PDF(Pubmed)
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.
摘要：
由中性鞘磷脂酶2（nSMase2）的活性产生的神经酰胺在哺乳动物细胞的应激反应中起关键作用。鞘脂代谢的失调与许多炎症相关的病理有关。然而,其对炎性细胞因子诱导的信号传导的影响尚不完全清楚.这里,我们使用邻近标记来探索nSMase2的质膜近端蛋白网络及其TNFα诱导的变化。我们建立了稳定表达nSMase2C末端与工程抗坏血酸过氧化物酶2（APEX2）融合的Jurkat细胞。去除过量的生物素苯酚显著改善了生物素化蛋白质的基于链霉亲和素的亲和纯化。使用我们优化的协议，我们通过定量质谱法测定了nSMase2-近端生物素化蛋白及其在TNFα刺激的前5分钟内的变化。我们观察到nSMase2微环境响应TNFα刺激的显着动态变化，与蛋白质网络的快速重塑一致。我们的数据证实了已知的nSMase2相互作用物，并表明大多数蛋白质的募集取决于nSMase2的酶活性。我们测量了与囊泡介导的转运相关的蛋白质的显着富集，包括循环内体的蛋白质，跨高尔基网络，在TNFα刺激的第一分钟内，酶活性nSMase2的近端体中的外细胞囊泡。因此，nSMase2近端网络及其TNFα诱导的变化为进一步研究nSMase2参与TNFα触发的早期信号通路提供了宝贵的资源。
