NIK promotes metabolic adaptation of glioblastoma cells to bioenergetic stress.

Cancers, including glioblastoma multiforme (GBM), undergo coordinated reprogramming of metabolic pathways that control glycolysis and oxidative phosphorylation (OXPHOS) to promote tumor growth in diverse tumor microenvironments. Adaptation to limited nutrient availability in the microenvironment is associated with remodeling of mitochondrial morphology and bioenergetic capacity. We recently demonstrated that NF-κB-inducing kinase (NIK) regulates mitochondrial morphology to promote GBM cell invasion. Here, we show that NIK is recruited to the outer membrane of dividing mitochondria with the master fission regulator, Dynamin-related protein1 (DRP1). Moreover, glucose deprivation-mediated metabolic shift to OXPHOS increases fission and mitochondrial localization of both NIK and DRP1. NIK deficiency results in decreased mitochondrial respiration, ATP production, and spare respiratory capacity (SRC), a critical measure of mitochondrial fitness. Although IκB kinase α and ß (IKKα/ß) and NIK are required for OXPHOS in high glucose media, only NIK is required to increase SRC under glucose deprivation. Consistent with an IKK-independent role for NIK in regulating metabolism, we show that NIK phosphorylates DRP1-S616 in vitro and in vivo. Notably, a constitutively active DRP1-S616E mutant rescues oxidative metabolism, invasiveness, and tumorigenic potential in NIK-/- cells without inducing IKK. Thus, we establish that NIK is critical for bioenergetic stress responses to promote GBM cell pathogenesis independently of IKK. Our data suggest that targeting NIK may be used to exploit metabolic vulnerabilities and improve therapeutic strategies for GBM.

Monomeric structure of an active form of bovine cytochrome c oxidase.

Cytochrome c oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a proton pump across the membrane. In all crystals reported to date, bovine CcO exists as a dimer with the same intermonomer contacts, whereas CcOs and related enzymes from prokaryotes exist as monomers. Recent structural analyses of the mitochondrial respiratory supercomplex revealed that CcO monomer associates with complex I and complex III, indicating that the monomeric state is functionally important. In this study, we prepared monomeric and dimeric bovine CcO, stabilized using amphipol, and showed that the monomer had high activity. In addition, using a newly synthesized detergent, we determined the oxidized and reduced structures of monomer with resolutions of 1.85 and 1.95 Å, respectively. Structural comparison of the monomer and dimer revealed that a hydrogen bond network of water molecules is formed at the entry surface of the proton transfer pathway, termed the K-pathway, in monomeric CcO, whereas this network is altered in dimeric CcO. Based on these results, we propose that the monomer is the activated form, whereas the dimer can be regarded as a physiological standby form in the mitochondrial membrane. We also determined phospholipid structures based on electron density together with the anomalous scattering effect of phosphorus atoms. Two cardiolipins are found at the interface region of the supercomplex. We discuss formation of the monomeric CcO, dimeric CcO, and supercomplex, as well as their role in regulation of CcO activity.

Mg(++) requirement for MtHK binding, and Mg(++) stabilization of mitochondrial membranes via activation of MtHK & MtCK and promotion of mitochondrial permeability transition pore closure: A hypothesis on mechanisms underlying Mg(++)'s antioxidant and cytoprotective effects.

Evidence points to magnesium's antioxidant, anti-necrotic, and anti-apoptotic effects in cardio- and neuroprotection. With magnesium being involved in over 300 biochemical reactions, the mechanisms underlying its cytoprotective and antioxidant effects have remained elusive. The profound anti-apoptotic, anabolic, and antioxidant effects of mitochondrion bound hexokinase (MtHk), and the anti-apoptotic, anti-necrotic, and antioxidant functions of mitochondrial creatine kinase (MtCK) have been established over the past few decades. As powerful regulators of the mitochondrial permeability transition pore (PTP), MtHK and MtCK promote anti-apoptosis and anti-necrosis by stabilizing mitochondrial outer and inner membranes. In this article, it is proposed that magnesium is essentially and directly involved in mitochondrial membrane stabilization via (i) Mg(++) ion requirement for the binding of mitochondrial hexokinase (ii) Mg(++)'s allosteric activation of mitochondrial bound hexokinase, and stimulation of mitochondrial bound creatine kinase activities, and (iii) Mg(++) inhibition of PTP opening by Ca(++) ions. These effects of Mg(++) ions are indirectly supplanted by the stimulatory effect of magnesium on the Akt kinase survival pathway. The "Magnesium/Calcium Yin Yang Hypothesis" proposes here that because of the antagonistic effects of Ca(++) and Mg(++) ions in the presence of high Ca(++) ion concentration at MtHK, MtCK, and PTP, magnesium supplementation may provide cytoprotective effects in the treatment of some degenerative diseases and cytopathies with high intracellular [Ca(++)]/ [Mg(++)] ratio at these sites, whether of genetic, developmental, drug induced, ischemic, immune based, toxic, or infectious etiology.

