Mycobacterium tuberculosis ClpC1 N-Terminal Domain Is Dispensable for Adaptor Protein-Dependent Allosteric Regulation.

ClpC1 hexamers couple the energy of ATP hydrolysis to unfold and, subsequently, translocate specific protein substrates into the associated ClpP protease. Substrate recognition by ATPases associated with various cellular activities (AAA+) proteases is driven by the ATPase component, which selectively determines protein substrates to be degraded. The specificity of these unfoldases for protein substrates is often controlled by an adaptor protein with examples that include MecA regulation of Bacillus subtilis ClpC or ClpS-mediated control of Escherichia coli ClpA. No adaptor protein-mediated control has been reported for mycobacterial ClpC1. Using pulldown and stopped-flow fluorescence methods, we report data demonstrating that Mycobacterium tuberculosis ClpC1 catalyzed unfolding of an SsrA-tagged protein is negatively impacted by association with the ClpS adaptor protein. Our data indicate that ClpS-dependent inhibition of ClpC1 catalyzed SsrA-dependent protein unfolding does not require the ClpC1 N-terminal domain but instead requires the presence of an interaction surface located in the ClpC1 Middle Domain. Taken together, our results demonstrate for the first time that mycobacterial ClpC1 is subject to adaptor protein-mediated regulation in vitro.

Evaluation of extracts prepared from 16 plants used in Yao ethnomedicine as potential anticancer agents.

ETHNOPHARMACOLOGICAL RELEVANCE: Medicines of the Yao ethnic group in China are a special branch of traditional Chinese medicine (TCM) and are well documented for use in disease prevention. According to an ethnopharmacological survey, there are 1392 species of medicinal plants that have been documented as Yao ethnomedicines and 104 of these species are used routinely. This study evaluated a partial collection of these 104 core plant species for their potential as anticancer agents. MATERIAL AND METHODS: A literature study of scientific journals and books in the local language was conducted. Based on an ethnopharmacological survey, 16 plant species widely used in Yao ethnomedicine were collected and 64 plant extracts were prepared from these plants. in vitro cytotoxicity screening was conducted with a panel of four human cancer cell lines, lung cancer A549, breast cancer BT20 and MCF-7, bone cancer U2OS. The potential toxicity of the extracts was evaluated using two normal human cell lines, human peripheral lung epithelial cells (HPL1A) and human umbilical vein endothelial cells (HUVEC). Additionally, the 10 extracts that demonstrated cytotoxicity in cancer cells with an IC50 of less than 25.0µg/mL were examined for the ability to induce apoptosis in U2OS cells. RESULTS: The up-to-date information regarding the traditional uses, pharmacological and biological activities, as well as the chemical constituents of the 16 plants are presented. Extracts from all 16 plants showed cytotoxicity against one to four of the human cancer cell lines and the cytotoxic effects of extracts from Melaleuca leucadendra, Stephania longa, Microsorium fortune and Bidens biternata were demonstrated for the first time. The highest anticancer potential was observed for extracts prepared from Melaleuca leucadendra Linn against all tested cancer cells (BT20, A549, U2OS, and MCF7) with an IC50 range of 3.1-32.7µg/mL. The selectivity index of the active samples varied from 0.1 to 25, and five extracts from Bidens biternata, Wedelia calendulacea, Stephania longa and Achras zapota showed significant selectivity against cancer cell lines versus normal cell lines. All tested extracts induced apoptosis in U2OS cells, and for the first time extracts from Melaleuca leucadendra and Microsorium fortune were shown to induce apoptosis. CONCLUSION: We demonstrated the in vitro anticancer efficacy and safety of 16 medicinal plants that have been historically used in Yao ethnomedicine. This study provides evidence to assist the clinical practice of Yao ethnomedicine and the development of chemotherapeutic agents from extracts prepared from these plants.