TBK1 inhibitors enhance transfection efficiency by suppressing p62/SQSTM1 phosphorylation

Genes Cells. 2023 Jan;28(1):68-77. doi: 10.1111/gtc.12987. Epub 2022 Nov 4.

Abstract

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.

Keywords: TBK1; TBK1 inhibitor; gene delivery; p62; p62 phosphorylation; selective autophagy.

MeSH terms

  • Animals
  • Autophagy* / genetics
  • DNA* / metabolism
  • Humans
  • Mice
  • Phosphorylation
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism
  • Sequestosome-1 Protein / genetics
  • Sequestosome-1 Protein / metabolism
  • Transfection

Substances

  • Sequestosome-1 Protein
  • DNA
  • SQSTM1 protein, human
  • TBK1 protein, human
  • Protein Serine-Threonine Kinases
  • Sqstm1 protein, mouse
  • Tbk1 protein, mouse