Voltage-Dependent Anion Channel 2 of Arabidopsis thaliana (AtVDAC2) Is Involved in ABA-Mediated Early Seedling Development

The voltage-dependent anion channel (VDAC) is the major transport protein in the outer membrane of mitochondria and plays crucial roles in energy metabolism, apoptosis, and metabolites transport. In plants, the expression of VDACs can be affected by different stresses, including drought, salinity and pathogen defense. In this study, we investigated the expression pattern of AtVDAC2 in A. thaliana and found ABA suppressed the accumulation of AtVDAC2 transcripts. Further, phenotype analysis of this VDAC deregulated-expression transgenic Arabidopsis plants indicated that AtVDAC2 anti-sense line showed an ABA-insensitivity phenotype during the early seedling development under ABA treatment. The results suggested that AtVDAC2 might be involved in ABA signaling in A. thaliana

Subcellular Localization of Hexokinases I and II Directs the Metabolic Fate of Glucose

The first step in glucose metabolism is conversion of glucose to glucose 6-phosphate (G-6-P) by hexokinases (HKs), a family with 4 isoforms. The two most common isoforms, HKI and HKII, have overlapping tissue expression, but different subcellular distributions, with HKI associated mainly with mitochondria and HKII associated with both mitochondrial and cytoplasmic compartments. Here we tested the hypothesis that these different subcellular distributions are associated with different metabolic roles, with mitochondrially-bound HK's channeling G-6-P towards glycolysis (catabolic use), and cytoplasmic HKII regulating glycogen formation (anabolic use).To study subcellular translocation of HKs in living cells, we expressed HKI and HKII linked to YFP in CHO cells. We concomitantly recorded the effects on glucose handling using the FRET based intracellular glucose biosensor, FLIPglu-600 mM, and glycogen formation using a glycogen-associated protein, PTG, tagged with GFP. Our results demonstrate that HKI remains strongly bound to mitochondria, whereas HKII translocates between mitochondria and the cytosol in response to glucose, G-6-P and Akt, but not ATP. Metabolic measurements suggest that HKI exclusively promotes glycolysis, whereas HKII has a more complex role, promoting glycolysis when bound to mitochondria and glycogen synthesis when located in the cytosol. Glycogen breakdown upon glucose removal leads to HKII inhibition and dissociation from mitochondria, probably mediated by increases in glycogen-derived G-6-P.These findings show that the catabolic versus anabolic fate of glucose is dynamically regulated by extracellular glucose via signaling molecules such as intracellular glucose, G-6-P and Akt through regulation and subcellular translocation of HKII. In contrast, HKI, which activity and regulation is much less sensitive to these factors, is mainly committed to glycolysis. This may be an important mechanism by which HK's allow cells to adapt to changing metabolic conditions to maintain energy balance and avoid injury

Hexokinase II Detachment from Mitochondria Triggers Apoptosis through the Permeability Transition Pore Independent of Voltage-Dependent Anion Channels

Type II hexokinase is overexpressed in most neoplastic cells, and it mainly localizes on the outer mitochondrial membrane. Hexokinase II dissociation from mitochondria triggers apoptosis. The prevailing model postulates that hexokinase II release from its mitochondrial interactor, the voltage-dependent anion channel, prompts outer mitochondrial membrane permeabilization and the ensuing release of apoptogenic proteins, and that these events are inhibited by growth factor signalling. Here we show that a hexokinase II N-terminal peptide selectively detaches hexokinase II from mitochondria and activates apoptosis. These events are abrogated by inhibiting two established permeability transition pore modulators, the adenine nucleotide translocator or cyclophilin D, or in cyclophilin D knock-out cells. Conversely, insulin stimulation or genetic ablation of the voltage-dependent anion channel do not affect cell death induction by the hexokinase II peptide. Therefore, hexokinase II detachment from mitochondria transduces a permeability transition pore opening signal that results in cell death and does not require the voltage-dependent anion channel. These findings have profound implications for our understanding of the pathways of outer mitochondrial membrane permeabilization and their inactivation in tumors

Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes

Poly(ADP-ribose) (pADPr) is a polymer assembled from the enzymatic polymerization of the ADP-ribosyl moiety of NAD by poly(ADP-ribose) polymerases (PARPs). The dynamic turnover of pADPr within the cell is essential for a number of cellular processes including progression through the cell cycle, DNA repair and the maintenance of genomic integrity, and apoptosis. In spite of the considerable advances in the knowledge of the physiological conditions modulated by poly(ADP-ribosyl)ation reactions, and notwithstanding the fact that pADPr can play a role of mediator in a wide spectrum of biological processes, few pADPr binding proteins have been identified so far. In this study, refined in silico prediction of pADPr binding proteins and large-scale mass spectrometry-based proteome analysis of pADPr binding proteins were used to establish a comprehensive repertoire of pADPr-associated proteins. Visualization and modeling of these pADPr-associated proteins in networks not only reflect the widespread involvement of poly(ADP-ribosyl)ation in several pathways but also identify protein targets that could shed new light on the regulatory functions of pADPr in normal physiological conditions as well as after exposure to genotoxic stimuli

