Biochemical characterization of human and yeast choline and ethanolamine kinases [QD415. S451 2006 f rb].

Kolina kinase (CK) dan etanolamina kinase (EK) merupakan enzim pertama di dalam biosintesis fosfatidilkolina dan fosfatidiletanolamina. Peningkatan aktiviti CK telah dikaitkan dengan tumor manusia. Oleh itu, perencatan CK telah dicadangkan sebagai strategi antikanser yang berpotensi. Choline kinase (CK) and ethanolamine kinase (EK) are the first enzymes in the biosynthesis of phosphatidylcholine and phosphatidylethanolamine. Increased activity of CK has been implicated in human tumors. CK inhibition has therefore been proposed as a potential anticancer strategy

Effects of protein kinase a phosphorylation on the biochemical properties and subcellular location of human choline kinase beta

Choline kinase is the most upstream enzyme in the CDP-choline pathway. It catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP and Mg2+ during the biosynthesis of phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase (CK) is encoded by two separate genes, cka and ckp, which produce three isoforms, CKa1, CKa2, and CKp. Previous studies have associated ckp with muscle development; however, the molecular mechanism underlying the transcriptional regulation of ckp has never been elucidated. In this report, the distal promoter region of the ckp gene was characterized. Mutational analysis of the promoter sequence and electrophoretic mobility shift assays (EMSA) showed that Ets and GATA transcription factors were essential for the repression of ckp promoter activity. Supershift and chromatin immunoprecipitation (ChIP) assays further identified that GATA3 but not GATA2 was bound to the GATA site of ckp promoter. In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckp promoter activity through Ets and GATA elements. PMA also decreased the ckp mRNA and protein levels about 12 hours after the promoter activity was down-regulated. EMSA further revealed that PMA treatment increased the binding of both Ets and GATA transcription factors to their respective DNA elements. The PMA-mediated repressive effect was abolished by chronic PMA treatment and by treatment with the PKC inhibitor PKC412, but not the PKC inhibitor Go 6983, suggesting PKCe or PKCq as the PKC isozyme involved in the PMA-mediated repression of ckp promoter. Further confirmation by using PKC isozyme specific inhibitors identified PKCe as the isozyme that mediated the PMA repression of ckp promoter

Biochemical Characterization Of Human And Yeast Choline And Ethanolamine Kinases

Kolina kinase (CK) dan etanolamina kinase (EK) merupakan enzim pertama di dalam biosintesis fosfatidilkolina dan fosfatidiletanolamina. Choline kinase (CK) and ethanolamine kinase (EK) are the first enzymes in the biosynthesis of phosphatidylcholine and phosphatidylethanolamine

Cloning, characterization and activity analysis of human choline kinase promoters

Choline kinase (CK) is the first enzyme in the COP-choline pathway, a de novo biosynthetic pathway for major phospholipid in the membrane of eukaryotic cells i.e phosphatidylcholine (Lykidis et a/., 2001 ). This enzyme catalyzes the phosphorylation of choline by ATP to form phosphocholine. In mammalian cells, choline kinase exists as three isoforms that are encoded by two separate genes named cka and ck/3. ck/3 codes for a single protein (CKJ3) while cka undergoes alternative splicing to produce CKu1 and CKu2 isoforms (Malito eta/., 2006). Increased activity of CK and its product, phosphocholine, have been implicated in human carcinogenesis. Elevated phosphocholine level is a common feature in cell lines derived from human tumors and this parameter seems to be able to distinguish malignant cell lines from normal cell lines irrespective of their proliferation rates (Bhakoo eta/., 1996; Aboagye and Bhujwalla, 1999). Overexpression of CK has been reported in a variety of human cancers such as lung, colo rectal as well as prostate adenocarcinomas (Nakagami et a/., 1999, Ramirez de Molina eta/., 2002a, Ramirez de Molina eta/., 2002b). In addition, studies had demonstrated the increased of CK activity upon induction of the H-ras oncogene in mouse fibroblast cell lines. Inhibition of CK has been proposed to be a potential antitumor strategy (Rodriguez-Gonzalez eta/., 2004). Rodriguez-Gonzalez et al. (2004) demonstrated that CK inhibitors could become potent antitumor drugs both in vitro and in vivo. Recently, CKu protein levels have been found to be drastically increased in both human tumors and cell lines derived from human tumor, when compared to normal tissues or appropriate human primary cells, respectively (Aoyama et a/., 2004). Increased levels of CKu mRNA but not CKJ3 in tumor-derived cell lines was also reported (Gallego-Ortega eta/., 2009)

