Identification and Characterization of Entamoeba histolytica Choline Kinase.

PURPOSE: Entamoeba histolytica is one of the death-causing parasites in the world. Study on its lipid composition revealed that it is predominated by phosphatidylcholine and phosphatidylethanolamine. Further study revealed that its phosphorylated metabolites might be produced by the Kennedy pathway. Here, we would like to report on the characterizations of enzymes from this pathway that would provide information for the design of novel inhibitors against these enzymes in future. METHODOLOGY: E. histolytica HM-1:IMSS genomic DNA was isolated and two putative choline/ethanolamine kinase genes (EhCK1 and EhCK2) were cloned and expressed from Escherichia coli BL21 strain. Enzymatic characterizations were further carried out on the purified enzymes. RESULTS: EhCK1 and EhCK2 were identified from E. histolytica genome. The deduced amino acid sequences were more identical to its homologues in human (35-48%) than other organisms. The proteins were clustered as ethanolamine kinase in the constructed phylogeny tree. Sequence analysis showed that they possessed all the conserved motifs in choline kinase family: ATP-binding loop, Brenner's phosphotransferase motif, and choline kinase motif. Here, the open reading frames were cloned, expressed, and purified to apparent homogeneity. EhCK1 showed activity with choline but not ethanolamine. The biochemical characterization showed that it had a Vmax of 1.9 ± 0.1 µmol/min/mg. Its Km for choline and ATP was 203 ± 26 µM and 3.1 ± 0.4 mM, respectively. In contrast, EhCK2 enzymatic activity was only detected when Mn2+ was used as the co-factor instead of Mg2+ like other choline/ethanolamine kinases. Highly sensitive and specific antibody against EhCK1 was developed and used to confirm the endogenous EhCK1 expression using immunoblotting. CONCLUSIONS: With the understanding of EhC/EK importance in phospholipid metabolism and their unique characteristic, EhC/EK could be a potential target for future anti-amoebiasis study.

Dysregulated expression of miR­367 in disease development and its prospects as a therapeutic target and diagnostic biomarker (Review).

MicroRNA (miR)-367 has a wide range of functions in gene regulation and as such plays a critical role in cell proliferation, differentiation and development, making it an essential molecule in various physiological processes. miR-367 belongs to the miR-302/367 cluster and is located in the intronic region of human chromosome 4 on the 4q25 locus. Dysregulation of miR-367 is associated with various disease conditions, including cancer, inflammation and cardiac conditions. Moreover, miR-367 has shown promise both as a tumor suppressor and a potential diagnostic biomarker for breast, gastric and prostate cancer. The elucidation of the essential role of miR-367 in inflammation, development and cardiac diseases emphasizes its versatility in regulating various physiological processes beyond cancer biology. However, further research is necessary to fully understand the complex regulatory mechanisms involving miR-367 in different physiological and pathological contexts. In conclusion, the versatility and significance of miR-367 makes it a promising candidate for further study and in the development of new diagnostic and therapeutic strategies.

Unusual metal ion cofactor requirement of Entamoeba histolytica choline and ethanolamine kinase isoforms.

