In vivo and in vitro characterization of the human ATG16L1 protein and its Crohn's disease associated mutant from T300A
Zusammenfassung der Projektergebnisse
The identification of the common coding variant T300A of the essential autophagy gene ATG16L1, as a risk allele for Crohn`s disease (CD) within genome-wide association studies represented a strong hint towards a role of autophagy in Inflammatory Bowel Disease (IBD). Indeed, an increasing number of genes with functions in autophagy and autophagy-dependent pathways emerge as susceptibility genes for CD. Autophagy (`self-digestion`) is well studied in yeast and best known as `the starvation response,’ enabling the generation of energy and building blocks (amino acids, etc.) under conditions of nutrient limitation/depletion. However, in higher organisms autophagy exceeds its function as a starvation response and appears as an underappreciated cellular process with roles in cell death, innate and adaptive immunity, neurodegeneration, cancer, aging, diabetes, and so forth. Using cell culture models I have demonstrated that both ATG16L1 variants (risk and protective, respectively) mediate basal autophagy (continuously active autophagy to remove damaged organelles, etc.) to a similar extent while the risk allele encoding T300A specifically leads to impairment in the clearance of intracellular Salmonella Typhimurium. Further, the T300A variant demonstrated reduced protein stability upon Salmonella infection. Those data strongly indicated a loss-of-function phenotype associated with the risk variant T300A and suggested that defective functions of T300A occur post-translational. Indeed, the Threonine/Alanine polymorphism is directly, C-terminal adjacent to the amino acid sequence DNVD suggesting that ATG16L1 may be a substrate for cleavage by caspases. In subsequent studies I could demonstrate that ATG16L1 can be a substrate for caspases 3 and 7 and that the efficacy of cleavage is regulated by the nature of the P1` amino acid: Threonine or Alanine, respectively. In brief, the risk variant T300A showed higher rates of cleavage by caspases 3 and 7 as compared to the protective variant. Further, an amino acid substitution of the critical aspartic acid (D) at position 299 to glutamic acid (E) abolished cleavage, further supporting specific cleavage by caspases. To study potential defects of the coding variant T300A in a physiological context I have generated T300A knock-in mice allowing a detailed analysis of immune cell populations, antigen presentation, survival, etc., in unchallenged or stressed conditions (e.g., DSS colitis). In addition, within this in vivo setting I will attempt to prove my hypothesis that T300A shows impaired protein stability under certain conditions. In addition, I have generated a targeted ES- clone possibly allowing the generation of ATG16L1-T300A-D299E mice. The comparison of wildtype, T300A, and T300A-D299E mice should help clarify if excessive cleavage of the risk variant T300A by caspase 3 and/or 7 represents a biochemical explanation for the association of the T300A polymorphism with IBD in vivo. Furthermore, T300A mice might serve as an in vivo model to test different environmental triggers of CD-pathogenesis, as well as new small molecule therapeutics designed to treat IBD via autophagy induction.
Projektbezogene Publikationen (Auswahl)
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Impaired autophagy of an intracellular pathogen induced by a Crohn`s disease associated ATG16L1 variant. PloS One, 2008. 3(10): e3391
Kuballa, P., et al.
