TBK1 inhibitors enhance transfection efficiency by suppressing p62/SQSTM1 phosphorylation
TBK1 inhibitors enhance transfection efficiency by suppressing p62/SQSTM1 phosphorylation
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DOI：
10.1111/gtc.12987
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发表时间：
2022-11-04
期刊：
影响因子：
2.1
通讯作者：
Ogawa，Hidesato
中科院分区：
文献类型：
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作者：
Tsuchiya，Megumi;Kong，Weixia;Ogawa，Hidesato
DNA转染是生命科学中的一项重要技术。非病毒转染试剂广泛用于基础科学中的转染。然而，低转染效率在某些细胞类型中是一个问题。这种低效率可以主要归因于转染的DNA通过p62依赖性选择性自噬的细胞内降解，特别是通过在S403残基处磷酸化的p62（p62-S403-P）。为了实现有效的DNA转染，我们专注于产生p62-S403-P的磷酸化过程，并研究抑制该过程是否会影响转染效率。磷酸化p62的激酶之一是TBK 1。通过基因组编辑消除鼠胚胎成纤维细胞中的TBK 1基因导致p62-S405-P（相当于人S403-P）的显著减少或丢失，并提高了转染效率，这表明TBK 1是在S403磷酸化p62的主要激酶。因此，TBK 1是药物治疗以提高转染效率的可行靶标。当用以下TBK 1抑制剂BX 795、MRT 67307或氨来沙诺之一处理细胞时，转染效率增强。当两种抑制剂组合使用时，这种效果协同改善。我们的研究结果表明，TBK 1抑制剂通过抑制p62磷酸化增强转染效率。
DNA transfection is an essential technique in the life sciences. Non‐viral transfection reagents are widely used for transfection in basic science. However, low transfection efficiency is a problem in some cell types. This low efficiency can be primarily attributed to the intracellular degradation of transfected DNA by p62‐dependent selective autophagy, specifically by p62 phosphorylated at the S403 residue (p62‐S403‐P). To achieve efficient DNA transfection, we focused on a phosphorylation process that generates p62‐S403‐P and investigated whether inhibition of this process affects transfection efficiency. One of the kinases that phosphorylate p62 is TBK1. The TBK1 gene depletion in murine embryonic fibroblast cells by genome editing caused a significant reduction or loss of p62‐S405‐P (equivalent to human S403‐P) and enhanced transfection efficiency, suggesting that TBK1 is a major kinase that phosphorylates p62 at S403. Therefore, TBK1 is a viable target for drug treatment to increase transfection efficiency. Transfection efficiency was enhanced when cells were treated with one of the following TBK1 inhibitors BX795, MRT67307, or amlexanox. This effect was synergistically improved when the two inhibitors were used in combination. Our results indicate that TBK1 inhibitors enhanced transfection efficiency by suppressing p62 phosphorylation.