Functional investigation of novel regulators of LC3-associated phagocytosis (LAP) and non-canonical autophagy in macrophages

• Grant number: 22/08177-6
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Effective date (Start): November 17, 2022
• Effective date (End): November 16, 2023
• Field of knowledge: Biological Sciences - Immunology - Cellular Immunology
• Principal Investigator: Larissa Dias da Cunha
• Grantee: Edismauro Garcia Freitas Filho
• Supervisor: Oliver Florey
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Research place: Babraham Institute, England
• Associated to the scholarship: 19/26040-5 - Identification and characterization of novel molecular determinants of LC3-associated phagocytosis (LAP) in macrophages, BP.PD

Abstract

The non-canonical pathway of autophagy known as LC3-associated phagocytosis (LAP) is a key cellular process that regulates the responses of innate immune cells, controlling the balance of inflammation, and innate responses that promote tissue protection and repair. LAP, an example of a CASM (Conjugation of ATG8 to Endolysosomal Single Membranes) event, utilizes some, but not all, components of the macroautophagy machinery to operate alternative conjugation of LC3 to phagosome membranes that ultimately results in cargo degradation. In this context, LAP guides the integration of phagocytosis of immunostimulatory particles into modulation of the gene expression and functional polarization of macrophages. While several constituents of the LAP cascade have been reported, the mechanisms underlying the fine orchestration of assembling of LAP complexes and their modulatory components remain not entirely understood. Therefore, this study aims to determine how novel components of LAP, identified during my postdoctoral fellowship, regulate the pathway. First, hypothesizing that the endosomal traffic may act as a hub in the targeting/organization of LAP cascade proteins onto the phagosome, we observed for the first time a positive and specific role of the RAB5c GTPase during LAP. Only the ablation of RAB5 isoform c, but not a or b, resulted in the reduction of LC3 lipidation onto the phagosome membrane in LAP-stimulated macrophages. Then, we also established the interactome profile of RUBCN, a critical component of LAP. We characterized this RUBCN interactome using a strategy of heterologous biotinylation labeling by proximity to the target proteins. Our unbiased proteomic data identified 202 RUBCN-binding partners, of which we selected some candidates to currently determine their impact on LAP. These candidates are involved in diverse immune processes and are associated with macrophage phagocytosis and early/later steps of phagosome biogenesis. Thus, we propose here a collaboration with Dr. Oliver Florey, a reference researcher in the non-canonical autophagy and CASM, focused on: 1) to investigate the Rab5c trafficking to phagosome and how the Rab5c-mediated early endosomal trafficking contributes to LAP; and 2) to characterize the role of promising targets in the organization of LAP-molecular complexes and the signaling pathways responsible for LAPosome maturation. For that, we will use the advanced real-time microscopic analyses provided by Babraham Institute, such as spinning-disk confocal microscopy, four-dimensional super-resolution, and video-light-electron microscopy, in association with molecular and biochemical approaches. Our findings may reveal new pharmacological targets capable of inducing or blocking LAP and help to elucidate how and why LAP diverges from macroautophagy and other CASM events, providing an important tool for investigating the immune response in pathologies wherein LAP participates. (AU)