New use for CETSA: monitoring innate immune receptor stability via post-translational modification by OGT.
New use for CETSA: monitoring innate immune receptor stability via post-translational modification by OGT.
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CETSA的新用途：通过OGT的翻译后修饰来监测先天免疫受体的稳定性。
DOI：
10.1007/s10863-018-9754-z
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发表时间：
2018-06
影响因子：
3
通讯作者：
Grimes CL
中科院分区：
文献类型：
--
作者：
Drake WR;Hou CW;Zachara NE;Grimes CL
O-GlcNAcylation是一种动态的、功能多样的翻译后修饰，可影响数千种蛋白质，包括先天免疫受体核苷酸结合寡聚化结构域蛋白2（Nod 2）。Nod 2的突变（R702 W、G908 R和100 fs）与克罗恩病相关，并且与野生型相比具有较低的稳定性。环己酰亚胺（CHX）-追踪半衰期测定已用于显示O-GlcNAc化增加野生型和克罗恩病变体Nod 2（R702 W）的稳定性和响应。需要一种更快速的方法来评估翻译后修饰提供的稳定性，以充分理解NLR稳定性和O-GlcNAc酰化之间的相关性。在这里，最近开发的通常用于证明蛋白质-配体结合的细胞热位移测定（CETSA）适于检测在Nod 2中增加O-GlcNAc化水平时蛋白质稳定性的变化。该试验用作预测其他克罗恩病相关Nod 2变体是否为O-GlcNAc酰化的方法，并且还鉴定了另一种NLR Nod 1上的修饰。经典的免疫沉淀和NF-κB转录测定被用来确认这些蛋白质上的这种修饰的存在和影响。结果表明，CETSA是一种简便的方法，可用于检测O-GlcNAc-transferase（OGT）客户蛋白的O-GlcNAc-Acylation稳定性效应，将成为研究翻译后修饰的有力工具。
O-GlcNAcylation is a dynamic and functionally diverse post-translational modification shown to affect thousands of proteins, including the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (Nod2). Mutations of Nod2 (R702W, G908R and 100fs) are associated with Crohn’s disease and have lower stabilities compared to wild type. Cycloheximide (CHX)-chase half-life assays have been used to show that O-GlcNAcylation increases the stability and response of both wild type and Crohn’s variant Nod2, R702W. A more rapid method to assess stability afforded by post-translational modifications is necessary to fully comprehend the correlation between NLR stability and O-GlcNAcylation. Here, a recently developed cellular thermal shift assay (CETSA) that is typically used to demonstrate protein-ligand binding was adapted to detect shifts in protein stabilization upon increasing O-GlcNAcylation levels in Nod2. This assay was used as a method to predict if other Crohn’s associated Nod2 variants were O-GlcNAcylated, and also identified the modification on another NLR, Nod1. Classical immunoprecipitations and NF-κB transcriptional assays were used to confirm the presence and effect of this modification on these proteins. The results presented here demonstrate that CETSA is a convenient method that can be used to detect the stability effect of O-GlcNAcylation on O-GlcNAc-transferase (OGT) client proteins and will be a powerful tool in studying post-translational modification.
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