The de novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine in Entamoeba histolytica is largely dependent on the CDP-choline and CDP-ethanolamine pathways. Although the first enzymes of these pathways, EhCK1 and EhCK2, have been previously characterized, their enzymatic activity was found to be low and undetectable, respectively. This study aimed to identify the unusual characteristics of these enzymes in this deadly parasite. The discovery that EhCKs prefer Mn2+ over the typical Mg2+ as a metal ion cofactor is intriguing for CK/EK family of enzymes. In the presence of Mn2+, the activity of EhCK1 increased by approximately 108-fold compared to that in Mg2+. Specifically, in Mg2+, EhCK1 exhibited a Vmax and K0.5 of 3.5 ± 0.1 U/mg and 13.9 ± 0.2 mM, respectively. However, in Mn2+, it displayed a Vmax of 149.1 ± 2.5 U/mg and a K0.5 of 9.5 ± 0.1 mM. Moreover, when Mg2+ was present at a constant concentration of 12 mM, the K0.5 value for Mn2+ was ~ 2.4-fold lower than that in Mn2+ alone, without affecting its Vmax. Although the enzyme efficiency of EhCK1 was significantly improved by about 25-fold in Mn2+, it is worth noting that its Km for choline and ATP were higher than in equimolar of Mg2+ in a previous study. In contrast, EhCK2 showed specific activity towards ethanolamine in Mn2+, exhibiting Michaelis-Menten kinetic with ethanolamine (Km = 312 ± 27 µM) and cooperativity with ATP (K0.5 = 2.1 ± 0.2 mM). Additionally, we investigated the effect of metal ions on the substrate recognition of human choline and ethanolamine kinase isoforms. Human choline kinase α2 was found to absolutely require Mg2+, while choline kinase ß differentially recognized choline and ethanolamine in Mg2+ and Mn2+, respectively. Finally, mutagenesis studies revealed that EhCK1 Tyr129 was critical for Mn2+ binding, while Lys233 was essential for substrate catalysis but not metal ion binding. Overall, these findings provide insight into the unique characteristics of the EhCKs and highlight the potential for new approaches to treating amoebiasis. Amoebiasis is a challenging disease for clinicians to diagnose and treat, as many patients are asymptomatic. However, by studying the enzymes involved in the CDP-choline and CDP-ethanolamine pathways, which are crucial for de novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine in Entamoeba histolytica, there is great potential to discover new therapeutic approaches to combat this disease.

DNA Methylation of Human Choline Kinase Alpha Promoter-Associated CpG Islands in MCF-7 Cells.

Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between -225 and -56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.

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.

Downregulation of human choline kinase α gene expression by miR-876-5p.

Choline kinase (CK) is the first enzyme in the CDP-choline pathway for the synthesis of phosphatidylcholine, the most abundant phospholipid in the mammalian cell membrane. This enzyme exists as three isozymes (α1, α2 and ß) and the CKα isozyme has been implicated in cancer pathogenesis. Inhibition of CK activity has been proposed for cancer therapies. MicroRNAs (miRNAs/miRs) are non­coding RNAs that serve important roles in diverse biological pathways and human diseases, including cancer. However, the regulation of CKα gene expression by miRNAs has never been investigated, to the best of the authors' knowledge. In the present study, two miRNA mimics, miR­876­5p and miR­646, were transfected into the HepG2 cell line and the effect of these miRNAs on the levels of CKα mRNA were determined by reverse transcription­quantitative polymerase chain reaction. Cells transfected with 25 nM miR­876­5p for 48 h exhibited significantly lower levels of CKα mRNA. Following optimization, miR­876­5p caused four times lower levels of CKα mRNA compared to the negative control. Effects of the miRNAs on HepG2 cell viability and cellular morphology were additionally analyzed using an MTT cell viability assay and scanning electron microscopy, respectively. HepG2 cells that were transfected with the optimum concentration of miR­876­5p for the optimum duration exhibited 25% lower viability than negative control and signs of apoptosis in electron micrographs. The results suggested miR­876­5p as a potential miRNA modulator of CKα expression in the cells, and may be relevant for the design of more effective anticancer strategy targeting CK.

Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition.

Choline kinase beta (CKß) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKß is important for normal mitochondrial function and muscle development as the lack of the ckß gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme's activity and its subcellular location. This study provides evidence for CKß phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKß by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKß phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKß was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKß residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKß phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKß to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKß, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis.

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

BACKGROUND: 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 EK2ß 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSION/SIGNIFICANCE: 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.

Systemic and coronary levels of CRP, MPO, sCD40L and PlGF in patients with coronary artery disease.

