Abstract
The protein barrier-to-autointegration factor (BAF) and its interaction partners, the LEM (LAP2B, emerin, MAN1)-domain proteins, constitute a powerful cytoplasmic DNA defense mechanism. Invading DNA molecules are quickly bound by the BAF system and trapped in membrane compartments. This decreases the nuclear uptake of DNA from the cytoplasm. Inhibition of the BAF system is therefore expected to enhance the efficacy of non-viral DNA transfection agents. In this study, we introduced a protocol for the recombinant expression of soluble BAF and developed two ELISA-type assays to discover small molecule inhibitors of BAF-dependent DNA retention by high throughput screening (HTS). The proton pump inhibitor rabeprazole as well as three compounds of the Maybridge library were identified as inhibitors of the LEM-BAF-DNA interaction chain. The inhibition was based on adduct formation with BAF cysteine residues. An enhancing effect of the compounds on cell culture transfection, however, was not observed, which may be attributed to the reducing environment of the cytoplasm that prevents the adduct formation with BAF cysteine residues. The novel assays developed here can provide new tools to further study the biological functions of the BAF system, and may lead to the identification of suitable BAF inhibitors in future HTS campaigns.
Highlights
The protein barrier-to-autointegration factor (BAF) and its interaction partners, the LEM (LAP2B, emerin, MAN1)-domain proteins, constitute a powerful cytoplasmic DNA defense mechanism
We developed two ELISA-type assays to find inhibitors of a powerful cellular defense system against double stranded DNA (dsDNA), which is constituted by the protein barrier-to-autointegration factor (BAF) and its interaction partners, the LEM-domain proteins
We introduced a protocol for the recombinant expression of soluble BAF and developed two ELISA-type assays for high throughput screening (HTS) of small molecule libraries to identify potential inhibitors of the BAF-dependent DNA clustering
Summary
The protein barrier-to-autointegration factor (BAF) and its interaction partners, the LEM (LAP2B, emerin, MAN1)-domain proteins, constitute a powerful cytoplasmic DNA defense mechanism. The small fraction of DNA that is taken up by the target cells must escape quickly from the endosomal compartments to avoid hydrolysis in the lysosomes, be transported through the highly crowded cytoplasmic e nvironment[3], and be smuggled into the nucleus[4,5] These hurdles are well known and delivery efforts in the field of gene therapy have focused on developing new complexing agents or modifying the existing ones, to increase serum stability, uptake, selectivity and endosomal escape of DNA6,7. We developed two ELISA-type assays to find inhibitors of a powerful cellular defense system against dsDNA, which is constituted by the protein barrier-to-autointegration factor (BAF) and its interaction partners, the LEM-domain proteins. This process eventually leads to the reassembly of the nuclear envelope, which makes BAF an indispensable player for the survival of dividing c ells[17]
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