Ligand-dependent active-site closure revealed in the crystal structure of Mycobacterium tuberculosis MenB complexed with product analogues

1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase catalyzes an essential intramolecular Claisen condensation in menaquinone biosynthesis and is an important target for the development of new antibiotics. This enzyme in Mycobacterium tuberculosis is cofactor-free and is classified as a type II DHNA-CoA synthase, differing from type I enzymes, which rely on exogenous bicarbonate for catalysis. Its crystal structures in complex with product analogues have been determined at high resolution to reveal ligand-dependent structural changes, which include the ordering of a 27-residue active-site loop (amino acids 107-133) and the reorientation of the carboxy-terminal helix (amino acids 289-301) that forms part of the active site from the opposing subunit across the trimer-trimer interface. These structural changes result in closure of the active site to the bulk solution, which is likely to take place through an induced-fit mechanism, similar to that observed for type I DHNA-CoA synthases. These findings demonstrate that the ligand-dependent conformational changes are a conserved feature of all DHNA-CoA synthases, providing new insights into the catalytic mechanism of this essential tubercular enzyme

Effect of Ultraviolet B Radiation on the Biosynthesis of Carotenoids in Foxtail Millet Grains

Foxtail millet is a vital nutritional cereal. The de-husked grain is usually yellow and mainly contains carotenoids, which directly reflects the millet quality. In this study, the impact of ultraviolet-B(UV-B) on millet color and carotenoid content was determined using two foxtail millet varieties, HuaJinZao (HJZ) and Qinhuang 2 (QH). The b* value at different stages of grain development and the content of carotenoids (primarily lutein and zeaxanthin) in foxtail millet grains decreased when the plants were exposed to low UV-B intensity. A total of 3113 and 96 differentially expressed genes were identified in HJZ and QH, respectively, and were found to be associated with the metabolism of tryptophan, starch, and sucrose as well as the biosynthesis of amino acids, which was relatively consistent with the functional annotation of differential metabolites. Furthermore, we evaluated the changes in the expression of seven and eight genes associated with carotenoid and starch metabolism, respectively, in the kernels of foxtail millet exposed to UV-B and found that appropriate UV-B intensity could promote the expression levels of genes involved in carotenoid synthesis and repress the expression of genes involved in carotenoid degradation. This study lays a theoretical foundation for cultivating new foxtail millet varieties with high carotenoid content

RhoB Acts as a Tumor Suppressor That Inhibits Malignancy of Clear Cell Renal Cell Carcinoma.

This study aims to investigate the biological role of RhoB in clear cell renal cell carcinoma (ccRCC). The expression of RhoB was examined in specimens of patients and cell lines by Western blot and Immunohistochemistry. The correlation between RhoB expression and clinicopathologic variables was also analyzed. The effects of RhoB on cell proliferation, cell cycle, cell apoptosis, and invasion/migration were detected by over-expression and knockdown of RhoB level in ccRCC cells via plasmids and RNAi. The results showed that RhoB was low-expressed in ccRCC surgical specimens and cell lines compared with adjacent normal renal tissues and normal human renal proximal tubular epithelial cell lines (HKC), and its protein expression level was significantly associated with the tumor pathologic parameter embracing tumor size(P = 0.0157), pT stage(P = 0.0035), TNM stage(P = 0.0024) and Fuhrman tumor grade(P = 0.0008). Further, over-expression of RhoB remarkably inhibited the cancer cell proliferation, colony formation and promoted cancer cell apoptosis, and aslo reduced the invasion and migration ability of ccRCC cells. Interestingly, up-regulation of RhoB could induce cell cycle arrest in G2/M phase and led to cell cycle regulators(CyclineB1,CDK1) and pro-apoptotic protein(casp3,casp9) aberrant expression. Moreover, knockdown of RhoB in HKC cells promoted cell proliferation and migration. Taken together, our study indicates that RhoB expression is decreased in ccRCC carcinogenesis and progression. Up-regulation of RhoB significantly inhibits ccRCC cell malignant phenotype. These findings show that RhoB may play a tumor suppressive role in ccRCC cells, raising its potential value in futural therapeutic target for the patients of ccRCC

Effects of RhoB on the abilities of cell proliferation of the tumor and normal kidney cell lines.

<p>The over-expression or down-expression efficiency was monitored at the protein level in Caki-1(<b>A</b>), A498 (<b>B</b>) and HKC (C) by western blot. Proliferation curve by MTS assay showing that Caki-1cells (D) transfected with pcDNA3.0-Flag-RhoB grew much more slowly than those transfected with empty vector, whereas cells transfected with si-RhoB and negative control oligo groups made no difference. MTS assay showed knockdown RhoB accelerated the proliferation velocity in A498 (E) and in HKC cells (<b>F</b>). (G) Effect of RhoB in colony formation of Caki-1 cells. Results were shown mean±SD *<i>p</i><0.05. ***<i>p</i><0.001. Each experiment was performed in triplicate.</p

Effects of RhoB in invasion and migration of 786-O cells.

