A Unique Mode of Coenzyme A Binding to the Nucleotide Binding Pocket of Human Metastasis Suppressor NME1.

Coenzyme A (CoA) is a key cellular metabolite which participates in diverse metabolic pathways, regulation of gene expression and the antioxidant defense mechanism. Human NME1 (hNME1), which is a moonlighting protein, was identified as a major CoA-binding protein. Biochemical studies showed that hNME1 is regulated by CoA through both covalent and non-covalent binding, which leads to a decrease in the hNME1 nucleoside diphosphate kinase (NDPK) activity. In this study, we expanded the knowledge on previous findings by focusing on the non-covalent mode of CoA binding to the hNME1. With X-ray crystallography, we solved the CoA bound structure of hNME1 (hNME1-CoA) and determined the stabilization interactions CoA forms within the nucleotide-binding site of hNME1. A hydrophobic patch stabilizing the CoA adenine ring, while salt bridges and hydrogen bonds stabilizing the phosphate groups of CoA were observed. With molecular dynamics studies, we extended our structural analysis by characterizing the hNME1-CoA structure and elucidating possible orientations of the pantetheine tail, which is absent in the X-ray structure due to its flexibility. Crystallographic studies suggested the involvement of arginine 58 and threonine 94 in mediating specific interactions with CoA. Site-directed mutagenesis and CoA-based affinity purifications showed that arginine 58 mutation to glutamate (R58E) and threonine 94 mutation to aspartate (T94D) prevent hNME1 from binding to CoA. Overall, our results reveal a unique mode by which hNME1 binds CoA, which differs significantly from that of ADP binding: the α- and ß-phosphates of CoA are oriented away from the nucleotide-binding site, while 3'-phosphate faces catalytic histidine 118 (H118). The interactions formed by the CoA adenine ring and phosphate groups contribute to the specific mode of CoA binding to hNME1.

Bacillus anthracis' PA63 Delivers the Tumor Metastasis Suppressor Protein NDPK-A/NME1 into Breast Cancer Cells.

Some highly metastatic types of breast cancer show decreased intracellular levels of the tumor suppressor protein NME1, also known as nm23-H1 or nucleoside diphosphate kinase A (NDPK-A), which decreases cancer cell motility and metastasis. Since its activity is directly correlated with the overall outcome in patients, increasing the cytosolic levels of NDPK-A/NME1 in such cancer cells should represent an attractive starting point for novel therapeutic approaches to reduce tumor cell motility and decrease metastasis. Here, we established the Bacillus anthracis protein toxins' transport component PA63 as transporter for the delivery of His-tagged human NDPK-A into the cytosol of cultured cells including human MDA-MB-231 breast cancer cells. The specifically delivered His6-tagged NDPK-A was detected in MDA-MB-231 cells via Western blotting and immunofluorescence microscopy. The PA63-mediated delivery of His6-NDPK-A resulted in reduced migration of MDA-MB-231 cells, as determined by a wound-healing assay. In conclusion, PA63 serves for the transport of the tumor metastasis suppressor NDPK-A/NME1 into the cytosol of human breast cancer cells in vitro, which reduced the migratory activity of these cells. This approach might lead to development of novel therapeutic options.

Analysis of 1- and 3-Phosphohistidine (pHis) Protein Modification Using Model Enzymes Expressed in Bacteria.

Despite the discovery of protein histidine (His) phosphorylation nearly six decades ago, difficulties in measuring and quantifying this unstable post-translational modification (PTM) have limited its mechanistic analysis in prokaryotic and eukaryotic signaling. Here, we describe reliable procedures for affinity purification, cofactor-binding analysis and antibody-based detection of phosphohistidine (pHis), on the putative human His kinases NME1 (NDPK-A) and NME2 (NDPK-B) and the glycolytic phosphoglycerate mutase PGAM1. By exploiting isomer-specific monoclonal N1-pHis and N3-pHis antibodies, we describe robust protocols for immunological detection and isomer discrimination of site-specific pHis, including N3-pHis on His 11 of PGAM1.

A novel photoelectrochemical immunosensor by integration of nanobody and ZnO nanorods for sensitive detection of nucleoside diphosphatase kinase-A.

Nucleoside diphosphatase kinase A (NDPK-A) is a metastasis-suppressor protein and a biomarker that act on a wide range cancer cells to inhibit the potential metastasis. Herein, we present a simple photoelectrochemical immunosensor based on ZnO nanorod arrays for the sensitive detection of NDPK-A. The ZnO nanorod arrays cosensitized with CdS nanoparticles and Mn2+ displayed a high and stable photocurrent response under irradiation. After anti-NPDK-A nanobodies were immobilized to the ZnO nanorod arrays, the proposed immunosensor can be utilized for detecting NPDK-A by monitoring the changes in the photocurrent signals of the electrode resulting from immunoreaction. Accordingly, the well-designed immunosensor exhibited a low limit of detection (LOD) of 0.3 pg mL-1 and a wide linear range from 0.5 pg mL-1 to 10 µg mL-1. The R2 of the regression curve is 0.99782. Meanwhile, the good stability, reproducibility and specificity of the resulting photoelectrochemical biosensor are demonstrated. In addition, the presented work would offer a novel and simple approach for the detection of immunoreactions and provide new insights in popularizing the diagnosis of NPDK-A.