Genome-Wide Identification of Cyclophilin Gene Family in Cotton and Expression Analysis of the Fibre Development in Gossypium barbadense.

Cyclophilins (CYPs) are a member of the immunophilin superfamily (in addition to FKBPs and parvulins) and play a significant role in peptidyl-prolyl cis-trans isomerase (PPIase) activity. Previous studies have shown that CYPs have important functions in plants, but no genome-wide analysis of the cotton CYP gene family has been reported, and the specific biological function of this gene is still elusive. Based on the release of the cotton genome sequence, we identified 75, 78, 40 and 38 CYP gene sequences from G. barbadense, G. hirsutum, G. arboreum, and G. raimondii, respectively; 221 CYP genes were unequally located on chromosomes. Phylogenetic analysis showed that 231 CYP genes clustered into three major groups and eight subgroups. Collinearity analysis showed that segmental duplications played a significant role in the expansion of CYP members in cotton. There were light-responsiveness, abiotic-stress and hormone-response elements upstream of most of the CYPs. In addition, the motif composition analysis revealed that 49 cyclophilin proteins had extra domains, including TPR (tetratricopeptide repeat), coiled coil, U-box, RRM (RNA recognition motif), WD40 (RNA recognition motif) and zinc finger domains, along with the cyclophilin-like domain (CLD). The expression patterns based on qRT-PCR showed that six CYP expression levels showed greater differences between Xinhai21 (long fibres, G. barbadense) and Ashmon (short fibres, G. barbadense) at 10 and 20 days postanthesis (DPA). These results signified that CYP genes are involved in the elongation stage of cotton fibre development. This study provides a valuable resource for further investigations of CYP gene functions and molecular mechanisms in cotton.

Proteomics-based identification of two novel direct targets of hypoxia-inducible factor-1 and their potential roles in migration/invasion of cancer cells.

Hypoxia-inducible factor-1 (HIF-1), consisting of oxygen-sensitive HIF-1alpha and constitutively expressed HIF-1beta subunits, is a master transcriptional activator for cellular response to hypoxia. To explore direct HIF-1 targets, here we used differential gel electrophoresis (DIGE) to compare the HIF-1-regulated proteins between leukemic U937T-cell line with and without conditional induction of HIF-1alpha protein by tetracycline-off system. Among the upregulated proteins identified, mRNA levels of annexin A1, macrophage-capping protein (CapG), S100 calcium-binding protein A4 (S100A4), S100A11, acyl-CoA-binding protein and calcyclin-binding protein also increased. The expressions of the annexin A1, CapG and S100A4 genes were significantly induced by hypoxia in five adherent cell lines tested besides U937 cells, while their expressions were blocked by the short hairpin RNA specifically against HIF-1alpha. Further luciferase reporter assay and chromatin immunoprecipitation showed that HIF-1alpha directly bound to three hypoxia-responsive elements located at intron 1 of S100A4 gene and hypoxia-responsive element at -350 to -346 of CapG gene, which are essential for HIF-1-induced expression. Additionally, the role of S100A4 expression in migration and invasion of cancer cells were also confirmed. These findings would provide new sights for understanding the molecular mechanisms underlying HIF-1 action.

NDRG1 is down-regulated in the early apoptotic event induced by camptothecin analogs: the potential role in proteolytic activation of PKC delta and apoptosis.

We previously reported that NSC606985, a new camptothecin analog, induces apoptosis of acute myeloid leukemic cells, which is triggered by proteolytic activation of protein kinase C delta (PKC delta). Here, we performed quantitative proteomic analysis of NSC606985-treated and untreated leukemic U937 cells with two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) in combination with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry. Thirty-three proteins were found to be deregulated. Then, we focused on N-myc downstream regulated gene 1 (NDRG1) down-regulated during apoptosis induction. The results demonstrated that the down-regulation of NDRG1 protein but not its mRNA was an early event prior to proteolytic activation of PKC delta in U937 cells under treatments of NSC606985 as well as other camptothecin analogs. With the ectopic expression of NDRG1, the proteolytic activation of PKC delta in NSC606985-treated leukemic cells was delayed and the cells were less sensitive to apoptosis. On the contrary, the suppression of NDRG1 expression by specific small interfering RNA significantly enhanced NSC606985-induced activation of PKC delta and apoptosis of U937 cells. In summary, our study suggests that the down-regulation of NDRG1 is involved in proteolytic activation of PKC delta during apoptosis induction, which would shed new light on the understanding the apoptotic process initiated by camptothecin.

NDRG1 contributes to retinoic acid-induced differentiation of leukemic cells.

N-Myc downstream-regulated gene 1 (NDRG1) protein has been shown to be up-regulated during leukemic cell differentiation induced by some differentiation-inducing agents such as all-trans retinoic acid (ATRA). However, the potential role of up-regulated NDRG1 in the event is greatly unknown. In this work, we show that inducible NDRG1 expression can drive leukemic U937 cells to undergo differentiation, while the knock-down of NDRG1 expression by specific small interfering RNA significantly antagonizes ATRA-induced differentiation of leukemic cells, proposing the role of NDRG1 in leukemic cell differentiation. Furthermore, our work shows that CCAAT/enhancer-binding protein beta (C/EBPbeta) and PU.1, which are important hematopoiesis-related transcription factors, may act as downstream effectors of NDRG1 in leukemic cell differentiation. Taking together, this study provides direct evidence for the role of NDRG1 protein in myeloid leukemic cell differentiation.

Comparative proteomic analysis of hypoxia-treated and untreated human leukemic U937 cells.

We reported recently that moderate hypoxia and hypoxia-mimetic agents could induce growth arrest and differentiation of leukemic cells via the mediation of hypoxia-inducible factor 1 alpha (HIF-1alpha), but the exact molecular mechanisms remain largely unknown. In this study, human acute promonocytic leukemic U937 cells were incubated under 2% O2 or in 50 microM of the hypoxia mimetic agent cobalt chloride (CoCl2) and normal oxygen for 24 h, and their protein expression profiles were compared by 2-DE coupled with MALDI-TOF/TOF MS/MS. We identified 62 and 16 proteins that were significantly deregulated by hypoxia and CoCl2 treatment, respectively. These proteins were mainly involved in metabolism, gene expression regulation, signal transduction, cell proliferation, differentiation and apoptosis. As an example, N-myc downstream regulated gene 1 (NDRG1), a putative differentiation-related gene, was up-regulated in both 2% O2- and CoCl2-treated U937 cells. Moreover, enforced HIF-1alpha expression also elevated NDRG1 mRNA and protein in U937 cells. These data will provide some clues for understanding mechanisms by which leukemic cells response to hypoxia.