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X-linked Charcot-Marie-Tooth Disease type 1(CMT1X) is the second most common form of inherited peripheral neuropathy that is caused by mutations in the gap junction beta-1 (GJB1) gene. Using targeted exome-sequencing, we investigated four CMT families from central-southern China and identified two novel missense variants (p.F31S and p.W44G) and two previously reported variants (p.R220Pfs*23 and p.R164Q) of GJB1. All four probands presented typical early-onset peripheral neuropathy, of which the R220Pfs*23 carrier also had neurologic manifestations in the central nervous system. We then constructed GJB1 expression vectors and performed cell biological analysis in vitro. Expression of FLAG-tagged GJB1 at various time points after transfection revealed evident protein aggregation with both wild-type and mutant forms, indicated with immunostaining and immunoblotting. Detergent-based sequential fractionation confirmed that all mutants were higher expressed and more prone to aggregate than the wild-type, whereas the R220Pfs*23mutant showed the greatest amount of SDS-solublemultimers and monomers among groups. Moreover, intracellular aggregation probably occurs in the endoplasmic reticulum compartment rather than the Golgi apparatus. Gap junction plaques were present in all groups and were only compromised in frameshift mutant. Further evidence reveals significant intracellular stress granule formation induced by mutated GJB1 and impaired cell viability indicative of cytotoxicity of self-aggregates. Together, our findings demonstrate novel GJB1 variants-induced cell stress and dysfunction and provide insights into understanding the pathomechanisms of GJB1-CMTX1 and other related disorders. [ABSTRACT FROM AUTHOR]

DNA topoisomerases are proved cancer therapeutic targets with clinically successful anticancer drugs for decades. However, the role of RNA topoisomerase (TOP3β) remained mysterious especially in cancer, and no targeted agent has been reported yet. In a target identification assay of anti-cancer compound using a modified DrugTargetSeqR strategy, mutation of TOP3B was detected in cancer cells acquired resistance to cinobufagin (CBG), a key compound of Huachansu that has been approved for cancer therapy in China. We demonstrated that CBG directly engaged with TOP3β, and promoted TOP3β depletion in wildtype but not mutant cancer cells. Notably, knockout of TOP3β in cancer cells significantly reduced tumor enlargement but not initiation, and inhibited colony formation upon nutrient deprivation. We also demonstrated that CBG induced formation of stress granule, RNA-loop and asymmetric DNA damages in cancer cells, and all these phenotypes were significantly attenuated in TOP3B knockout cells. Of note, examination of a panel of cancer cell lines revealed associations among cell growth inhibition and induction of DNA damage as well as TOP3B depletion upon CBG treatment. Our findings not only highlighted TOP3β as a promising therapeutic target of cancer, but also identified CBG as a lead chemical inhibitor of TOP3β for cancer therapy. [Display omitted] [ABSTRACT FROM AUTHOR]