cKMT1 is a New Lysine Methyltransferase That Methylates the Ferredoxin-NADP(+) Oxidoreductase and Regulates Energy Transfer in Cyanobacteria.

Lysine methylation is a conserved and dynamic regulatory posttranslational modification performed by lysine methyltransferases (KMTs). KMTs catalyze the transfer of mono-, di-, or tri-methyl groups to substrate proteins and play a critical regulatory role in all domains of life. To date, only one KMT has been identified in cyanobacteria. Here, we tested all of the predicted KMTs in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), and we biochemically characterized sll1526 that we termed cKMT1 (cyanobacterial lysine methyltransferase 1) and determined that it can catalyze lysine methylation both in vivo and in vitro. Loss of cKMT1 alters photosynthetic electron transfer in Synechocystis. We analyzed cKMT1-regulated methylation sites in Synechocystis using a timsTOF Pro instrument. We identified 305 class I lysine methylation sites within 232 proteins, and of these, 80 methylation sites in 58 proteins were hypomethylated in ΔcKMT1 cells. We further demonstrated that cKMT1 could methylate ferredoxin-NADP(+) oxidoreductase (FNR) and its potential sites of action on FNR were identified. Amino acid residues H118 and Y219 were identified as key residues in the putative active site of cKMT1 as indicated by structure simulation, site-directed mutagenesis, and KMT activity measurement. Using mutations that mimic the unmethylated forms of FNR, we demonstrated that the inability to methylate K139 residues results in a decrease in the redox activity of FNR and affects energy transfer in Synechocystis. Together, our study identified a new KMT in Synechocystis and elucidated a methylation-mediated molecular mechanism catalyzed by cKMT1 for the regulation of energy transfer in cyanobacteria.

Proteomic analysis of the regulatory networks of ClpX in a model cyanobacterium Synechocystis sp. PCC 6803.

Protein homeostasis is tightly regulated by protein quality control systems such as chaperones and proteases. In cyanobacteria, the ClpXP proteolytic complex is regarded as a representative proteolytic system and consists of a hexameric ATPase ClpX and a tetradecameric peptidase ClpP. However, the functions and molecular mechanisms of ClpX in cyanobacteria remain unclear. This study aimed to decipher the unique contributions and regulatory networks of ClpX in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We showed that the interruption of clpX led to slower growth, decreased high light tolerance, and impaired photosynthetic cyclic electron transfer. A quantitative proteomic strategy was employed to globally identify ClpX-regulated proteins in Synechocystis cells. In total, we identified 172 differentially expressed proteins (DEPs) upon the interruption of clpX. Functional analysis revealed that these DEPs are involved in diverse biological processes, including glycolysis, nitrogen assimilation, photosynthetic electron transport, ATP-binding cassette (ABC) transporters, and two-component signal transduction. The expression of 24 DEPs was confirmed by parallel reaction monitoring (PRM) analysis. In particular, many hypothetical or unknown proteins were found to be regulated by ClpX, providing new candidates for future functional studies on ClpX. Together, our study provides a comprehensive ClpX-regulated protein network, and the results serve as an important resource for understanding protein quality control systems in cyanobacteria.

Systematic Survey of the Regulatory Networks of the Long Noncoding RNA BANCR in Cervical Cancer Cells.

Long noncoding RNAs (lncRNAs) are increasingly recognized as functional regulators of human cancers. BRAF-activated noncoding RNA (BANCR), an oncogenic lncRNA, has a carcinogenic effect on many types of cancers. However, the clinical significance and molecular mechanisms of BANCR in cervical cancer are still unclear. Here, we generated BANCR knockout cell lines via CRISPR/Cas9 editing and revealed that BANCR plays roles in the apoptosis, proliferation, and migration of HeLa cells. A quantitative proteomics strategy was employed to globally identify BANCR-regulated proteins in HeLa cells. In total, we identified 569 differentially expressed proteins (DEPs) upon knockout of BANCR in HeLa cells. Bioinformatic analysis revealed that these DEPs were involved in diverse cellular pathways. Functional studies revealed that BANCR exerts its effects on the proliferation and apoptosis of HeLa cells through the regulation of cAMP-responsive element binding protein 1 (CREB1) expression. Mechanistically, BANCR could inhibit the expression of miR-582-5p, and CREB1 is a direct target of miR-582-5p; therefore, BANCR may exert its function by regulating CREB1 expression via targeting miR-582-5p in cervical cancer cells. Collectively, this study established a proteome-wide BANCR regulatory network, which provides novel insight into the molecular pathogenesis of cervical cancer and can serve as a basis for the development of targeted therapies.

