Identification of important residues for catalysis and substrate specificity in human choline kinase by site directed mutagenesis

Phosphatidylcholine (PtdCho) is a prominent constituent of eukaryotic and some prokaryotic membranes. Choline kinase (CK), the initial enzyme of the COP-choline pathway, mediates the conversion of choline to phosphorylcholine and is localized in the supernatant fraction of cells. CK has been recognized as a new target for anticancer therapy. To identify the amino acid residue of human CK (hCK) that is important for catalysis, conserved aspartate at position 342 in hCKa2 was mutated to asparagine. The mutant construct was successfully cloned into pET14b vector and overexpressed in E. coli BL21 (DE3). The mutant protein D342NhCKa2 showed dramatic loss of activity, only 12.46% of wild type protein activity remained. The Km for choline of the mutant protein increased 1.18 folds while Km for ethanolamine increased 598 folds compared to wild type. The Km and V max for ATP of mutant protein increased 3 and 4 folds respectively. The increased Km suggested that the residue is important for the binding of the substrates and play a role in catalysis. Mutation of aspartate 342 might also cause the activity inhibition or disruption of homo-dimer complex in hCKa2

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

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

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

Eating with a purpose: development and motivators for consumption of superfood

This research aimed to examine an integrated and modified Health Belief Model by encapsulating the factors influencing consumer likelihood to consume superfoods as adjusted to the Malaysian population. It was conducted in Peninsular Malaysia from May 2019 until October 2019 using a sample size of 1,000 individuals obtained via purposive sampling, whereby the data were analysed by using structural equation modelling. The result showed that consumer likelihood to consume superfoods was positively influenced by perceived benefits and perceived susceptibility, while negatively influenced by perceived barrier. The cue to action had a direct influence on perceived susceptibility, perceived seriousness, and perceived benefits. Surprisingly, the cue to action was not too influential on perceived barrier and likelihood to consume superfoods. Nevertheless, the proposed modified Health Belief Model fitted the data better than the original model. This implied that it is important to focus on the cue to action especially in the superfood-buying context as opposed to the original Health Belief Model which neglected the cue to action

Concurrent immunoblot detection of CKα1 and α2 isoforms in MCF-7 (lane 1) and HepG2 (lane 3) cell lysates.

<p>Only CKα1 was detected in the HCT-116 cell lysate (lane 2). 5 ng of each purified CKα1 and α2 were loaded as references (lane +). 50 µg of each cell lysate were loaded and detection was performed with 10000-fold dilution of CKα antiserum. Results are representative of triplicate experiments with similar results.</p

Immunoblot detection of human and baker's yeast choline and ethanolamine kinases showing isoform specificity of CKα antiserum.

<p>Detection of purified hCKα1 (1), hCKα2 (2), hCKβ (3), Δ89N-hEK1 (4), hEK2α (5), Δ49N-hCKα2 (6), Δ84N-hCKα2 (7), yCK (8) and yEK (9) were performed with 10000-fold dilution of CKα antiserum. Each lane was loaded with 50 ng of purified protein. Lane M is ChemiBlot molecular weight marker.</p

Immunoblot detection showing specificity of CKα antiserum in HeLa (A) and mouse embryonic stem cell (B) lysates.

<p>The detection was performed with different dilutions of CKα antiserum for HeLa cell lysate. 6 ng of purified hCKα2 was used as the positive control (+). 10000-fold dilution of CKα antiserum was used for detection of mouse CKα in 50 µg of mouse embryonic stem cell protein lysate (E), using 50 µg of HeLa cell protein extract as the positive control (H). M: ChemiBlot molecular weight marker. Results are representative of triplicate experiments with similar results.</p

Differential expression of CKα in human normal and tumor tissues.

<p>(A) Levels of total CKα in 30 human normal and tumor tissues. The signal intensities (arbitrary unit) were normalized with respect to the corresponding GAPDH signals. (B) Pairwise comparison of tumor/normal CKα levels. Only tissues showing detectable levels of CKα in both tumor and normal samples were analyzed. Positive and negative fold changes indicate higher and lower expression in tumor, respectively. (C) CKα signal detected on the panel by using 20000-fold diluted CKα antiserum. (D) GAPDH detection assay provided by the supplier; it was used for signal normalization. (E) Identities of protein spots on the panel.</p