Characterization of human VDAC isoforms: a peculiar function for VDAC3?

VDACs are a family of pore-forming proteins mainly located in the mitochondrial outer membrane. In mammals three isoforms exist. In this work we review the information available about them with the addition of new results. We have compared the human VDACs transformed in a yeast strain lacking the endogenous porin. VDAC1 and 2 are able to complement the lack of porin in mitochondrial respiration and modulation of ROS. VDAC3 has a limited ability to support the mitochondrial respiration and has no influence in the control of ROS production. The over-expression of VDAC isoforms in wild type yeast strain led to a dramatic sensitivity to oxidative stress, especially for VDAC3, and a shorter lifespan in respiratory conditions. Real-time PCR comparison of the isoforms indicated that in HeLa cells VDAC1 is 10 times more abundant than VDAC2 and 100 times than VDAC3. The over-expression of any single isoform caused a 10 times increase of the transcripts of VDAC2 and VDAC3, while VDAC1 is not changed by the over-expression of the other isoforms. Models of VDAC2 and VDAC3 isoform structure showed that they could be made of a 19-strand beta-barrel and an N-terminal sequence with variable features. In this work we show for the first time a functional characterization of VDAC3 in a cellular context.

Neurotoxic properties of the anabolic androgenic steroids nandrolone and methandrostenolone in primary neuronal cultures.

Anabolic-androgenic steroid (AAS) abuse is associated with multiple neurobehavioral disturbances. The sites of action and the neurobiological sequels of AAS abuse are unclear at present. We investigated whether two different AASs, nandrolone and methandrostenolone, could affect neuronal survival in culture. The endogenous androgenic steroid testosterone was used for comparison. Both testosterone and nandrolone were neurotoxic at micromolar concentrations, and their effects were prevented by blockade of androgen receptors (ARs) with flutamide. Neuronal toxicity developed only over a 48-hr exposure to the steroids. The cell-impermeable analogues testosterone-BSA and nandrolone-BSA, which preferentially target membrane-associated ARs, were also neurotoxic in a time-dependent and flutamide-sensitive manner. Testosterone-BSA and nandrolone-BSA were more potent than their parent compounds, suggesting that membrane-associated ARs were the relevant sites for the neurotoxic actions of the steroids. Unlike testosterone and nandrolone, toxicity by methandrostenolone and methandrostenolone-BSA was insensitive to flutamide, but it was prevented by the glucocorticoid receptor (GR) antagonist RU-486. Methandrostenolone-BSA was more potent than the parent compound, suggesting that its toxicity relied on the preferential activation of putative membrane-associated GRs. Consistently with the evidence that membrane-associated GRs can mediate rapid effects, a brief challenge with methandrostenolone-BSA was able to promote neuronal toxicity. Activation of putative membrane steroid receptors by nontoxic (nanomolar) concentrations of either nandrolone-BSA or methandrostenolone-BSA became sufficient to increase neuronal susceptibility to the apoptotic stimulus provided by ß-amyloid (the main culprit of AD). We speculate that AAS abuse might facilitate the onset or progression of neurodegenerative diseases not usually linked to drug abuse.

Live cell interactome of the human voltage dependent anion channel 3 (VDAC3) revealed in HeLa cells by affinity purification tag technique.

In higher eukaryotes three different VDAC genes encode three homologous proteins which do not show the same activity. VDAC1 and VDAC2 isoforms have been characterized while VDAC3 isoform is still elusive. To explore VDAC3 protein interactions, we have established a stable cell line expressing a fluorescent and dual-tagged construct. This clone expresses a stable amount of VDAC3. Live cell imaging shows that fluorescent VDAC3 localizes in the mitochondria. Proteins interacting with VDAC3 have been separated by tandem-affinity purification and 2-D gel electrophoresis and identified by mass spectrometry. In the list of putative interacting proteins, there are cytosolic, mitochondrial, cytoskeletal and ER proteins. Coherent pathways like cell redox homeostasis, response to stress, formation/rearrangement of disulfide bonds, response to unfolded proteins or protein folding have been found to be related to clusters of proteins identified in this experiment. The list of associated proteins has been validated by immunoprecipitation experiments utilizing specific antibodies. Likely biological and pathological processes have been analyzed. Cytosolic proteins associated with VDAC3 include tubulins and cytoskeletal proteins, stress sensors, chaperones and proteasome components, redox-mediating enzymes such as protein disulphide isomerase. The overall picture points to a role for VDAC3 as mediator for the organization of protein complexes and regulator of the traffic of misfolded or non-folded proteins evoked from different stimuli.

