Investigating the role of neutral sphingomyelinases in membrane vesicle trafficking

Evidence collected over the years has established small extracellular vesicle (sEV) or exosome secretion as a novel paradigm for intercellular communication under both physiological and pathological conditions. SEVs are generated as intraluminal vesicles (ILVs) during multivesicular body (MVB) maturation in the endocytic pathway. Upon fusion of MVBs with the plasma membrane, ILVs are released into the extracellular space as sEVs. The molecular mechanisms that underlies ILV generation and the subsequent sorting of these secretory MVBs from the conventional degradative MVBs remain largely unraveled. Neutral sphingomyelinase 2 (nSMase2, encoded by SMPD3) activity drives intraluminal vesicle (ILV) generation for sEV secretion. In addition to its role in producing ceramide required for membrane invagination to form ILVs, here, we report that nSMase2 regulates sEV secretion through modulation of vacuolar H+ -ATPase (V-ATPase) activity. Specifically, we show that nSMase2 inhibition induces V-ATPase complex assembly that drives MVB lumen acidification and consequently reduces sEV secretion. Conversely, we demonstrate that stimulating nSMase2 activity with the inflammatory cytokine TNFα decreases acidification and increases sEV secretion. Thus, we unravel that nSMase2 activity affects MVB membrane lipid composition to counteract V-ATPase-mediated endosome acidification, and thereby shift MVB fate towards sEV secretion. The second part of this thesis focuses on neutral sphingomyelinase 1 (nSMase1, encoded by SMPD2), which also belongs to the sphingomyelinase enzyme family. The molecular characterization and biological function of nSMase1 remain poorly studied. Here, we show that SMPD2 knockdown (KD) reduces LAMP1 at the mRNA levels and is required for initiating a full-potential unfolded protein response under ER stress. Additionally, SMPD2 KD dramatically reduces the global protein translation rate. We further show that SMPD2 KD cells are arrested in the G1 phase of the cell cycle and that two important cell cycle regulating processes - PI3K/Akt pathway and Wnt signaling pathway are altered. Taken together, we propose a role for nSMase1 in buffering ER stress and modulating cellular fitness via cell cycle regulation.2022-07-2

Phosphorylation of Ykt6 SNARE Domain Regulates Its Membrane Recruitment and Activity

Sensitive factor attachment protein receptors (SNARE) proteins are important mediators of protein trafficking that regulate the membrane fusion of specific vesicle populations and their target organelles. The SNARE protein Ykt6 lacks a transmembrane domain and attaches to different organelle membranes. Mechanistically, Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. We identified phosphorylation sites in the SNARE domain of Ykt6 that mediate Ykt6 membrane recruitment and are essential for cellular growth. Using proximity-dependent labeling and membrane fractionation, we found that phosphorylation regulates Ykt6 conversion from a closed to an open conformation. This conformational switch recruits Ykt6 to several organelle membranes, where it functionally regulates the trafficking of Wnt proteins and extracellular vesicle secretion in a concentration-dependent manner. We propose that phosphorylation of its SNARE domain leads to a conformational switch from a cytosolic, auto-inhibited Ykt6 to an active SNARE at different membranes