Abstract Background Dysregulation of autophagy, a key cellular multi-step process for degrading damaged organelles and intracellular pathogens has been implicated in Crohn’s disease (CD) and revealed by various susceptibility genes including ATG16L1. We recently identified altered autophagy flux in patients carrying the variants CD-related ATG16L1 T300A1. We hypothesize that this defect compromises autophagic cargo clearance, thereby impairing cellular homeostasis and promoting inflammation. Methods We developed a proteomic approach to analyse the nature of the content of autophagic vesicles, known as autophagosomes isolated from HeLa cell lines either expressing or not the ATG16L1 T300A variant. Cells were transfected with a plasmid encoding the fusion protein APEX2–LC3B, where LC3B serves as an autophagosome-specific marker and APEX2 as a peroxidase for proximity labelling. After autophagosomes enrichment, biotinylated autophagosomal proteins were isolated and identified by quantitative mass spectrometry. Analyses were conducted under both basal conditions and after bacterial strain exposure (CD-related AIEC-LF82 pathobiont or E. coli MG1655, as control). Results Proteomic profiling of autophagosomes revealed that expression of the ATG16L1 T300A variant results in a pronounced reduction in both the quantity and diversity of autophagic cargo, an effect that is further amplified following bacterial exposure. Notably, 31 cargoes were uniquely detected in T300A-expressing cells, including several proteins with anti-inflammatory or antimicrobial functions, such as members of the BPIF (bactericidal permeability-increasing fold–containing) family. These findings indicate that T300A-expressing cells possess distinct cargo signatures, suggesting a potential adaptive shift in the selection of substrates targeted for degradation. Conclusion Our data indicate that the ATG16L1 T300A variant reshapes the autophagosomal proteome, selectively enriching proteins with antimicrobial and anti-inflammatory functions. This modified cargo landscape offers new mechanistic support for a role of autophagy-related polymorphisms in regulating inflammatory responses in CD. Reference: Quiniou G, Andromaque L, Duclaux-Loras R, et al. Impaired reprogramming of the autophagy flux in maturing dendritic cells from crohn disease patients with core autophagy gene-related polymorphisms. Autophagy. 2024;20(8):1837-1853. doi:10.1080/15548627.2024.2338574 Conflict of interest: Ms. Andromaque, Leslie: No conflict of interest Duclaux-Loras, Rémi: No conflict of interest Viret, Christophe: No conflict of interest Faure, Mathias: No conflict of interest Nancey, Stéphane: board membership and lecturing fees from Abbvie, Takeda, Celltrion Healthcare, Pfizer, Galapagos, Johnson & Jonshon, Lilly, Fresenius, Amgen, Medac, MSD. Rozieres, Aurore: None
Andromaque et al. (Thu,) studied this question.