Amyloid-β Triggers the Release of Neuronal Hexokinase 1 from Mitochondria

Brain accumulation of the amyloid-β peptide (Aβ) and oxidative stress underlie neuronal dysfunction and memory loss in Alzheimer's disease (AD). Hexokinase (HK), a key glycolytic enzyme, plays important pro-survival roles, reducing mitochondrial reactive oxygen species (ROS) generation and preventing apoptosis in neurons and other cell types. Brain isozyme HKI is mainly associated with mitochondria and HK release from mitochondria causes a significant decrease in enzyme activity and triggers oxidative damage. We here investigated the relationship between Aβ-induced oxidative stress and HK activity. We found that Aβ triggered HKI detachment from mitochondria decreasing HKI activity in cortical neurons. Aβ oligomers further impair energy metabolism by decreasing neuronal ATP levels. Aβ-induced HKI cellular redistribution was accompanied by excessive ROS generation and neuronal death. 2-deoxyglucose blocked Aβ-induced oxidative stress and neuronal death. Results suggest that Aβ-induced cellular redistribution and inactivation of neuronal HKI play important roles in oxidative stress and neurodegeneration in AD

Yeast Mitochondrial Interactosome Model: Metabolon Membrane Proteins Complex Involved in the Channeling of ADP/ATP

The existence of a mitochondrial interactosome (MI) has been currently well established in mammalian cells but the exact composition of this super-complex is not precisely known, and its organization seems to be different from that in yeast. One major difference is the absence of mitochondrial creatine kinase (MtCK) in yeast, unlike that described in the organization model of MI, especially in cardiac, skeletal muscle and brain cells. The aim of this review is to provide a detailed description of different partner proteins involved in the synergistic ADP/ATP transport across the mitochondrial membranes in the yeast Saccharomyces cerevisiae and to propose a new mitochondrial interactosome model. The ADP/ATP (Aacp) and inorganic phosphate (PiC) carriers as well as the VDAC (or mitochondrial porin) catalyze the import and export of ADP, ATP and Pi across the mitochondrial membranes. Aacp and PiC, which appear to be associated with the ATP synthase, consist of two nanomotors (F0, F1) under specific conditions and form ATP synthasome. Identification and characterization of such a complex were described for the first time by Pedersen and co-workers in 2003

In self-defence: hexokinase promotes voltage-dependent anion channel closure and prevents mitochondria-mediated apoptotic cell death.

In tumour cells, elevated levels of mitochondria-bound isoforms of hexokinase (HK-I and HK-II) result in the evasion of apoptosis, thereby allowing the cells to continue proliferating. The molecular mechanisms by which bound HK promotes cell survival are not yet fully understood. Our studies relying on the purified mitochondrial outer membrane protein VDAC (voltage-dependent anion channel), isolated mitochondria or cells in culture suggested that the anti-apoptotic activity of HK-I occurs via modulation of the mitochondrial phase of apoptosis. In the present paper, a direct interaction of HK-I with bilayer-reconstituted purified VDAC, inducing channel closure, is demonstrated for the first time. Moreover, HK-I prevented the Ca(2+)-dependent opening of the mitochondrial PTP (permeability transition pore) and release of the pro-apoptotic protein cytochrome c. The effects of HK-I on VDAC activity and PTP opening were prevented by the HK reaction product glucose 6-phosphate, a metabolic intermediate in most biosynthetic pathways. Furthermore, glucose 6-phosphate re-opened both the VDAC and the PTP closed by HK-I. The HK-I-mediated effects on VDAC and PTP were not observed using either yeast HK or HK-I lacking the N-terminal hydrophobic peptide responsible for binding to mitochondria, or in the presence of an antibody specific for the N-terminus of HK-I. Finally, HK-I overexpression in leukaemia-derived U-937 or vascular smooth muscle cells protected against staurosporine-induced apoptosis, with a decrease of up to 70% in cell death. These results offer insight into the mechanisms by which bound HK promotes tumour cell survival, and suggests that its overexpression not only ensures supplies of energy and phosphometabolites, but also reflects an anti-apoptotic defence mechanism