Abstract
A single nucleotide polymorphism in Atg16L1, an autophagy-related gene (ATG), is a risk factor for Crohn disease, a major form of chronic inflammatory bowel disease. However, it is still unknown how the Atg16L1 variant contributes to disease development. The Atg16L1 protein possesses a C-terminal WD repeat domain whose function is entirely unknown, and the Crohn disease-associated mutation (T300A) is within this domain. To elucidate the function of the WD repeat domain, we established an experimental system in which a WD repeat domain mutant of Atg16L1 is stably expressed in Atg16L1-deficient mouse embryonic fibroblasts. Using the system, we show that the Atg16L1 complex forms a dimeric complex and that the total Atg16L1 protein level is strictly maintained, possibly by the ubiquitin proteasome system. Furthermore, we show that an Atg16L1 WD repeat domain deletion and the T300A mutant have little impact on canonical autophagy and autophagy against Salmonella enterica serovar Typhimurium. Therefore, we propose that Atg16L1 T300A is differentially involved in Crohn disease and canonical autophagy.
Highlights
Macroautophagy, referred to here as autophagy, is an intracellular process in which the cytosol and organelles are sequestered within double membrane-bound structures, called autophagosomes, which deliver their contents to the lysosome/ vacuole for degradation [1]
We show that an Atg16L1 WD repeat domain deletion and the T300A mutant have little impact on canonical autophagy and autophagy against Salmonella enterica serovar Typhimurium
The MEFs derived from Atg16L1⌬/⌬ mice express deleted forms of the Atg16L1 protein that lack the entire coiled-coil domain, which is involved in Atg16L1 multimerization and is essential for Atg16L1 function [15] (Fig. 1A)
Summary
Reagents and Antibodies—Cell culture reagents were purchased from Invitrogen. The following antibodies were used: anti-human Atg5 [16], anti-mouse Atg16L1 [4], anti-p62. Recombinant retroviruses were prepared as described previously [17]. Cells were lysed in phosphate-buffered saline containing 2% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor mixture Fluorescence Microscopy—Cells were cultured on coverslips and fixed with 3% paraformaldehyde in phosphate-buffered saline. Gel Filtration—Gel-filtration analysis was performed as previously described [4]. Sucrose Density Gradient Centrifugation—The cytosol fraction was prepared as described in the gel-filtration section. Twohundred microliters of the cytosol fraction was loaded on the top of discontinuous stepwise sucrose gradients. The samples were sedimented for 16 h at 100,000 ϫ g, and 0.6-ml fractions were collected from the top of the gradient. Bulk Protein Degradation Assay—The bulk protein degradation assay was performed as previously described [20]. Bacterial infections were performed as previously described [21]. Statistics—All values shown in the figures are represented with standard deviation
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