Biotin-Lys-His Blocks Aggregation of RNA-binding Protein TLS, a Cause of Amyotrophic Lateral Sclerosis

Abstract

RNA-binding protein TLS/FUS is a causative gene for amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TLS mutations induce the propensity of TLS to form aggregates in motor neurons causing neuronal degenerative lesions to their necrosis. TLS is prone to be precipitated in high concentration around 10 mg/ml, while mutated TLS is suspected to be precipitated even lower or physiological concentration in the motor neurons. An unidentified agent from infections would cause formation of the precipitation of TLS through their surface antigens. The precipitation driven with agents might be attribute to a small compound appeared on the surface. Biotinylated isoxazole (BISOX) has been reported to be precipitated with divergent RNA-binding proteins including TLS in nuclear extracts of cultured mammalian cells. We have published a molecular model for crystal formation of BISOX with TLS providing a plat form for searching novel regulators to precipitate TLS in neuronal disorders. Because BISOX is an artificial compound, we have further explored to obtain naturally occurring agents to induce the TLS precipitation and generated a conceivable biological compound, biotin-Lys-His (BLH). We have examined the precipitation of BLH with HeLa cell nuclear extract, but did not detect any TLS signal. Then, we add BLH to reaction of BISOX with TLS, and serendipitously observed a robust inhibitory effect of BLH on the formation of crystal of BISOX with TLS. We employed in silico analysis to show how BLH blocks the crystal formation of BISOX with TLS. The computational analysis of the events presented a model that BLH should be incorporated into the crystal formation of BISOX but some steric hindrance placed by BLH blocks growing of the crystal of BISOX and TLS. These results provide the potentiality that BLH should block the aggregate formation of TLS in ALS, leading to a seed for drug discovery against ALS, although it needs future endeavor to find more compounds to have effect on the TLS aggregation.

Versatility of RNA-Binding Proteins in Living Cells

“…RNase treatment also facilitated phase separation of all three TLS/FUS. We finally tested the effect of bisox, a reagent induces TLS aggregation [38], and similar dipeptide named bLH on TLS/FUS fibrilization. As expected, bisox induced fibrilization of all three TLS/FUS examined, and interestingly, bLH inhibited the TLS/FUS aggregation formed by bisox.…”

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

“…A biotinylated compound, biotinylated isoxazole (BISOX) was produced to capture binding molecules to isoxazole. Unexpectedly, BISOX induced precipitation of proteins from cultured cell lysates [11], and the precipitation contained more than hundreds of RBPs [11][12][13][14]. Many of these proteins are components of RNA-protein assemblies forming in nuclei of living cells, called RNA granules or membraneless organelles.…”