Mechanisms of MAVS Regulation at the Mitochondrial Membrane

Mitochondria have emerged as critical platforms for antiviral innate immune signaling. This is due in large part to the mitochondrial localization of the innate immune signaling adaptor MAVS (mitochondrial antiviral signaling protein), which coordinates signals received from two independent cytosolic pathogen recognition receptors (PRRs) to induce antiviral genes. The existence of a shared adaptor for two central PRRs presents an ideal target by which the host cell can prevent cellular damage induced by uncontrolled inflammation through alteration of MAVS expression and/or signaling. In this review, we focus on the MAVS regulome and review the cellular factors that regulate MAVS by (1) protein–protein interactions, (2) alterations in mitochondrial dynamics, and/or (3) post-translational modifications.     

Desynchronisation of Glycolytic Oscillations in Yeast Cell Populations

Glycolytic oscillations of intact yeast cells of the strain Saccharomyces carlsbergensis were investigated at both the levels of cell populations and of individual cells. Individual cells showed glycolytic oscillations even at very low cell densities (e.g. 1.010⁵ cells/ml). By contrast, the collective behaviour on the population level was cell density-dependent: at high cell densities it is oscillatory, but below the threshold density of 1.010⁶ cells/ml the collective dynamics becomes quiescent. We demonstrate that the transition in the collective dynamics is caused by the desynchronisation of the oscillations of individual cells. This is characteristic for a Kuramoto transition. Spatially resolved measurements at low cell densities revealed that even cells that adhere to their neighbours oscillated with their own, independent frequencies and phases.     

Metabolite Concentrations and Phase Relations in d-Fructose Induced Glycolytic Oscillations

The frequency of glycolytic oscillations in yeast cells is only slightly modified by the consumption rate of either glucose or fructose, but it is reduced to 1 /2 to 2/3 if the ketohexose is the only substrate. Phase relations between NADH, adenine nucleotides and sugar phosphates (G-6-P, F-6-P, FDP, DAP) are independent of the hexose fermented. With fructose as a substrate the amplitudes of adenylates and hexose phosphates are distinctly smaller than with glucose. The maximum of G-6-P is higher with glucose and the minimum concentration of FDP is higher with fructose. In the transition to anaerobiosis FDP, adenosine 5′-diphosphate and adenosine 5′-phosphate are extremely high, whereas G-6-P, F-6-P and ATP concentrations are lower if fructose is the substrate fermented. The results are indicative for different control characteristics of the phosphofructokinase step, but on their basis it cannot be distinguished between a direct interaction of fructose (or one of its derivatives) on the phosphofructokinase kinetics or the existence of a bypass for fructose which might be able to withdraw a part of the substrate from the control point at the phosphofructokinase.     

Glycolytic ATP Fuels the Plasma Membrane Calcium Pump Critical for Pancreatic Cancer Cell Survival

Pancreatic cancer is an aggressive cancer with poor prognosis and limited treatment options. Cancer cells rapidly proliferate and are resistant to cell death due, in part, to a shift from mitochondrial metabolism to glycolysis. We hypothesized that this shift is important in regulating cytosolic Ca²⁺ ([Ca²⁺]_i), as the ATP-dependent plasma membrane Ca²⁺ ATPase (PMCA) is critical for maintaining low [Ca²⁺]_i and thus cell survival. The present study aimed to determine the relative contribution of mitochondrial versus glycolytic ATP in fuelling the PMCA in human pancreatic cancer cells. We report that glycolytic inhibition induced profound ATP depletion, PMCA inhibition, [Ca²⁺]_i overload, and cell death in PANC1 and MIA PaCa-2 cells. Conversely, inhibition of mitochondrial metabolism had no effect, suggesting that glycolytic ATP is critical for [Ca²⁺]_i homeostasis and thus survival. Targeting the glycolytic regulation of the PMCA may, therefore, be an effective strategy for selectively killing pancreatic cancer while sparing healthy cells.     