<p>The invasion (<b>A</b>) and migration (<b>B</b>) of 786-O cells transfected with pcDNA3.0-Flag-RhoB decreased nearly 4.59-fold (for invasion) and 5-fold (for migration) after transfection with pc-DNA3.0-Flag-RhoB compared with the empty vector. However, the invasion and migration number in the si-RhoB group presented no significant difference compared to the negative control group. (<b>C</b>) 786-O cells transfected with pcDNA3.0-Flag-RhoB exhibited an obvious decrease in migration rate compared to cells transfected with empty vector, si-RhoB and negative control groups at 24h point. The data shown were mean±SD, ***<i>p</i><0.001. Each experiment was performed in triplicate.</p

The Correlation between RhoB expression and clinicopathological parameters of ccRCC parients.

<p>The Correlation between RhoB expression and clinicopathological parameters of ccRCC parients.</p

RhoB expression is downregulated in ccRCC tissues and cell lines.

<p>(<b>A</b>) RhoB protein expression was detected in surgical specimens of ccRCC and their corresponding adjacent non-tumorous tissues by western blot analysis. (<b>B</b>) Expression of RhoB in the ccRCC-derived cell lines A498, Caki-2,786-O, 769-P, Caki-1 and human renal proximal tubular cell lines (HKC and HK2) was detecte by western blot. β-action was used as an internal control. (<b>C</b>) RhoB protein expression in ccRCC tissues and the matched noncancerous counterpart of the T1 stage ccRCC tissues. (200×c1, 200×c2, the T1 stage of ccRCC tissues and its corresponding noncancerous tissues; 200×c3,the T2 stage of ccRCC tissues; 200×c4, the T3 stage of ccRCC tissues).</p

Influence of RhoB in 786-O cells and Caki-1 cells on cell cycle distribution.

<p>786-O (<b>A</b>) and Caki-1(<b>C</b>) cells transfected with pcDNA3.0-Flag-RhoB, compared to empty vector showed a higher percentage in G2/M phase. Cells transfected with si-RhoB and negative control group showed no difference in cycling phase distribution. (<b>B, D</b>) The data were shown mean±SD of three independent experiments, each performed in triplicate. (<b>E</b>) The expression levels of cyclin B1 and CDK1 were significantly decreased in 786-O cells transfected with pcDNA3.0-Flag-RhoB (<i>p</i><0.05). (<b>F</b>) Protein relative expression level is shown in mean±SD of triplicate independent experiments with similar numbers.</p

Attenuation of Krüppel-Like Factor 4 Facilitates Carcinogenesis by Inducing G1/S Phase Arrest in Clear Cell Renal Cell Carcinoma

<div><p>Krüppel-like factor 4 (KLF4) is a transcription factor with diverse functions in various cancer types; however, the function of KLF4 in clear cell renal cell carcinoma (ccRCC) carcinogenesis remains unknown. In this study, we initially examined KLF4 expression by using a cohort of surgically removed ccRCC specimens and cell lines. Results indicated that the transcription and translation of KLF4 were lower in ccRCC tissues than in patient-matched normal tissues. Furthermore, the KLF4 expression was significantly downregulated in the five ccRCC cell lines at protein and mRNA levels compared with that in normal renal proximal tubular epithelial cell lines (HKC). KLF4 downregulation was significantly correlated with tumor stage and tumor diameter. Promoter hypermethylation may contribute to its low expression. In addition, <i>in vitro</i> studies indicated that the KLF4 overexpression significantly inhibited proliferation in human ccRCC cell lines 786-O and ACHN. Moreover, the KLF4 overexpression arrested the cell cycle progress at the G1/S phase transition by upregulating p21<i>^WAF1/CIP1</i> expression and downregulating cyclin D1 expression, KLF4 knockdown in HKC cells did the opposite. <i>In vivo</i> studies confirmed the anti-proliferative effect of KLF4. Our results suggested that KLF4 had an important function in suppressing the growth of ccRCC.</p> </div

KLF4 was frequently methylated in human primary ccRCC.

<p>(A) KLF4 mRNA downregulation in ccRCC tissues. KLF4 expression was quantified by ΔΔCt method, in which PPIA was used as a control sample. N: normal; T: tumor (<i>P</i><0.01). (B). Schematic structure of the KLF4 promoter CpG island. Analysis of exon 1, CpG, transcription start, and MSP sites. (C) MSP analysis of KLF4 methylation in primary ccRCC tissues and normal tissues. Ten cases with a methylated allele are shown. The methylated allele was observed in 11 of 25 (44%) primary ccRCC tissues but was absent in the corresponding normal tissues. Normal blood lymphocyte DNA (NL), in vitro methylated DNA (IVD), and water were used as the negative control treatment, the positive control, and the blank sample, respectively. N: normal; T: tumor; M: methylated; U: unmethylated.</p