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

Autor:	Schöl M; Institute for Virology and Immunobiology, University of Wuerzburg, Würzburg, Germany., Schempp R; Institute for Virology and Immunobiology, University of Wuerzburg, Würzburg, Germany., Hennig T; Institute for Virology and Immunobiology, University of Wuerzburg, Würzburg, Germany., Wigger D; Institute of Pharmacy, Department of Pharmacology & Toxicology, Freie Universität Berlin, Berlin, Germany., Schumacher F; Institute of Pharmacy, Department of Pharmacology & Toxicology, Freie Universität Berlin, Berlin, Germany., Kleuser B; Institute of Pharmacy, Department of Pharmacology & Toxicology, Freie Universität Berlin, Berlin, Germany., Stigloher C; Imaging Core Facility, Biocenter, University of Wuerzburg, Würzburg, Germany., van Ham M; Cellular Proteome Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany., Jänsch L; Cellular Proteome Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany., Schneider-Schaulies S; Institute for Virology and Immunobiology, University of Wuerzburg, Würzburg, Germany., Dölken L; Institute of Virology, Medizinische Hochschule Hannover, Hannover, Germany., Avota E; Institute for Virology and Immunobiology, University of Wuerzburg, Würzburg, Germany.
Jazyk:	angličtina
Zdroj:	Frontiers in immunology [Front Immunol] 2024 Jul 09; Vol. 15, pp. 1435701. Date of Electronic Publication: 2024 Jul 09 (Print Publication: 2024).
DOI:	10.3389/fimmu.2024.1435701
Abstrakt:	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. Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2024 Schöl, Schempp, Hennig, Wigger, Schumacher, Kleuser, Stigloher, van Ham, Jänsch, Schneider-Schaulies, Dölken and Avota.)
Databáze:	MEDLINE
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