Decabromodiphenyl Ethane Mainly Affected the Muscle Contraction and Reproductive Endocrine System in Female Adult Zebrafish.

The novel brominated flame retardant decabromodiphenyl ethane (DBDPE) has become a widespread environmental pollutant. However, the target tissue and toxicity of DBDPE are still not clear. In the current study, female zebrafish were exposed to 1 and 100 nM DBDPE for 28 days. Chemical analysis revealed that DBDPE tended to accumulate in the brain other than the liver and gonad. Subsequently, tandem mass tag-based quantitative proteomics and parallel reaction monitoring verification were performed to screen the differentially expressed proteins in the brain. Bioinformatics analysis revealed that DBDPE mainly affected the biological process related to muscle contraction and estrogenic response. Therefore, the neurotoxicity and reproductive disruptions were validated via multilevel toxicological endpoints. Specifically, locomotor behavioral changes proved the potency of neurotoxicity, which may be caused by disturbance of muscular proteins and calcium homeostasis; decreases of sex hormone levels and transcriptional changes of genes related to the hypothalamic-pituitary-gonad-liver axis confirmed reproductive disruptions upon DBDPE exposure. In summary, our results suggested that DBDPE primarily accumulated in the brain and evoked neurotoxicity and reproductive disruptions in female zebrafish. These findings can provide important clues for a further mechanism study and risk assessment of DBDPE.

CBX6 Promotes HCC Metastasis Via Transcription Factors Snail/Zeb1-Mediated EMT Mechanism.

PURPOSE: Hepatocellular carcinoma (HCC) is the most common malignant tumor worldwide with high morbidity and mortality rates. We aimed to examine the expression of chromobox 6 (CBX6) in HCC and analyze its correlation with clinicopathological features of HCC patients. Moreover, the role of CBX6 in the HCC cell proliferation, invasion and metastasis and the potential mechanism underlying HCC metastasis were also investigated. METHODS: We used quantitative polymerase chain reaction (qRT-PCR) and Western blot to evaluate the expression levels of CBX6 in HCC cell lines. Furthermore, the expression of CBX6 in HCC and the adjacent non-tumor tissues was assessed by immunohistochemistry (IHC). Cell proliferation was evaluated using MTT assay, cell migration and invasion were measured using wound healing and transwell assays. Finally, we detected the expression of target proteins in HCC cell lines transfected with CBX6 overexpression plasmid or CBX6 shRNA plasmid by Western blot. RESULTS: We found that the expression of CBX6 was increased in 280 cases of HCC tissues compared that in adjacent non-tumor tissues. HCC patients with high CBX6 expression had a higher tendency to have high growth rate, strong invasion ability, high clinical stage and poor tumor differentiation. Functional study demonstrated that the upregulation of CBX6 promotes proliferation, migration and invasion of HCC cells while silencing CBX6 in HCC cells significantly inhibited cell proliferation, migration and invasion. Furthermore, CBX6 could accelerate the EMT process in HCC cells by upregulating the expression of snail and zeb1. CONCLUSION: CBX6 played an important role in the process of tumorigenesis and progression in HCC and enhanced the invasion and metastasis ability of HCC cells through regulating transcription factors snail/zeb1-mediated EMT mechanism, which indicated that the protein could serve as a novel therapeutic target for the treatment of HCC.

Trans-arterial chemoembolization combined with Jinlong capsule for advanced hepatocellular carcinoma: a PRISMA-compliant meta-analysis in a Chinese population.

CONTEXT: Jinlong capsule (JLC) is an animal-derived traditional Chinese medical preparation for advanced hepatocellular carcinoma (HCC). However, its clinical efficacy is still not well investigated. OBJECTIVE: This study summarizes the efficacy and safety of JLC combined with trans-arterial chemoembolization (TACE) for patients with HCC. METHODS: The databases PubMed, Cochrane Library, Web of Science, EMBASE, Medline, China National Knowledge Infrastructure, Wanfang Database, Chinese Scientific Journal Database and Chinese Biological Medicine Database were systematically searched from the date of their inception until February 2020. Jinlong capsule, trans-arterial chemoembolization, and hepatocellular carcinoma were the key terms searched. Randomized controlled trials and high-quality prospective cohort trials comparing the combined use of JLC and TACE versus TACE for HCC were included. Data were pooled using random or fixed effect models depending on heterogeneity. RESULTS: Data from 19 articles with 1,725 HCC patients were analysed. Compared with TACE treatment alone, the combination of TACE and JLC significantly prolonged patients' 6-36 month overall survival (p < 0.05), and markedly improved the overall response rate (RR = 1.37, 95% CI = 1.24-1.52, p < 0.00001) and disease control rate (RR = 1.11, 95% CI = 1.06-1.17, p < 0.0001) of patients. The liver function, quality of life, and immune function of patients were significantly improved; the partial adverse events related to TACE were also effectively relieved after the combination treatment. CONCLUSION: This meta-analysis suggests that the combination of TACE and JLC is more effective in the treatment of HCC than treatment with TACE alone.