BACKGROUND: Biomarkers play a pivotal role in the diagnosis and management of patients with acute coronary syndrome. This study aimed to investigate the differences in level of several biomarkers, i.e. C-reactive protein, myeloperoxidase, soluble CD40 ligand and placental growth factor, between acute coronary syndrome and chronic stable angina patients. The relationship between these biomarkers in the coronary circulation and systemic circulation was also investigated. METHODS: A total of 79 patients were recruited in this study. The coronary blood was sampled from occluded coronary artery, while the peripheral venous blood was withdrawn from antecubital fossa. The serum concentrations of C-reactive protein, soluble CD40 ligand and placental growth factor and plasma concentration of myeloperoxidase were measured using ELISA method. RESULTS: The systemic level of the markers measured in the peripheral venous blood was significantly increased in acute coronary syndrome compared to chronic stable angina patients. The concentrations of the C-reactive protein, myeloperoxidase and soluble CD40 ligand taken from peripheral vein were closely similar to the concentration found in coronary blood of ACS patients. The level of placental growth factor was significantly higher in coronary circulation than its systemic level. CONCLUSION: The concentration of these C-reactive protein, myeloperoxidase, soluble CD40 ligand and placental growth factor were significantly increased in acute coronary syndrome patients. The concentration of the markers measured in the systemic circulation directly reflected those in the local coronary circulation. Thus, these markers have potential to become a useful tool in predicting plaque vulnerability in the future.

Ets and GATA transcription factors play a critical role in PMA-mediated repression of the ckß promoter via the protein kinase C signaling pathway.

BACKGROUND: 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, ckα and ckß, which produce three isoforms, CKα1, CKα2, and CKß. Previous studies have associated ckß with muscle development; however, the molecular mechanism underlying the transcriptional regulation of ckß has never been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: In this report, the distal promoter region of the ckß 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 ckß promoter activity. Supershift and chromatin immunoprecipitation (ChIP) assays further identified that GATA3 but not GATA2 was bound to the GATA site of ckß promoter. In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckß promoter activity through Ets and GATA elements. PMA also decreased the ckß 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 PKCε or PKCη as the PKC isozyme involved in the PMA-mediated repression of ckß promoter. Further confirmation by using PKC isozyme specific inhibitors identified PKCε as the isozyme that mediated the PMA repression of ckß promoter. CONCLUSION/SIGNIFICANCE: These results demonstrate the participation of the PKC signaling pathway in the regulation of ckß gene transcription by Ets and GATA transcription factors.

Development of a thermostabilized, one-step, nested, tetraplex PCR assay for simultaneous identification and differentiation of Entamoeba species, Entamoeba histolytica and Entamoeba dispar from stool samples.

Entamoeba histolytica is the only Entamoeba species that causes amoebiasis in humans. Approximately 50 million people are infected, with 100, 000 deaths annually in endemic countries. Molecular diagnosis of Entamoeba histolytica is important to differentiate it from the morphologically identical Entamoeba dispar to avoid unnecessary medication. Conventional molecular diagnostic tests require trained personnel, cold-chain transportation and/or are storage-dependent, which make them user-unfriendly. The aim of this study was to develop a thermostabilized, one-step, nested, tetraplex PCR assay for the detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba species in cold-chain-free and ready-to-use form. The PCR test was designed based on the Entamoeba small subunit rRNA (SSU-rRNA) gene, which detects the presence of any Entamoeba species, and simultaneously can be used to differentiate Entamoeba histolytica from Entamoeba dispar. In addition, a pair of primers was designed to serve as an internal amplification control to help identify inhibitors in the samples. All PCR reagents together with the designed primers were thermostabilized by lyophilization and were stable at 24 °C for at least 6 months. The limit of detection of the tetraplex PCR was found to be 39 pg DNA or 1000 cells for Entamoeba histolytica and 78 pg DNA or 1000 cells for Entamoeba dispar, and the specificity was 100 %. In conclusion, this cold-chain-free, thermostabilized, one-step, nested, multiplex PCR assay was found to be efficacious in differentiating Entamoeba histolytica from other non-pathogenic Entamoeba species.

Balance of human choline kinase isoforms is critical for cell cycle regulation: implications for the development of choline kinase-targeted cancer therapy.