Glucose ameliorates the metabolic profile and mitochondrial function of platelet concentrates during storage in autologous plasma.

BACKGROUND: It is essential that the quality of platelet metabolism and function remains high during storage in order to ensure the clinical effectiveness of a platelet transfusion. New storage conditions and additives are constantly evaluated in order to achieve this. Using glucose as a substrate is controversial because of its potential connection with increased lactate production and decreased pH, both parameters triggering the platelet lesion during storage. MATERIALS AND METHODS: In this study, we analysed the morphological status and metabolic profile of platelets stored for various periods in autologous plasma enriched with increasing glucose concentrations (13.75, 27.5 and 55 mM). After 0, 2, 4, 6 and 8 days, high energy phosphates (ATP, GTP, ADP, AMP), oxypurines (hypoxanthine, xanthine, uric acid), lactate, pH, mitochondrial function, cell lysis and morphology, were evaluated. RESULTS: The data showed a significant dose-dependent improvement of the different parameters in platelets stored with increasing glucose, compared to what detected in controls. Interestingly, this phenomenon was more marked at the highest level of glucose tested and in the period of time generally used for platelet transfusion (0-6 days). CONCLUSION: These results indicate that the addition of glucose during platelet storage ameliorates, in a dose-dependent manner, the biochemical parameters related to energy metabolism and mitochondrial function. Since there was no correspondence between glucose addition, lactate increase and pH decrease in our experiments, it is conceivable that platelet derangement during storage is not directly caused by glucose through an increase of anaerobic glycolysis, but rather to a loss of mitochondrial functions caused by reduced substrate availability.

Beta-amyloid monomer and insulin/IGF-1 signaling in Alzheimer's disease.

Alzheimer's disease is the most common form of dementia among older people and is still untreatable. While ß-amyloid protein is recognized as the disease determinant with a pivotal role in inducing neuronal loss and dementia, an impaired brain insulin signaling seems to account in part for the cognitive deficit associated with the disease. The origin of this defective signaling is uncertain. Accumulating toxic species of ß-amyloid, the so-called oligomers, has been proposed to be responsible for downregulation of neuronal insulin receptors. We have found that the nontoxic form of ß-amyloid, the monomer, is able to activate insulin/insulin-like growth factor-1 (IGF-1) receptor signaling and thus behaves as a neuroprotectant agent. Our suggestion is that depletion of ß-amyloid monomers, occurring in the preclinical phase of Alzheimer's disease, might be the cause of early insulin/IGF-1 signaling disturbances that anticipate cognitive decline.

Outer membrane VDAC1 controls permeability transition of the inner mitochondrial membrane in cellulo during stress-induced apoptosis.

Voltage-dependent anion channel (VDAC)1 is the main channel of the mitochondrial outer membrane (MOM) and it has been proposed to be part of the permeability transition pore (PTP), a putative multiprotein complex candidate agent of the mitochondrial permeability transition (MPT). Working at the single live cell level, we found that overexpression of VDAC1 triggers MPT at the mitochondrial inner membrane (MIM). Conversely, silencing VDAC1 expression results in the inhibition of MPT caused by selenite-induced oxidative stress. This MOM-MIM crosstalk was modulated by Cyclosporin A and mitochondrial Cyclophilin D, but not by Bcl-2 and Bcl-X(L), indicative of PTP operation. VDAC1-dependent MPT engages a positive feedback loop involving reactive oxygen species and p38-MAPK, and secondarily triggers a canonical apoptotic response including Bax activation, cytochrome c release and caspase 3 activation. Our data thus support a model of the PTP complex involving VDAC1 at the MOM, and indicate that VDAC1-dependent MPT is an upstream mechanism playing a causal role in oxidative stress-induced apoptosis.

An intracellular wave of cytochrome c propagates and precedes Bax redistribution during apoptosis.

