Algae undergo a complete metabolic transformation under stress by arresting cell growth, inducing autophagy and hyper-accumulating biofuel precursors such as triacylglycerols and starch. However, the regulatory mechanisms behind this stress-induced transformation are still unclear. Here, we use biochemical, mutational, and "omics" approaches to demonstrate that PI3K signaling mediates the homeostasis of energy molecules and influences carbon metabolism in algae. In Chlamydomonas reinhardtii, the inhibition and knockdown (KD) of algal class III PI3K led to significantly decreased cell growth, altered cell morphology, and higher lipid and starch contents. Lipid profiling of wild-type and PI3K KD lines showed significantly reduced membrane lipid breakdown under nitrogen starvation (−N) in the KD. RNA-seq and network analyses showed that under −N conditions, the KD line carried out lipogenesis rather than lipid hydrolysis by initiating de novo fatty acid biosynthesis, which was supported by tricarboxylic acid cycle down-regulation and via acetyl-CoA synthesis from glycolysis. Remarkably, autophagic responses did not have primacy over inositide signaling in algae, unlike in mammals and vascular plants. The mutant displayed a fundamental shift in intracellular energy flux, analogous to that in tumor cells. The high free fatty acid levels and reduced mitochondrial ATP generation led to decreased cell viability. These results indicate that the PI3K signal transduction pathway is the metabolic gatekeeper restraining biofuel yields, thus maintaining fitness and viability under stress in algae. This study demonstrates the existence of homeostasis between starch and lipid synthesis controlled by lipid signaling in algae and expands our understanding of such processes, with biotechnological and evolutionary implications.
Background : Bcl-2 family proteins play a central role in regulating apoptosis. In human, over 20 members of this family have been identified to date. Bfl-1, a member of the Bcl-2 family, has been known to retard apoptosis in various cell lines. However, the function of Bfl-1 remains unclear. Methods : In order to investigate the Bfl-1 function, we employed yeast two-hybrid system to identify the proteins which are capable of interacting with Bfl-1. The interaction of inhibitor kappaB kinase-(IKK-) and Bfl-1 was confirmed using glutathione S-transferase pull down assays. To determine which regions of IKK- were required for interaction with Bfl-1, we constructed 12 deletion mutants of IKK- and 5 deletion mutants of Bfl-1. Results : Bfl-1 interacted with the C-terminal region of IKK- which is a subunit of IKK complex, and IKK- activity is very important in the NF-B related pathway. In addition, the amino acids 673-745 of IKK- were important for Bfl-1 interactions, and amino acids 1-484 of Bfl-1, including Bcl-2 homology domains (BH1, BH2, BH3, BH4), were crucial for IKK- interactions. Conclusion : IKK C-terminus contains many serine residues as binding partner of Bfl-1. Our results suggested that Bfl-1 is involved in the NF-B activation through interaction of IKK- and Bfl-1. Further studies need to be performed to understand functions of the IKK- and Bfl-1 associated with the regulation of the NF-B activation pathway.
Radiotherapy is currently applied in the treatment of human cancers. We studied whether genistein would enhance the radiosensitivity and explored its precise molecular mechanism in cervical cancer cells. After co-treatment with genistein and irradiation, the viability, cell cycle analysis, and apoptosis signaling cascades were elucidated in CaSki cells. The viability was decreased by co-treatment with genistein and irradiation compared with irradiation treatment alone. Treatment with only gamma-irradiation led to cell cycle arrest at the G1 phase. On the other hand, co-treatment with genistein and gamma-irradiation caused a decrease in the G1 phase and a concomitant increase up to 56% in the number of G2 phase. In addition, cotreatment increased the expression of p53 and p21, and Cdc2- tyr-15-p, supporting the occurrence of G2/M arrest. In general, apoptosis signaling cascades were activated by the following events: release of cytochrome c, upregulation of Bax, downregulation of Bcl-2, and activation of caspase-3 and -8 in the treatment of genistein and irradiation. Apparently, co-treatment downregulated the transcripts of E6*I, E6*II, and E7. Genistein also stimulated irradiation-induced intracellular reactive oxygene, species (ROS) production, and co-treatment-induced apoptosis was inhibited by the antioxidant N-acetylcysteine, suggesting that apoptosis has occurred through the increase in ROS by genistein and gamma-irradiation in cervical cancer cells. Gamma-irradiation increased cyclooxygenase-1 (COX-2) expression, whereas the combination with genistein and gamma-irradiation almost completely prevented irradiation-induced COX-2 expression and PGE2 production. Co-treatment with genistein and gamma-irradiation inhibited proliferation through G2/M arrest and induced apoptosis via ROS modulation in the CaSki cancer cells.
Previously, we reported that overexpression of Opa (Neisseria gonorrhoeae opacity-associated)-interacting protein 5 (OIP5) caused multi-septa formation and growth defects, both of which are considered cancer-related phenotypes. To evaluate OIP5 as a possible cancer therapeutic target, we examined its expression level in 66 colorectal cancer patients. OIP5 was upregulated about 3.7-fold in tumors and over 2-fold in 58 out of 66 colorectal cancer patients. Knockdown of OIP5 expression by small interfering RNA specific to OIP5 (siOIP5) resulted in growth inhibition of colorectal and gastric cancer cell lines. Growth inhibition of SNU638 by siOIP5 caused an increase in sub-G1 DNA content, as measured by flow cytometry, as well as an apoptotic gene expression profile. These results indicate that knockdown of OIP5 may induce apoptosis in cancer cells. Therefore, we suggest that OIP5 might be a potential cancer therapeutic target, although the mechanisms of OIP5-induced carcinogenesis should be elucidated.
Specific monoclonal antibodies(mAbs) against bis-indole alkaloids related to vinblastine were established to develop a simple and specific immunoassay system for the quantitation of Vinca alkaloids. Vinca alkaloids were extracted from tissue cultured cells of periwinkle plant (Vinca rosea L.). Spleen cells from Balb/c mice immunized with vinblastine-bovine serum albumin(VBL-BSA) conjugate as immunogen were fused with myeloma cells(Sp2/0-Ag.14) in the presence of polyethylene glycol. In the preliminary experiments, 32 clones which highly reacted with VBL-BSA conjugate were selected by ELISA(Enzyme-linked immunosorbent assay). These clones were further analyzed by inhibition assay of ELISA. The results obtained with two typical monoclonal antibodies, KN-1 and KN-2, were described. KN-1 exhibited considerable reactivities with soluble dimeric bis-indole alkaloids, whereas no cross reacted with monomeric bis-indole alkaloids. However KN-2 showed cross reactivity with mono- and di-meric bis-indole alkaloids. Furthermore, KN-1 was applied to the immunoassay system for determining the VBL amounts of in vitro cultured cell extracts. This assay system could detect dimeric vinca alkaloid as low as 0.05 nM.
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