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Interactome of CD63 in WT and ATG16L1-KO cells

ABSTRACT: We use an MS-based proteomic approach to demonstrate the physical interaction between the exosomal marker CD63 and mutant huntingtin, likely occurring upon transfer of the latter within the MVBs compartment, confirming how such pathway diversion can indeed regard autophagy substrate proteins (and other cytosolic cargoes) upon autophagy compromise or in response to other stimuli. In order to do so, we did perform the same experiments, namely transiently over-expressing GFP/CD63 and FLAG/mutant huntingtin in WT and ATG16L1-KO and performed GFP-trap experiments starting from four different samples (per genotype). Immuno-isolated samples were then subjected to mass spectrometry analysis. Interestingly, upon comparative analysis and gene identification, around 5700 proteins were detected. Out of these, we could report 126 proteins interacting differentially with CD63 in autophagy-dependent manner. Finally, the MS-approach was also instrumental in confirming, the lack of any significant change in the interaction between CD63 and mutant huntingtin as a function of the different autophagy background.

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ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Michele Costanzo

LAB HEAD: Michele Costanzo

PROVIDER: PXD076375 | Pride | 2026-05-13

REPOSITORIES: Pride

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Project description:Susceptibility to Crohn's disease, a complex inflammatory disease involving the small intestine, is controlled by over 30 loci. One Crohn's disease risk allele is in ATG16L1, a gene homologous to the essential yeast autophagy gene ATG16 (ref. 2). It is not known how ATG16L1 or autophagy contributes to intestinal biology or Crohn's disease pathogenesis. To address these questions, we generated and characterized mice that are hypomorphic for ATG16L1 protein expression, and validated conclusions on the basis of studies in these mice by analysing intestinal tissues that we collected from Crohn's disease patients carrying the Crohn's disease risk allele of ATG16L1. Here we show that ATG16L1 is a bona fide autophagy protein. Within the ileal epithelium, both ATG16L1 and a second essential autophagy protein ATG5 are selectively important for the biology of the Paneth cell, a specialized epithelial cell that functions in part by secretion of granule contents containing antimicrobial peptides and other proteins that alter the intestinal environment. ATG16L1- and ATG5-deficient Paneth cells exhibited notable abnormalities in the granule exocytosis pathway. In addition, transcriptional analysis revealed an unexpected gain of function specific to ATG16L1-deficient Paneth cells including increased expression of genes involved in peroxisome proliferator-activated receptor (PPAR) signalling and lipid metabolism, of acute phase reactants and of two adipocytokines, leptin and adiponectin, known to directly influence intestinal injury responses. Importantly, Crohn's disease patients homozygous for the ATG16L1 Crohn's disease risk allele displayed Paneth cell granule abnormalities similar to those observed in autophagy-protein-deficient mice and expressed increased levels of leptin protein. Thus, ATG16L1, and probably the process of autophagy, have a role within the intestinal epithelium of mice and Crohn's disease patients by selective effects on the cell biology and specialized regulatory properties of Paneth cells. Experiment Overall Design: 4 Samples: 2 replicates of Atg16-hypomorph Paneth cells and 2 replicates of Wildtype Paneth cells.

			Action	DRS
	DIANN_ID.xlsx	Xlsx
	DIA_IP_CD63_n4_KO_1.d.7z	Other
	DIA_IP_CD63_n4_KO_2.d.7z	Other
	DIA_IP_CD63_n4_KO_3.d.7z	Other
	DIA_IP_CD63_n4_KO_4.d.7z	Other

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Interactome of CD63 in WT and ATG16L1-KO cells

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