The mitochondrially-localized nucleoside diphosphate kinase D (NME4) is a novel metastasis suppressor.

BACKGROUND: Mitochondrial nucleoside diphosphate kinase (NDPK-D, NME4, NM23-H4) is a multifunctional enzyme mainly localized in the intermembrane space, bound to the inner membrane. RESULTS: We constructed loss-of-function mutants of NDPK-D, lacking either NDP kinase activity or membrane interaction and expressed mutants or wild-type protein in cancer cells. In a complementary approach, we performed depletion of NDPK-D by RNA interference. Both loss-of-function mutations and NDPK-D depletion promoted epithelial-mesenchymal transition and increased migratory and invasive potential. Immunocompromised mice developed more metastases when injected with cells expressing mutant NDPK-D as compared to wild-type. This metastatic reprogramming is a consequence of mitochondrial alterations, including fragmentation and loss of mitochondria, a metabolic switch from respiration to glycolysis, increased ROS generation, and further metabolic changes in mitochondria, all of which can trigger pro-metastatic protein expression and signaling cascades. In human cancer, NME4 expression is negatively associated with markers of epithelial-mesenchymal transition and tumor aggressiveness and a good prognosis factor for beneficial clinical outcome. CONCLUSIONS: These data demonstrate NME4 as a novel metastasis suppressor gene, the first localizing to mitochondria, pointing to a role of mitochondria in metastatic dissemination.

Membrane trafficking. Nucleoside diphosphate kinases fuel dynamin superfamily proteins with GTP for membrane remodeling.

Dynamin superfamily molecular motors use guanosine triphosphate (GTP) as a source of energy for membrane-remodeling events. We found that knockdown of nucleoside diphosphate kinases (NDPKs) NM23-H1/H2, which produce GTP through adenosine triphosphate (ATP)-driven conversion of guanosine diphosphate (GDP), inhibited dynamin-mediated endocytosis. NM23-H1/H2 localized at clathrin-coated pits and interacted with the proline-rich domain of dynamin. In vitro, NM23-H1/H2 were recruited to dynamin-induced tubules, stimulated GTP-loading on dynamin, and triggered fission in the presence of ATP and GDP. NM23-H4, a mitochondria-specific NDPK, colocalized with mitochondrial dynamin-like OPA1 involved in mitochondria inner membrane fusion and increased GTP-loading on OPA1. Like OPA1 loss of function, silencing of NM23-H4 but not NM23-H1/H2 resulted in mitochondrial fragmentation, reflecting fusion defects. Thus, NDPKs interact with and provide GTP to dynamins, allowing these motor proteins to work with high thermodynamic efficiency.

Metal sensing and signal transduction by CnrX from Cupriavidus metallidurans CH34: role of the only methionine assessed by a functional, spectroscopic, and theoretical study.

When CnrX, the periplasmic sensor protein in the CnrYXH transmembrane signal transduction complex of Cupriavidus metallidurans CH34, binds the cognate metal ions Ni(II) or Co(II), the ECF-type sigma factor CnrH is made available in the cytoplasm for the RNA-polymerase to initiate transcription at the cnrYp and cnrCp promoters. Ni(II) or Co(II) are sensed by a metal-binding site with a N3O2S coordination sphere with octahedral geometry, where S stands for the thioether sulfur of the only methionine (Met123) residue of CnrX. The M123A-CnrX derivative has dramatically reduced signal propagation in response to metal sensing while the X-ray structure of Ni-bound M123A-CnrXs showed that the metal-binding site was not affected by the mutation. Ni(II) remained six-coordinate in M123A-CnrXs, with a water molecule replacing the sulfur as the sixth ligand. H32A-CnrXs, the soluble model of the wild-type membrane-anchored CnrX, was compared to the double mutants H32A-M123A-CnrXs and H32A-M123C-CnrXs to spectroscopically evaluate the role of this unique ligand in the binding site of Ni or Co. The Co- and Ni-bound forms of the protein display unusually blue-shifted visible spectra. TD-DFT calculations using structure-based models allowed identification and assignment of the electronic transitions of Co-bound form of the protein and its M123A derivative. Among them, the signature of the S-Co transition is distinguishable in the shoulder at 530 nm. In vitro affinity measurements point out the crucial role of Met123 in the selectivity for Ni or Co, and in vivo data support the conclusion that Met123 is a trigger of the signal transduction.