Dynamic Changes in the Proximitome of Neutral Sphingomyelinase-2 (NSM2) in TNFα Stimulated Jurkat Cells

ABSTRACT: Ceramides generated by the activity of the neutral sphingomyelinase 2 (NSM2) 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 NSM2 and TNFα-induced changes thereof. We established Jurkat cells stably expressing NSM2 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 NSM2-proximal biotinylated proteins and their changes within the first 5 min of TNFα stimulation by quantitative mass spectrometry. We observed significant dynamic changes in the NSM2 microenvironment in response to TNFαstimulation consistent with rapid remodeling of protein networks. Our data confirmed known NSM2 interactors and revealed that the recruitment of most proteins depended on NSM2 enzymatic activity. We measured significant enrichment of proteins related to vesicle-mediated transport, including proteins of recycling endosomes, trans-Golgi network, and exocytic vesicles in the proximitome of enzymatically active NSM2 within the first minutes of TNFα stimulation. Hence, the NSM2 proximal network and its TNFα-induced changes provide a valuable resource for further investigations into the involvement of NSM2 in the early signaling pathways triggered by TNFα.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Lothar Jaensch  

LAB HEAD: Lothar Jaensch

PROVIDER: PXD052930 | Pride | 2024-07-05

REPOSITORIES: Pride