Background

DJ-1 is a redox-sensitive protein with multiple roles in cell homeostasis, levels of which are altered in patients with mast cell (MC)–related disorders. However, whether DJ-1 can regulate human MC function is unknown.

Objective

We sought to investigate the potential role of DJ-1 in the responses of human MCs to antigen stimulation.

Methods

DJ-1 was silenced in human CD34⁺-derived MCs and in the LAD2 MC line by using lentiviral short hairpin RNA constructs. Release of β-hexosaminidase, prostaglandin D₂, and GM-CSF and changes in reactive oxygen species levels were measured after FcεRI engagement. Enzymatic assays, sucrose density gradient centrifugation, immunoprecipitation, dot and Western blotting, and confocal imaging were performed for signaling, cellular localization, and coassociation studies.

Results

DJ-1 knockdown substantially reduced mediator release, as well as Lyn kinase and spleen tyrosine kinase activation and signaling through mechanisms that appeared largely unrelated to DJ-1 antioxidant activity. Following FcεRI activation, nonoxidized rather than oxidized DJ-1 translocated to lipid rafts, where it associated with Lyn, an interaction that appeared critical for maximal Lyn activation and initiation of signaling. Using purified recombinant proteins, we demonstrated that DJ-1 directly bound to Lyn but not to other Src kinases, and this interaction was specific for human but not mouse proteins. In addition, DJ-1 reduced Src homology 2 domain–containing phosphatase 2 phosphatase activity by scavenging reactive oxygen species, thus preventing spleen tyrosine kinase dephosphorylation and perpetuating MC signaling.

Conclusion

We demonstrate a novel role for DJ-1 in the early activation of Lyn by FcεRI, which is essential for human MC responses and provides the basis for an alternative target in allergic disease therapy.

中文翻译：

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DJ-1与Lyn的相互作用对于IgE介导的人类肥大细胞刺激至关重要

背景

DJ-1是一种氧化还原敏感蛋白，在细胞稳态中具有多种作用，在患有肥大细胞（MC）相关疾病的患者中其水平会改变。但是，DJ-1是否可以调节人的MC功能尚不清楚。

客观的

我们试图研究DJ-1在人类MC对抗原刺激的反应中的潜在作用。

方法

通过使用慢病毒短发夹RNA构建体，在人类CD34 ⁺衍生的MC和LAD2 MC系中使DJ-1沉默。FcεRI参与后，测量β-己糖胺酶，前列腺素D ₂和GM-CSF的释放以及活性氧水平的变化。进行了酶促测定，蔗糖密度梯度离心，免疫沉淀，斑点和蛋白质印迹以及共聚焦成像，以进行信号传导，细胞定位和共缔合研究。

结果

DJ-1敲低大大减少了介质的释放，以及Lyn激酶和脾酪氨酸激酶的激活和信号转导，其机制与DJ-1抗氧化剂的活性无关。FcεRI活化后，未氧化的DJ-1（而不是氧化的DJ-1）易位到脂质筏中，并与Lyn结合，这种相互作用对于最大的Lyn活化和信号启动至关重要。使用纯化的重组蛋白，我们证明DJ-1直接与Lyn结合，但不与其他Src激酶结合，并且这种相互作用是特异性针对人类而非小鼠蛋白的。此外，DJ-1通过清除活性氧来降低Src同源性2结构域的磷酸酶2磷酸酶的活性，从而防止脾酪氨酸激酶的去磷酸化并延长MC信号传导。

结论

我们展示了DJ-1在由FcεRI激活Lyn的早期激活中的新作用，这对于人类MC反应至关重要，并为过敏性疾病治疗中的替代靶标提供了基础。