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 CKβ 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 CKβ and its interaction with CKα. With the aim of profiling the intracellular roles of CKα and CKβ, 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 β 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, CKβ‐ and CKα + CKβ‐silenced cells had no aberrant phenotype. Therefore, our results support the hypothesis that the balance of the α and β 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 CKβ.

BackgroundCholine 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 FindingsIn 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/SignificanceApart 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.

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.

Klebsiella pneumoniae causes neonatal sepsis and nosocomial infections. One of the strains, K. pneumoniae MGH 78578, shows high level of resistance to multiple microbial agents. In this study, domain family, amino acid sequence and topology analyses were performed on one of its hypothetical protein, YggG (KPN_03358). Structural bioinformatics approaches were used to predict the structure and functionality of YggG protein. The open reading frame (ORF) of yggG, which was a putative metalloprotease gene, was also cloned, expressed and characterized. The ORF was PCR amplified from K. pneumoniae MGH 78578 genomic DNA and cloned into a pET14-b vector for heterologous expression in Escherichia coli. The purified YggG protein was subsequently assayed for casein hydrolysis under different conditions. This protein was classified as peptidase M48 family and subclan gluzincin. It was predicted to contain one transmembrane domain by TMpred. Optimal protein expression was achieved by induction with 0.6 mM isopropyl thiogalactoside (IPTG) at 25 °C for six hours. YggG was purified as soluble protein and confirmed to be proteolytically active under the presence of 1.25 mM zinc acetate and showed optimum activity at 37 °C and pH 7.4. We confirmed for the first time that the yggG gene product is a zinc-dependent metalloprotease.