Bax is considered to be pivotal in inducing cytochrome c release (CCR) from mitochondria during apoptosis. Indeed, Bax redistributes to the mitochondrial outer membrane (MOM) upon activation and forms homo-multimers that are capable of permeabilizing the MOM. Our attempts to image this sequence of events in single live cells resulted in unexpected observations. Bax redistribution exhibited two distinct components: an early minor redistribution that was silent in terms of homo-multimerization and a major late redistribution that was synchronous with the formation of Bax multimers, but that proceeded belatedly, i.e. only after caspase 3/7 (C3/7) had already been activated. Intriguingly, neither of these two components of redistribution correlated with CCR, which turned out to be spatially organized, propagating as a traveling wave at constant velocity. Strikingly, propagation of the CCR wave (1) preceded signs of in situ Bax conformational activation; (2) appeared to be independent of autocatalytic loops involving a positive feedback of either C3/7, Ca(2+) mobilization or mitochondrial permeability transition; and (3) was triggered by diffuse stimulation with the synthetic Bak activator BH3I-1 but then proceeded independently of Bak activation. Thus, the CCR wave not only questions the exact role of Bax redistribution in cell death, but also indicates the existence of yet unidentified positive-feedback loops that ensure a spatiotemporal control of apoptosis at the subcellular scale.

Determination of the conformation of the human VDAC1 N-terminal peptide, a protein moiety essential for the functional properties of the pore.

Mitochondrial porin or VDAC (voltage-dependent anion-selective channel) is the most abundant protein in the mitochondrial outer membrane. The structure of VDAC has been predicted to be a transmembrane beta-barrel with an alpha-helix at the N terminus. It is a matter of debate as to whether this putative alpha-helix plays a structural role as a component of the pore walls or a function in the pore activity. We have synthesised the human VDAC1 (HVDAC1) N-terminal peptide Ac-AVPPTYADLGKSARDVFTK-NH2 (Prn2-20) and determined its structure by CD and NMR spectroscopy. CD studies show that the Prn2-20 peptide exists in aqueous solvent as an unstructured peptide with no stable secondary structure. In membrane-mimetic SDS micelles or water/trifluoroethanol, however, it assumes an amphipathic alpha-helix conformation between Tyr5 and Val16, as deduced from NMR. No ordered structure was observed in dodecyl beta-maltoside. Differential scanning calorimetric measurements were carried out in order to examine the membrane affinity of the peptide. Upon interaction with the negatively charged 1,2 dipalmitoyl-sn-glycero-3-phosphoserine membrane, Prn2-20 exhibited distinctive behaviour, suggesting that electrostatics play an important role. Interaction between the peptide and artificial bilayers indicates that the peptide lies on the membrane surface. Recombinant HVDAC1 deletion mutants, devoid of seven or 19 N-terminal amino acids, were used for transfection of eukaryotic cells. Over-expression of HVDAC1 increases the number of Cos cells with depolarised mitochondria, and this effect is progressively reduced in cells transfected with HVDAC1 lacking those seven or 19 amino acids. The mitochondrial targeting of the deletion mutants is unaffected. The overall picture emerging from our experiments is that the VDAC N-terminal peptide plays a role in the proper function of this protein during apoptotic events.

Regulation of peroxiredoxins by nitric oxide in immunostimulated macrophages.

Reactive oxygen species and nitric oxide (NO) are capable of both mediating redox-sensitive signal transduction and eliciting cell injury. The interplay between these messengers is quite complex, and intersection of their signaling pathways as well as regulation of their fluxes requires tight control. In this regard, peroxiredoxins (Prxs), a recently identified family of six thiol peroxidases, are central because they reduce H2O2, organic peroxides, and peroxynitrite. Here we provide evidence that endogenously produced NO participates in protection of murine primary macrophages against oxidative and nitrosative stress by inducing Prx I and VI expression at mRNA and protein levels. We also show that NO prevented the sulfinylation-dependent inactivation of 2-Cys Prxs, a reversible overoxidation that controls H2O2 signaling. In addition, studies using macrophages from sulfiredoxin (Srx)-deficient mice indicated that regeneration of 2-Cys Prxs to the active form was dependent on Srx. Last, we show that NO increased Srx expression and hastened Srx-dependent recovery of 2-Cys Prxs. We therefore propose that modulation by NO of Prx expression and redox state, as well as up-regulation of Srx expression, constitutes a novel pathway that contributes to antioxidant response and control of H2O2-mediated signal transduction in mammals.