Complex I Controls Mitochondrial and Plasma Membrane Potentials in Nerve Terminals

Reductions in the activities of mitochondrial electron transport chain (ETC) enzymes have been implicated in the pathogenesis of numerous chronic neurodegenerative disorders. Maintenance of the mitochondrial membrane potential (Δψ_m) is a primary function of these enzyme complexes, and is essential for ATP production and neuronal survival. We examined the effects of inhibition of mitochondrial ETC complexes I, II/III, III and IV activities by titrations of respective inhibitors on Δψ_m in synaptosomal mitochondria. Small perturbations in the activity of complex I, brought about by low concentrations of rotenone (1–50 nM), caused depolarisation of Δψ_m. Small decreases in complex I activity caused an immediate and partial Δψ_m depolarisation, whereas inhibition of complex II/III activity by more than 70% with antimycin A was required to affect Δψ_m. A similarly high threshold of inhibition was found when complex III was inhibited with myxothiazol, and inhibition of complex IV by more than 90% with KCN was required. The plasma membrane potential (Δψ_p) had a complex I inhibition threshold of 40% whereas complex III and IV had to be inhibited by more than 90% before changes in Δψ_p were registered. These data indicate that in synaptosomes, both Δψ_m and Δψ_p are more susceptible to reductions in complex I activity than reductions in the other ETC complexes. These findings may be of relevance to the mechanism of neuronal cell death in Parkinson’s disease in particular, where such reductions in complex I activity are present.

     

Plasma Membrane Ca-ATPase of Radish Seedlings : II. Regulation by Calmodulin

The effect of calmodulin on the activity of the plasma membrane Ca-ATPase was investigated on plasma membranes purified from radish (Raphanus sativus L.) seedlings. Calmodulin stimulated the hydrolytic activity and the transport activity of the plasma membrane Ca-ATPase to comparable extents in a manner dependent on the free Ca²⁺ concentration. Stimulation was marked at low, nonsaturating Ca²⁺ concentrations and decreased increasing Ca²⁺, so that the effect of calmodulin resulted in an increase of the apparent affinity of the enzyme for free Ca²⁺. The pattern of calmodulin stimulation of the plasma membrane Ca-ATPase activity was substantially the same at pH 6.9 and 7.5, in the presence of ATP or ITP, and when calmodulin from radish seeds was used rather than that from bovine brain. At pH 6.9 in the presence of 5 micromolar free Ca²⁺, stimulation of the plasma membrane Ca-ATPase was saturated by 30 to 50 micrograms per milliliter bovine brain calmodulin. The calmodulin antagonist calmidazolium inhibited both basal and calmodulin-stimulated plasma membrane Ca-ATPase activity to comparable extents.     

Influence of Severe Hypoglycemia on Mitochondrial and Plasma Membrane Function in Rat Brain

Abstract: Previous experiments have shown that severe hypoglycemia disrupts cerebral energy state in spite of a maintained cerebral oxygen consumption, suggesting uncoupling of oxidative phosphorylation. Other studies have demonstrated that hypoglycemia leads to loss of cerebral cortical phospholipids and phospholipid-bound fatty acids. The objective of the present study was, therefore, to study respiratory characteristics of brain mitochondria during severe hypoglycemia and to correlate respiratory activity to mitochondrial phospholipid composition. Mitochondria were isolated after 30 or 60 min of hypoglycemia with ceased EEG activity, and after a 90-min recovery period, and their resting (state 4) and ADP-stimulated (state 3) oxygen consumption rates and phospholipids and phospholipid-bound fatty acid content were measured. After 30 min of hypoglycemia, state 3 respiration decreased without any increase in state 4 respiration or change in ADP/O ratio. This decrease, which occurred with glutamate plus malate—but not with succinate—as substrates, was partly reversed by addition of bovine serum albumin and KCI. Chemical analyses of isolated mitochondria did not reveal changes in their phospholipid or fatty acid content. The results thus failed to account for the dissociation of cerebral energy state and oxygen consumption. It is emphasized, though, that uncoupling may well occur in vivo due to accumulation of free fatty acids and “futile cycling” of K⁺ and Ca²⁺. After 60 min of hypoglycemia, a moderate decrease in state 3 respiration was observed also with succinate as substrate, and there was some decrease in ADP/O ratios in KCI-containing media. However, the changes in ADP/O ratios were more conspicuous during recovery; in addition, state 4 respiration increased significantly. It is concluded that changes in mitochondrial function after 30 min of hypoglycemia are potentially reversible but that true mitochondrial failure develops in the recovery period following 60 min of hypoglycemia. This conclusion was corroborated by results demonstrating incomplete recovery of cerebral energy state. Since EEG and sensory evoked potentials return after 30 min but not after 60 min of hypoglycemia it seemed difficult to explain failure of return of electrophysiological function after 60 min of hypoglycemia solely by mitochondrial dysfunction; plasma membrane function was therefore assessed by measurements of extracellular potassium activity ([K⁺]_e). The results showed that whereas [K⁺]_e remained close to control in the recovery period following 30 min of hypoglycemia it rose progressively during recovery following 60 min of hypoglycemia. Possibly, inhibition of Na⁺ K⁺–activated ATPase could contribute to the permanent loss of spontaneous or evoked electrical activity.     

