biostudies-literatureUnknown15Schol MCeramides generated by the activity of the neutral sphingomyelinase 2 (nSMase2) play a pivotal role in stress responses in mammalian cells. Dysregulation of sphingolipid metabolism has been implicated in numerous inflammation-related pathologies. However, its influence on inflammatory cytokine-induced signaling is yet incompletely understood. Here, we used proximity labeling to explore the plasma membrane proximal protein network of nSMase2 and TNFα-induced changes thereof. We established Jurkat cells stably expressing nSMase2 C-terminally fused to the engineered ascorbate peroxidase 2 (APEX2). Removal of excess biotin phenol substantially improved streptavidin-based affinity purification of biotinylated proteins. Using our optimized protocol, we determined nSMase2-proximal biotinylated prFrontiers in immunology1435701https://www.ebi.ac.uk/biostudies/studies/S-EPMC11263205biostudies-literatureDynamic changes in the proximitome of neutral sphingomyelinase-2 (nSMase2) in TNFα stimulated Jurkat cells.PMC11263205Schol MHennig TWigger DSchumacher FJansch LSchempp RSchneider-Schaulies SKleuser BStigloher CDolken Lvan Ham MAvota EfalseDynamic changes in the proximitome of neutral sphingomyelinase-2 (nSMase2) in TNFα stimulated Jurkat cells.Ceramides generated by the activity of the neutral sphingomyelinase 2 (nSMase2) play a pivotal role in stress responses in mammalian cells. Dysregulation of sphingolipid metabolism has been implicated in numerous inflammation-related pathologies. However, its influence on inflammatory cytokine-induced signaling is yet incompletely understood. Here, we used proximity labeling to explore the plasma membrane proximal protein network of nSMase2 and TNFα-induced changes thereof. We established Jurkat cells stably expressing nSMase2 C-terminally fused to the engineered ascorbate peroxidase 2 (APEX2). Removal of excess biotin phenol substantially improved streptavidin-based affinity purification of biotinylated proteins. Using our optimized protocol, we determined nSMase2-proximal biotinylated pr2024-01-01T00:00:00Z20242026-05-02T12:27:55.758Z2025-06-01T01:16:41.479ZS-EPMC112632053904482810.3389/fimmu.2024.1435701