Highly Specific Antibodies for Co-Detection of Human Choline Kinase α1 and α2 Isoforms

BACKGROUND: Choline kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKα1, α2, and β). Specific inhibition of CKα has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CKα used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the α1 or the α2 isoform. METHODOLOGY/PRINCIPAL FINDINGS: In this study, an antiserum against CKα was produced by immunizing rabbits with denatured, purified recombinant CKα2 full-length protein. This antiserum was highly specific for CKα when tested with extracts from different cell lines, and there was no cross reactivity with purified CKβ and other related proteins like human ethanolamine kinases (EK) and yeast choline or ethanolamine kinases. The antiserum simultaneously detected both CKα1 and α2 isoforms in MCF-7 and HepG2 cell extracts, but not in HeLa, HCT-116, and mouse embryonic stem cell extracts. Subsequent protein dot blot assay of total CKα in a human normal/tumor protein array of 30 tissue samples by using the antiserum showed that CKα was not overexpressed in all tumor tissues when compared to their normal counterparts. Most striking differences between tumor and normal CKα expression levels were observed in kidney (11-fold higher in tumor) and liver (15-fold lower in tumor) samples. CONCLUSION/SIGNIFICANCE: Apart from its high sensitivity and specificity, the antiserum produced in this work, which does not require further purification, has the advantage of co-detecting both α1 and α2 isoforms in cell extracts for direct comparison of their expression levels

AP4 transcription factor binding site is a repressor element in ek2 promoter of human liver carcinoma cell line, HepG2

Ethanolamine kinase (EK) is the first enzyme in the Kennedy pathway for the biosynthesis of phosphatidylethanolamine. Although EK has been reported to be involved in phospholipid biosynthesis, carcinogenesis, cell growth, muscle development and sex determination during embryonic development, little is known about its transcriptional regulation by endogenous or exogenous signals. Human EK exists as EK1, EK2α and EK2β isoforms, encoded by two separate genes, named ek1 and ek2. Compared to ek1 gene, ek2 is expressed at a higher level in liver and EK2 isoforms also accept choline as substrate besides ethanolamine, which could contribute to liver carcinogenesis. The main aim of this study was to analyze and characterize the human ek2 promoter in cultured mammalian cells. Human ek2 (2011 bp) promoter was cloned into reporter vector, pGL4.10 [luc2] and the promoter activities were studied in human liver carcinoma (HepG2 cells). Sequence analyses showed that ek2 promoter contains numerous putative transcription factor binding sites including AP4 and it is devoid of a recognizable consensus TATA box but it contains a high number of guanine (G) and cytosine (C) nucleotides. PCR mutagenesis of three nucleotides at E-box motif of AP4 transcription binding site located between -293 and -276 of ek2 promoter was successfully performed to show that AP4 transcription factor binding site acts as a repressive element in the regulation of ek2 expression. AP4 upregulation has been implicated in bad prognosis of carcinoma, therefore the regulatory role of AP4 binding site reported in this study could be a link between ek2 and carcinogenesis. Although further studies need to be carried out to understand and to determine the repression mechanism of AP4 in ek2 promoter, the characterization and analysis of ek promoter performed in this study provide important understanding of its basal transcriptional regulation which would allow us to control ek expression levels in pathologic conditions that involve this gene

DNA methylation of human choline kinase alpha gene

Increased level of choline kinase (CK) is a common feature in cancers and inhibition of this enzyme has been applied as anticancer strategy. DNA methylation of gene promoter especially at CpG island is associated with suppression of gene expression. Despite the importance of CK especially the alpha isoform in cancer pathogenesis, epigenetic regulation of ckα expression has not been investigated. Hence, this study aimed to determine the effect of DNA methylation on ckα promoter activity and gene expression by using hypomethylating (5-aza) and methylating (budesonide) agents. The level of DNA methylation in the second CpG island of ckα promoter was determined by PCR-based method. 5-aza and budesonide increased the methylation of the selected CpG island compared to untreated control. Treatment with the drugs produced opposite effect, with 5-aza induced ckα promoter activity and gene expression while budesonide suppressed the promoter activity and mRNA level of this gene. Deletion of a region containing the second CpG island on ckα promoter resulted in significantly lower promoter activity. In conclusion, this study showed that DNA methylation could be one of the mechanisms that regulate the expression of ckα gene