OsNF-YC10, a seed preferentially expressed gene regulates grain width by affecting cell proliferation in rice.

Grain size and shape are important factors in determining the grain yield. In this study, OsNF-YC10, a member of the NF-Y transcription factor family encoding a putative histone transcription factor, was cloned and characterized. qRT-PCR and mRNA in situ hybridization analysis revealed that OsNF-YC10 was highly expressed in endosperm and spikelet hull at late developmental stages. The results showed that OsNF-YC10 was a nuclear protein showing transcription activation activity. The osnf-yc10 lines, produced using CRISPR/Cas9 technology, showed narrow, thin and light grains. Cytological experiments revealed significantly reduced cell number of spikelet hull in osnf-yc10 lines compared with that in WT. Narrow, thin, and light grains were found consistently in OsNF-YC10 RNAi transgenic lines. Moreover, the number of cells decreased in the grain-width direction than WT. These results indicated that OsNF-YC10 plays an important role in determining grain size and shape. OsNF-YC10 was further revealed to influence the expression of GW8 (a positive regulator of grain width), GW7 (a negative regulator of grain width) and cell cycle-regulated genes CYCD4, CYCA2.1, CYCB2.1, CYCB2.2, E2F2. Taken together, it is suggested that OsNF-YC10 regulates the grains size and shape by influencing the cell proliferation of spikelet hulls.

Regeneration of Solanum nigrum by somatic embryogenesis, involving frog egg-like body, a novel structure.

A new protocol was established for the regeneration of Solanum nigrum by frog egg-like bodies (FELBs), which are novel somatic embryogenesis (SE) structures induced from the root, stem, and leaf explants. The root, stem, and leaf explants (93.33%, 85.10%, and 100.00%, respectively) were induced to form special embryonic calli on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid, under dark condition. Further, special embryonic calli from the root, stem, and leaf explants (86.97%, 83.30%, and 99.47%, respectively) were developed into FELBs. Plantlets of FELBs from the three explants were induced in vitro on MS medium supplemented with 5.0 mg/L 6-benzylaminopurine and 0.1 mg/L gibberellic acid, and 100.00% plantlet induction rates were noted. However, plantlet induction in vivo on MS medium supplemented with 20 mg/L thidiazuron showed rates of 38.63%, 15.63%, and 61.30% for the root, stem, and leaf explants, respectively, which were lower than those of the in vitro culture. Morphological and histological analyses of FELBs at different development stages revealed that they are a novel type of SE structure that developed from the mesophyll (leaf) or cortex (stem and root) cells of S. nigrum.

Arsenic trioxide induces G2/M arrest in hepatocellular carcinoma cells by increasing the tumor suppressor PTEN expression.

Arsenic trioxide (As(2)O(3)), an effective agent against acute promyelocytic leukemia, has been reported to inhibit the viability of solid tumors cell lines recently. The detailed molecular mechanism underlying the As(2)O(3)-induced inactivation of the cdc2 and possible functional role of PTEN in the observed G2/M arrest has yet to be elucidated. Here, we assessed the role of PTEN in regulation of As(2)O(3)-mediated G2/M cell cycle arrest in Hepatocellular carcinoma cell lines (HepG2 and SMMC7721). After 24 h following treatment, As(2)O(3) induced a concentration-dependent accumulation of cells in the G2/M phase of the cell cycle. The sustained G2/M arrest by As(2)O(3) is associated with decreased cdc2 protein and increased phospho-cdc2(Tyr15). As(2)O(3) treatment increased Wee1 levels and decreased phospho-Wee1(642). Moreover, As(2)O(3) substantially decreased the Ser473 and Thr308 phosphorylation of Akt and upregulated PTEN expression. Downregulation of PTEN by siRNA in As(2)O(3) -treated cells increased phospho-Wee1(Ser642) while decreased phospho-cdc2(Tyr15), resulting in decreased the G2/M cell cycle arrest. Therefore, induction of G2/M cell cycle arrest by As(2)O(3) involved upregulation of PTEN.