In search of novel genes required for phagocytosis of apoptotic cells by macrophages, we took an unbiased approach and carried out a deficiency screen of the Drosophila autosomes (NC Franc and K White, unpublished data). We identified pallbearer (pall), a gene essential for efficient phagocytosis of apoptotic cells by embryonic macrophages (Silva et al. 2007, Immunity 4:541-4). The pall gene encodes a novel Drosophila F-Box protein. F-Box proteins generally provide substrate specificity to Skp/Cullin/F-box (SCF) complexes, which act as E3 Ubiquitin ligases promoting ubiquitylation of phosphorylated subtrates and their subsequent degradation via the 26S proteasome.

We found that, as for other F-box proteins found in yeast and mammalian systems, the Pallbearer (PALL) F-Box protein acts as an E3 Ubiquitin ligase by physically interacting with SkpA, dCullin1 (also known as Lin19) and the E2 Ubiquitin ligase UbcD1 (also known as Effete) to form an SCF/E2 ligase complex. We showed that all components of this complex are required for efficient phagocytosis of apoptotic cells in vivo. Furthermore, we found that macrophage-specific expression of dominant-negative forms of genes encoding two proteasome subunits significantly reduced the ability of macrophages to clear apoptotic corpses. With these results, we demonstrated a direct role for ubiquitylation and proteasomal degradation in phagocytosis of apoptotic corpses in vivo.

Our goal is now to understand how ubiquitylation and proteasomal degradation promotes phagocytosis. To do so, we have initiated a search for the Pall substrate(s) using a biochemical approach to identify potential interactors. Identifying the molecular nature and physiological role of the Pall substrates that needs to be degraded for efficient phagocytosis to proceed may help us understand how these substrates may keep the phagocytic machinery in check.

Some obligate intracellular pathogens use and subvert the phagocytic machinery to hide within the phagocytes where it can proliferate and eventually propagate throughout the body. Thus our studies may give us some clues as to how we may develop new therapeutic approaches to fight infection by such pathogens, by preventing further uptake of such pathogens following the first signs of infection and thus limiting their ability to proliferate within the phagocytes.

We will pursue this project along the following three lines:

1) Characterize the expression and localization of PALL during the course of apoptotic cell clearance in wild-type macrophages, and phagocytosis-defective mutant macrophages;

2) Identify the subtrate(s) of the PALL F-box protein, which needs to be degraded for efficient phagocytosis of apoptotic cells to proceed, characterize and study the inhibitory role of the PALL substrate(s) in apoptotic cell clearance;

3) Study the in vivo localization of PALL and all other components of the phagocytosis machinery identified thus far to try and understand the dynamics in time and space of these molecules during phagocytosis in wild-types and phagocytosis-defective mutant macrophages.