Dynamic changes in the proximitome of neutral sphingomyelinase-2 (nSMase2) in TNFα stimulated Jurkat cells

Front Immunol. 2024 Jul 9:15:1435701. doi: 10.3389/fimmu.2024.1435701. eCollection 2024.

Abstract

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 proteins and their changes within the first 5 min of TNFα stimulation by quantitative mass spectrometry. We observed significant dynamic changes in the nSMase2 microenvironment in response to TNFα stimulation consistent with rapid remodeling of protein networks. Our data confirmed known nSMase2 interactors and revealed that the recruitment of most proteins depended on nSMase2 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 nSMase2 within the first minutes of TNFα stimulation. Hence, the nSMase2 proximal network and its TNFα-induced changes provide a valuable resource for further investigations into the involvement of nSMase2 in the early signaling pathways triggered by TNFα.

Keywords: APEX2; ceramide; neutral sphingomyelinase 2 (nSMase2); plasma membrane (PM); protein dynamic; proteomics; proximity labeling; tumor necrosis factor-alpha (TNF-alpha).

MeSH terms

  • Cell Membrane / metabolism
  • Humans
  • Jurkat Cells
  • Signal Transduction
  • Sphingomyelin Phosphodiesterase* / metabolism
  • Tumor Necrosis Factor-alpha* / metabolism
  • Tumor Necrosis Factor-alpha* / pharmacology

Substances

  • Tumor Necrosis Factor-alpha
  • Sphingomyelin Phosphodiesterase
  • SMPD3 protein, human

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The work was funded through the German Research Foundation (DFG). MS, RS and DW were supported by the Research Training Group “Metabolism, topology and compartmentalization of membrane-proximal lipid and signaling components in infection” (RTG 2581-417857878). Grant holders were EA, SS-S, BK, LD, CS The JEOL JEM-1400 Flash transmission electron microscopy was funded by DFG grant 426173797 (INST 93/1003-1 FUGG).