Structural Modeling and Biochemical Characterization of Recombinant KPN_02809, a Zinc-Dependent Metalloprotease from Klebsiella pneumoniae MGH 78578

Klebsiella pneumoniae is a Gram-negative, cylindrical rod shaped opportunistic pathogen that is found in the environment as well as existing as a normal flora in mammalian mucosal surfaces such as the mouth, skin, and intestines. Clinically it is the most important member of the family of Enterobacteriaceae that causes neonatal sepsis and nosocomial infections. In this work, a combination of protein sequence analysis, structural modeling and molecular docking simulation approaches were employed to provide an understanding of the possible functions and characteristics of a hypothetical protein (KPN_02809) from K. pneumoniae MGH 78578. The computational analyses showed that this protein was a metalloprotease with zinc binding motif, HEXXH. To verify this result, a ypfJ gene which encodes for this hypothetical protein was cloned from K. pneumoniae MGH 78578 and the protein was overexpressed in Escherichia coli BL21 (DE3). The purified protein was about 32 kDa and showed maximum protease activity at 30 °C and pH 8.0. The enzyme activity was inhibited by metalloprotease inhibitors such as EDTA, 1,10-phenanthroline and reducing agent, 1,4-dithiothreitol (DTT). Each molecule of KPN_02809 protein was also shown to bind one zinc ion. Hence, for the first time, we experimentally confirmed that KPN_02809 is an active enzyme with zinc metalloprotease activity

ChoK-ing the Pathogenic Bacteria: Potential of Human Choline Kinase Inhibitors as Antimicrobial Agents

Novel antimicrobial agents are crucial to combat antibiotic resistance in pathogenic bacteria. Choline kinase (ChoK) in bacteria catalyzes the synthesis of phosphorylcholine, which is subsequently incorporated into the cell wall or outer membrane. In certain species of bacteria, phosphorylcholine is also used to synthesize membrane phosphatidylcholine. Numerous human ChoK inhibitors (ChoKIs) have been synthesized and tested for anticancer properties. Inhibition of S. pneumoniae ChoK by human ChoKIs showed a promising effect by distorting the cell wall and retarded the growth of this pathogen. Comparison of amino acid sequences at the catalytic sites of putative choline kinases from pathogenic bacteria and human enzymes revealed striking sequence conservation that supports the potential application of currently available ChoKIs for inhibiting bacterial enzymes. We also propose the combined use of ChoKIs and nanoparticles for targeted delivery to the pathogen while shielding the human host from any possible side effects of the inhibitors. More research should focus on the verification of putative bacterial ChoK activities and the characterization of ChoKIs with active enzymes. In conclusion, the presence of ChoK in a wide range of pathogenic bacteria and the distinct function of this enzyme has made it an attractive drug target. This review highlighted the possibility of “choking” bacterial ChoKs by using human ChoKIs

Sp1 and Sp3 Are the Transcription Activators of Human ek1 Promoter in TSA-Treated Human Colon Carcinoma Cells.

Ethanolamine kinase (EK) catalyzes the phosphorylation of ethanolamine, the first step in the CDP-ethanolamine pathway for the biosynthesis of phosphatidylethanolamine (PE). Human EK exists as EK1, EK2α and EK2β isoforms, encoded by two separate genes, named ek1 and ek2. EK activity is stimulated by carcinogens and oncogenes, suggesting the involvement of EK in carcinogenesis. Currently, little is known about EK transcriptional regulation by endogenous or exogenous signals, and the ek gene promoter has never been studied.In this report, we mapped the important regulatory regions in the human ek1 promoter. 5' deletion analysis and site-directed mutagenesis identified a Sp site at position (-40/-31) that was essential for the basal transcription of this gene. Treatment of HCT116 cells with trichostatin A (TSA), a histone deacetylase inhibitor, significantly upregulated the ek1 promoter activity through the Sp(-40/-31) site and increased the endogenous expression of ek1. Chromatin immunoprecipitation assay revealed that TSA increased the binding of Sp1, Sp3 and RNA polymerase II to the ek1 promoter in HCT116 cells. The effect of TSA on ek1 promoter activity was cell-line specific as TSA treatment did not affect ek1 promoter activity in HepG2 cells.In conclusion, we showed that Sp1 and Sp3 are not only essential for the basal transcription of the ek1 gene, their accessibility to the target site on the ek1 promoter is regulated by histone protein modification in a cell line dependent manner