Autophagic exocitosis of ATP: Rab and SNARES proteins

FADER CM

Paris

Conferencia; framework of the U1134 conferences; 2023

Broussais Hospital. Université Paris Cité

ATP exocytosis has emerged as an important autocrine/paracrine signal to triggerresponses regarding platelet aggregation, inter-astrocytes communication, cellmigration, differentiation, etc. The molecular mechanisms underlying this processhave not been well defined so far. In addition, autophagy-dependent ATP releasewas shown to be an important process involved in immunogenic cell death andinflammation. Autophagic exocytosis has been also implicated in IL-1β, IL-18,galectin-3 secretion and presentation of antigens to the major histocompatibilitycomplex. The regulation of this process is poorly understood. Our group haspreviously shown that there is an important role of the v-SNARE protein VAMP7 inthe autophagic ATP exocytosis. Since there is a close connection betweenVAMP7, the small GTP-ase Rab21 and autophagy we decided to investigate therole of this Rab protein, its guanine-nucleotide exchange factor VARP and anotherRab related to VARP (Rab32) in the autophagic exocytosis of ATP. HeLa cellswere transiently transfected with GFP-Vector, YFP-Rab32wt, YFP-Rab32T39N(negative dominant mutant), GFP-Rab21wt, GFP-Rab21T33N (negative dominantmutant) and/or RFP-LC3. Then we incubated this cells in full medium, resveratrol50nM and rapamycin 50nM for 4 hours or starvation medium for 2 hours. We usedLC3B or VARP antibodies to detect endogenous proteins. Lysotracker red was put30 minutes before the incubation finished. For ATP release assays, we used theluciferin-luciferase kit to measure the amounts of ATP in the extracellular medium.We took samples at 0, 30, 60, 90, 120, 180 and 240 minutes after the autophagicstimulus began. We found that autophagy leads to a redistribution of Rab32 orRab21 molecules to the cell periphery, where they colocalized with LC3 and VARP.ATP release was significantly increased in starvation and in less content inresveratrol condition. Our results suggest a role for Rab21, Rab32 and VARP inthe autophagic exocytosis of ATP.