The enzyme choline kinase (CK), which catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP, has an essential role in the biosynthesis of phosphatidylcholine, the major constituent of all mammalian cell membranes. CK is encoded by two separate genes expressing the three isoforms CKα1, CKα2 and CKß that are active as homodimeric or heterodimeric species. Metabolic changes observed in various cancer cell lines and tumors have been associated with differential and marked up-regulation of the CKα genes, and specific inhibition of CKα activity has been proposed as a potential anti-cancer strategy. As a result, less attention has been given to CKß and its interaction with CKα. With the aim of profiling the intracellular roles of CKα and CKß, we used RNA interference (RNAi) as a molecular approach to down-regulate the expression of CK in HeLa cells. Individual and simultaneous RNAi-based silencing of the CK α and ß isoforms was achieved using different combinations of knockdown strategies. Efficient knockdown was confirmed by immunodetection using our isoform-specific antibodies and by quantitative real-time PCR. Our analyses of the phenotypic consequences of CK depletion showed the expected lethal effect of CKα knockdown. However, CKß- and CKα + CKß-silenced cells had no aberrant phenotype. Therefore, our results support the hypothesis that the balance of the α and ß isoforms is critical for cancer cell survival. The suppression of the cancer cell killing effect of CKα silencing by simultaneous knockdown of both isoforms implies that a more effective CK-based anti-cancer strategy can be achieved by reducing cross-reactivity with CKß.

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.

Novel 4-amino bis-pyridinium and bis-quinolinium derivatives as choline kinase inhibitors with antiproliferative activity against the human breast cancer SKBR-3 cell line.

Choline kinase (ChoK) is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. Human ChoK has three isoforms: ChoKα1, α2, and ß. Specific inhibition of ChoKα has been reported to selectively kill tumor cells. In this study, ten new symmetrical bis-pyridinium and bis-quinolinium derivatives were synthesized and tested for their ability to inhibit human ChoKα2. These compounds have electron-releasing groups at position 4 of the pyridinium or quinolinium rings. 1,1'-[(Butane-1,3-diylbis(benzene-1,4-diylmethylene)]bis[4-(4-bromo-N-methylanilino)pyridinium)] dibromide and 1,1'-(biphenyl-3,3'-diylmethylene)bis[7-chloro-4-(perhydroazepine-1-yl)quinolinium] dibromide were identified as highly potent ChoK inhibitors with IC(50) values of 80 nM. Kinetic enzymatic assays indicated a mixed and predominantly competitive mechanism of inhibition for these compounds, which exhibited strong antiproliferative activity (EC(50) 1 µM) against the human breast cancer SKBR3 cell line.

UBC and YWHAZ as suitable reference genes for accurate normalisation of gene expression using MCF7, HCT116 and HepG2 cell lines.

Relative quantification of in vitro gene expression using real-time PCR requires stably expressed reference gene for normalisation. In this study, total RNA from MCF7, HCT116 and HepG2 cells were extracted and converted to cDNA using commercially available kit, and real-time PCR was then performed to analyse the expression levels of twelve reference genes to select the most ideal reference gene for accurate normalisation in gene expression study. geNorm and NormFinder software were used to analyse the stabilities of the reference genes, which showed a wide range of C(t) values. The geNorm analysis showed the following ranking for stability of genes: UBC, YWHAZ > RPLP > TBP > ACTB > HPRT1 > PPIA > GAPDH > GUSB > B2M > TUBB > RRN18S. A similar ranking of reference genes was obtained by NormFinder, and the four most stable reference genes were identical using both approaches. UBC and YWHAZ were proposed to be the two most suitable reference genes based on the above analyses. To further assess the stabilities of the UBC and YWHAZ in a formal experiment, MCF7, HCT116 and HepG2 cell lines were subjected to treatments with 5-aza-dC and TSA. Both UBC and YWHAZ exhibited stable expression levels across control and treatment groups. Therefore, we propose that UBC and YWHAZ are the two most suitable reference genes for our gene expression studies using MCF7, HCT116 and HepG2 cell lines.