ATP produced by oxidative phosphorylation is channeled toward hexokinase bound to mitochondrial porin (VDAC) in beetroots (Beta vulgaris).

Mitochondrial porins or voltage-dependent anion channels (VDAC) are the main route for solute transport through outer mitochondrial membranes (OMM). In mammals, hexokinase (HK) binds to VDAC, which allows the channeling of ATP synthesized by oxidative phosphorylation toward HK. In plants, although HK has been found associated with OMM, evidence for an interaction with VDAC is scarce. Thus, in this work, we studied the physical and functional interaction between these proteins in beetroot mitochondria. To observe a physical interaction between HK and VDAC, OMM presenting HK activity were prepared from purified mitochondria. Protein complexes were solubilized from OMM with mild detergents and separated by centrifugation in glycerol gradients. Both HK activity and immunodetected VDAC were found in small (9S-13S) and large (>40S) complexes. OMM proteins were also separated according to their hydropathy by serial phase partitioning with Triton X-114. Most of HK activity was found in hydrophobic fractions where VDAC was also present. These results indicated that HK could be bound to VDAC in beetroot mitochondria. The functional interaction of HK with VDAC was demonstrated by observing the effect of apyrase on HK-catalyzed glucose phosphorylation in intact mitochondria. Apyrase, which hydrolyzes freely soluble ATP, competed efficiently with hexokinase for ATP when it was produced outside mitochondria (with PEP and pyruvate kinase), but not when it was produced inside mitochondria by oxidative phosphorylation. These results suggest that HK closely interacts with VDAC in beetroot mitochondria, and that this interaction allows the channeling of respiratory ATP toward HK through VDAC.

Macromolecular organization of ATP synthase and complex I in whole mitochondria.

We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F(1) heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.

Stabilization of mitochondrial and microsomal function by polysaccharide of Ulva lactuca on D-Galactosamine induced hepatitis in rats.

In this study we used liver mitochondrial and microsomal fraction from rats pretreated with seaweed Ulva lactuca polysaccharide extract (ULP - 200mg/kg body weight, daily for 21 days, oral gavage) on D-Galactosamine (500mg/kg body weight, intraperitoneally) challenge. Effectiveness of ULP was determined based on functional status of trichloro acetic acid (TCA), urea cycle, and microsomal enzymes. The composition of sulfate polysaccharide content such as total sugars, sulfate and uronic acid were examined. In addition the fine ultra structural changes were examined using electron microscopy (EM). We observed significant (p<0.001) mitochondrial and microsomal abnormalities during liver damage by D-Galactosamine, consequently altering enzymes of energy metabolism. Electron microscopy of D-Galactosamine intoxicated rat liver tissue revealed the swelling and loss of mitochondrial cristae. Conversely the rats pretreated with ULP against D-Galactosamine challenge prevented (p<0.05) the significant abnormality of TCA, microsomal enzymes and severity of mitochondria as observed in EM study in rats injected with D-Galactosamine alone. However no effective prevention was observed in urea cycle enzymes among D-Galactosamine and treatment group rats. These results showed the effectiveness of ULP in stabilizing the functional status of mitochondrial and microsomal membrane which might be due to the presence of sulfated polysaccharide that could prevented the oxidative stress induced by D-Galactosamine intoxication.

Sulfide : quinone oxidoreductase (SQR) from the lugworm Arenicola marina shows cyanide- and thioredoxin-dependent activity.

The lugworm Arenicola marina inhabits marine sediments in which sulfide concentrations can reach up to 2 mM. Although sulfide is a potent toxin for humans and most animals, because it inhibits mitochondrial cytochrome c oxidase at micromolar concentrations, A. marina can use electrons from sulfide for mitochondrial ATP production. In bacteria, electron transfer from sulfide to quinone is catalyzed by the membrane-bound flavoprotein sulfide : quinone oxidoreductase (SQR). A cDNA from A. marina was isolated and expressed in Saccharomyces cerevisiae, which lacks endogenous SQR. The heterologous enzyme was active in mitochondrial membranes. After affinity purification, Arenicola SQR isolated from yeast mitochondria reduced decyl-ubiquinone (K(m) = 6.4 microm) after the addition of sulfide (K(m) = 23 microm) only in the presence of cyanide (K(m) = 2.6 mM). The end product of the reaction was thiocyanate. When cyanide was substituted by Escherichia coli thioredoxin and sulfite, SQR exhibited one-tenth of the cyanide-dependent activity. Six amino acids known to be essential for bacterial SQR were exchanged by site-directed mutagenesis. None of the mutant enzymes was active after expression in yeast, implicating these amino acids in the catalytic mechanism of the eukaryotic enzyme.