INTERACTOME OF RUBICON-TurboID IN MACROPHAGES UNVEILS MOLECULAR COMPONENTS OF LC3-ASSOCIATED PHAGOCYTOSIS (LAP)

LC3-associated phagocytosis (LAP) is a noncanonical pathway of conjugation of ATG8 proteins, such as LC3, onto a phagosome membrane, regulating phagosome maturation, cargo degradation, and signal transduction. LAP is triggered by the recognition and engulfment of dead cells, immune complexes, and pathogens, thus contributing to homeostasis maintenance and immune responses to infection. Rubicon (RUBCN) is an early component of the LAP cascade. Due to the central role of RUBCN in LAP, herein we aim to characterize novel molecular components of LAP identified by proteomics. We previously expressed RUBCN fused with biotin ligase BirA derived from Escherichia coli (RUBCN-TurboID) in RAW 264.7 cells and identified 126 RUBCN-interacting candidates during LAP induction. Among those, two target proteins with unknown role in LAP, mitochondrial outer membrane protein Voltage-Dependent Anion Channel 2 (VDAC2) and the phosphatase Src Homology 2 (SH2)- containing Inositol 5’-Phosphatase 1 (SHIP-1), were selected. We found that VDAC2 is recruited to the vicinity of zymosan-containing phagosomes. To test the role of VDAC2 in LAP, we used in vitro knockout by CRISPR in bone marrow-derived macrophages (BMDM) of Cas9-expressing mice. Immunofluorescence and immunoblot analysis confirmed the efficient depletion of VDAC2 in BMDM. Of note, we found an increase of LC3⁺ phagosomes in Vdac2^-/- BMDM treated with zymosan. Conversely, we did not detect any SHIP-1 recruitment to phagosomes by immunostaining of fixed cells, indicating a transient involvement of SHIP-1 in LAP. Using a similar knockout strategy as above, we found decreased LC3 lipidation onto the phagosome in Ship-1^-/- BMDM. Thus, our preliminary data suggest a negative role for VDAC2 and a positive role for SHIP-1 in LAP. The next steps include to evaluate the role of both targets for the response against Aspergillus fumigatus to determine the significance of these novel putative components of LAP for macrophages.