 in lipid transport and may explain increased susceptibility to viral infection observed in mice lacking the ATG16L1 WD domain.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User: Chris White
Date Deposited: 30 May 2024 07:25
Last Modified: 30 May 2024 07:25

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