Potential Inhibitor Against Phase Separation, 1,6-hexanediol Specifically Binds to Beta Actin in Nuclear Extract of Human Cell Line

Abstract

RNA-binding protein (RBP) TLS/FUS plays a major role in induction of phase separation/phase transition and aggregation in relation to familial amyotrophic lateral sclerosis (ALS). Recently, organelles without lipid-bilayer membrane including stress granule, Cajal body, and nucleolus are found to be formed by the phase separation. The phase separation is an event that solutions with two solvents separate into two distinctive phases. The phase separation is prone to have solid phase and forms harmful precipitation or aggregation against living cells, indicating that the phase separation has both benefit and risk on cellular programs. Thus, it is essential for utilization of the phase separation in divergent cellular programs to control or inhibit the undesirable precipitation in living cells. Here, we analyze an inhibitory mechanism of the phase separation and precipitation. Inhibition of the phase separation is one of a critical regulatory step to prevent dysregulation of the phase separation and resulting deleterious precipitations. An inhibitory agent against the phase separation, amphiphilic alcohol, 1,6-hexanediol (1,6-HD) has been reported to examine function of phase separations. Thus, affinity chromatography of biotinylated 1,6-HD is employed to identify an initial event induced by 1,6-HD. Upon successful synthesis of biotinylated compounds of 1,6-HD (bio-1,6-HD), the affinity chromatography with bio-1,6-HD has been established at this study. The bio-1,6-HD captured protein bands on SDS-PAGE gel from HeLa cell nuclear extracts. The bands were analyzed with mass spectrometric analyses, showing that the proteins should be cytoskeleton related proteins. Further analysis using specific antibodies revealed one of the bands as human beta actin. It has been shown that beta actin is involved in divergent cellular activities including the phase separation and also neuronal functions like long-term potentiation. Therefore, beta actin might initiate the 1,6-HD-induced inhibition of the precipitation, although more experiments should be required to test whether it actually works in living cells.

m6A Modified Short RNA Fragments Inhibit Cytoplasmic TLS/FUS Aggregation Induced by Hyperosmotic Stress

“…Cytoplasmic aggregates of RBPs are often observed in neurons of neurodegenerative disease patients, and are speculated to be the cause of the diseases [17,52,53]. Dissolving these aggregates is anticipated to lead to the treatment of the diseases, and 1,6-hexanediol is one of the most widely used reagents in order to disperse LLPS of RBPs in vitro [54,55]. Nevertheless, 1,6-hexanediol can provoke defects in cellular functions, such as impairment of kinase and phosphatase activities, and immobilization of chromatins [56,57].…”