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Stability of Smyd1 in endothelial cells is controlled by PML-dependent SUMOylation upon cytokine stimulation
Biochemical Journal ( IF 4.1 ) Pub Date : 2021-01-15 , DOI: 10.1042/bcj20200603
Samuel Becker 1 , Gustav Steinemann 1 , Weronika Karle 1 , Kerrin Roos 1 , Celine Huajia Liem 1 , Shalini Muralikumar 2 , Andrea Volkamer 2 , Barbara Munz 3 , Andreas Zakrzewicz 1 , Janine Berkholz 1, 4
Affiliation  

Smyd1 is an epigenetic modulator of gene expression that has been well-characterized in muscle cells. It was recently reported that Smyd1 levels are modulated by inflammatory processes. Since inflammation affects the vascular endothelium, this study aimed to characterize Smyd1 expression in endothelial cells. We detected Smyd1 in human endothelial cells (HUVEC and EA.hy926 cells), where the protein was largely localized in PML nuclear bodies (PML-NBs). By transfection of EA.hy926 cells with expression vectors encoding Smyd1, PML, SUMO1, active or mutant forms of the SUMO protease SuPr1 and/or the SUMO-conjugation enzyme UBC9, as well as Smyd1- or PML-specific siRNAs, in the presence or absence of the translation blocker cycloheximide or the proteasome-inhibitor MG132, and supported by computational modeling, we show that Smyd1 is SUMOylated in a PML-dependent manner and thereby addressed for degradation in proteasomes. Furthermore, transfection with Smyd1-encoding vectors led to PML up-regulation at the mRNA level, while PML transfection lowered Smyd1 protein stability. Incubation of EA.hy926 cells with the pro-inflammatory cytokine TNF-α resulted in a constant increase in Smyd1 mRNA and protein over 24 h, while incubation with IFN-γ induced a transient increase in Smyd1 expression, which peaked at 6 h and decreased to control values within 24 h. The IFN-γ-induced increase in Smyd1 was accompanied by more Smyd1 SUMOylation and more/larger PML-NBs. In conclusion, our data indicate that in endothelial cells, Smyd1 levels are regulated through a negative feedback mechanism based on SUMOylation and PML availability. This molecular control loop is stimulated by various cytokines.

中文翻译:

Smyd1在内皮细胞中的稳定性受细胞因子刺激后PML依赖性SUMOylation的控制

Smyd1是基因表达的表观遗传调节剂,已在肌肉细胞中得到充分表征。最近有报道说,Smyd1水平是由炎症过程调节的。由于炎症影响血管内皮,因此本研究旨在表征Smyd1在内皮细胞中的表达。我们在人内皮细胞(HUVEC和EA.hy926细胞)中检测到Smyd1,该蛋白质主要位于PML核小体(PML-NBs)中。通过存在编码Smyd1,PML,SUMO1,SUMO蛋白酶SuPr1和/或SUMO偶联酶UBC9的活性或突变形式以及Smyd1或PML特异性siRNA的表达载体转染EA.hy926细胞是否存在翻译阻滞剂环己酰亚胺或蛋白酶体抑制剂MG132,并得到计算模型的支持,我们表明,Smyd1以PML依赖性方式被SUMOylated,从而解决了蛋白酶体的降解。此外,用Smyd1编码载体转染导致PML在mRNA水平上调,而PML转染降低了Smyd1蛋白的稳定性。将EA.hy926细胞与促炎细胞因子TNF-α一起孵育会导致Smyd1 mRNA和蛋白质在24小时内持续增加,而与IFN-γ孵育会导致Smyd1表达瞬时增加,在6小时达到峰值,然后下降在24小时内控制值。IFN-γ诱导的Smyd1增加伴随着更多的Smyd1 SUMOylation和更多/更大的PML-NBs。总之,我们的数据表明在内皮细胞中,Smyd1水平通过基于SUMOylation和PML可用性的负反馈机制进行调节。
更新日期:2021-01-15
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