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TBK1 inhibitors enhance transfection efficiency by suppressing p62/SQSTM1 phosphorylation
Megumi Tsuchiya1, Weixia Kong1, Yasushi Hiraoka1
1Graduate School of Frontier Biosciences, Osaka University, Suita, Japan.
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.
Insights
Inhibiting TBK1 kinase, which causes p62 phosphorylation and DNA degradation, significantly enhances DNA transfection efficiency. This discovery offers a new strategy for improving gene delivery in life sciences research.
Area of Science:
- Molecular Biology
- Cell Biology
- Biotechnology
Background:
- Non-viral DNA transfection is crucial in life sciences but often suffers from low efficiency in certain cell types.
- This inefficiency is linked to the degradation of transfected DNA via p62-dependent selective autophagy, specifically involving p62 phosphorylated at S403 (p62-S403-P).
Purpose of the Study:
- To investigate the role of p62 phosphorylation in DNA transfection efficiency.
- To identify kinases involved in p62 phosphorylation and explore their potential as targets for enhancing transfection.
Main Methods:
- Utilized genome editing to deplete TBK1 (TANK-binding kinase 1) in murine embryonic fibroblast cells.
- Assessed levels of p62-S405-P (murine equivalent of human S403-P) and measured DNA transfection efficiency.
- Treated cells with TBK1 inhibitors (BX795, MRT67307, amlexanox) and assessed their impact on transfection.
Main Results:
- TBK1 gene depletion significantly reduced p62-S405-P levels and markedly enhanced DNA transfection efficiency.
- Treatment with TBK1 inhibitors, individually or in combination, also improved transfection efficiency.
- These findings indicate TBK1 is a key kinase phosphorylating p62, and its inhibition boosts transfection.
Conclusions:
- TBK1 is a major kinase responsible for p62 phosphorylation at S403, a process that hinders DNA transfection.
- Inhibiting TBK1 activity is a promising strategy to overcome low transfection efficiency.
- TBK1 inhibitors represent a viable therapeutic approach to enhance gene delivery in biological research.
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