In humans, mutations in ATGL lead to TG accumulation in LDs of most tissues and cells, including peripheral blood leukocytes. This pathologic condition is called Jordans’ anomaly, in which functional consequences have not been investigated. In the present study, we tested the hypothesis that ATGL plays a role in leukocyte LD metabolism and immune cell function. Similar to humans with loss-of-function mutations in ATGL, we found that global and myeloid-specific Atgl−/− mice exhibit Jordans’ anomaly with increased abundance of intracellular TG-rich LDs in neutrophil granulocytes. In a model of inflammatory peritonitis, lipid accumulation was also observed in monocytes and macrophages but not in eosinophils or lymphocytes. Neutrophils from Atgl−/− mice showed enhanced immune responses in vitro, which were more prominent in cells from global compared with myeloid-specific Atgl−/− mice. Mechanistically, ATGL−/− as well as pharmacological inhibition of ATGL led to an impaired release of lipid mediators from neutrophils. These findings demonstrate that the release of lipid mediators is dependent on the liberation of precursor molecules from the TG-rich pool of LDs by ATGL. Our data provide mechanistic insights into Jordans’ anomaly in neutrophils and suggest that ATGL is a potent regulator of immune cell function and inflammatory diseases.
Lysosome-associated protein transmembrane-4b (LAPTM4B) associates with poor prognosis in several cancers, but its physiological function is not well understood. Here we use novel ceramide probes to provide evidence that LAPTM4B interacts with ceramide and facilitates its removal from late endosomal organelles (LEs). This lowers LE ceramide in parallel with and independent of acid ceramidase-dependent catabolism. In LAPTM4B-silenced cells, LE sphingolipid accumulation is accompanied by lysosomal membrane destabilization. However, these cells resist ceramide-driven caspase-3 activation and apoptosis induced by chemotherapeutic agents or gene silencing. Conversely, LAPTM4B overexpression reduces LE ceramide and stabilizes lysosomes but sensitizes to drug-induced caspase-3 activation. Together, these data uncover a cellular ceramide export route from LEs and identify LAPTM4B as its regulator. By compartmentalizing ceramide, LAPTM4B controls key sphingolipid-mediated cell death mechanisms and emerges as a candidate for sphingolipid-targeting cancer therapies.
Membrane
proteins are functionally regulated by the composition
of the surrounding lipid bilayer. The late endosomal compartment is
a central site for the generation of ceramide, a bioactive sphingolipid,
which regulates responses to cell stress. The molecular interactions
between ceramide and late endosomal transmembrane proteins are unknown.
Here, we uncover in atomistic detail the ceramide interaction of Lysosome
Associated Protein Transmembrane 4B (LAPTM4B), implicated in ceramide-dependent
cell death and autophagy, and its functional relevance in lysosomal
nutrient signaling. The ceramide-mediated regulation of LAPTM4B depends
on a sphingolipid interaction motif and an adjacent aspartate residue
in the protein’s third transmembrane (TM3) helix. The interaction
motif provides the preferred contact points for ceramide while the
neighboring membrane-embedded acidic residue confers flexibility that
is subject to ceramide-induced conformational changes, reducing TM3
bending. This facilitates the interaction between LAPTM4B and the
amino acid transporter heavy chain 4F2hc, thereby controlling mTORC
signaling. These findings provide mechanistic insights into how transmembrane
proteins sense and respond to ceramide.
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