Selective Regulation of Maize Plasma Membrane Aquaporin Trafficking and Activity by the SNARE SYP121

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K⁺ channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K⁺ channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis.     

线粒体膜完整性对细胞命运的调控

线粒体双层膜的完整性是细胞存活的关键因素,其遭到破坏后会使细胞发生凋亡、焦亡或炎症。线粒体膜的破坏包括线粒体外膜通透、线粒体内膜通透、通透性转换,三者可通过调控不同的信号通路导致不同的细胞命运。然而,这些信号通路之间存在交叉关联,使得线粒体膜对细胞命运的调控错综复杂,导致人们对其机制缺乏清晰的认识。本综述首先分析了不同程度线粒体外膜通透在细胞存活、癌变或凋亡中的作用,接着讨论了线粒体内膜通透通过引发线粒体DNA释放促进炎症发生的分子机制,然后阐述了线粒体通透性转换引发焦亡的作用机制,最后总结出线粒体膜完整性影响细胞命运决策的内在关联。深入了解线粒体膜完整性调控细胞命运的分子动力学机制,有助于为癌症和神经退行性疾病的诊疗提供思路。     

The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP

Evidence suggests that the plasma membrane Ca²⁺-ATPase (PMCA), which is critical for maintaining a low intracellular Ca²⁺ concentration ([Ca²⁺]_i), utilizes glycolytically derived ATP in pancreatic ductal adenocarcinoma (PDAC) and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition, and an irreversible [Ca²⁺]_i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype toward mitochondrial metabolism and assessing the effects of mitochondrial versus glycolytic inhibitors on ATP depletion, PMCA inhibition, and [Ca²⁺]_i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitizing cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca²⁺]_i in α-ketoisocaproate- and galactose-cultured cells, suggesting that the glycolytic dependence of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.     

The Plasma Membrane Ca2+ ATPase and the Plasma Membrane Sodium Calcium Exchanger Cooperate in the Regulation of Cell Calcium

Calcium is an ambivalent signal: it is essential for the correct functioning of cell life, but may also become dangerous to it. The plasma membrane Ca²⁺ ATPase (PMCA) and the plasma membrane Na⁺/Ca²⁺ exchanger (NCX) are the two mechanisms responsible for Ca²⁺ extrusion. The NCX has low Ca²⁺ affinity but high capacity for Ca²⁺ transport, whereas the PMCA has a high Ca²⁺ affinity but low transport capacity for it. Thus, traditionally, the PMCA pump has been attributed a housekeeping role in maintaining cytosolic Ca²⁺, and the NCX the dynamic role of counteracting large cytosolic Ca²⁺ variations (especially in excitable cells). This view of the roles of the two Ca²⁺ extrusion systems has been recently revised, as the specific functional properties of the numerous PMCA isoforms and splicing variants suggests that they may have evolved to cover both the basal Ca²⁺ regulation (in the 100 nM range) and the Ca²⁺ transients generated by cell stimulation (in the μM range).Ca²⁺ controls critical cellular responses in all eukaryotic organisms. It controls both short-term biological processes that occur in milliseconds, such as muscle contraction, as well as long-term processes that require longer times, such as cell proliferation and organ development. The specificity of cellular Ca²⁺ signals is controlled by a sophisticated “toolkit” comprising numerous ion channels, pumps, and exchangers that drive the fluxes of Ca²⁺ ions across the plasma membrane and across the membranes of intracellular organelles (Berridge et al. 2003).The plasma membrane contains several types of channels that mediate Ca²⁺ entry from the extracellular ambient, and two systems for Ca²⁺ extrusion: a low affinity, high capacity Na⁺/Ca²⁺ exchanger (NCX), and a high-affinity, low-capacity Ca²⁺-ATPase (the plasma membrane Ca²⁺ pump (PMCA)) (Fig. 1). The type of channels and the relative proportions of NCX and PMCA vary with the cell type, the NCX being particularly abundant in excitable tissues, e.g., heart and brain. The regulated opening of the Ca²⁺ channels by either voltage gating, interaction with ligands or the emptying of intracellular stores, allows a limited amount of Ca²⁺ to enter the cell to transmit signals to its designated targets. Thereafter, the Ca²⁺ transients must be dissipated: its extrusion from the cell is mediated by the NCX and the PMCA pump, but Ca²⁺ is also restored to basal levels by sequestration in the endo/sarcoplasmic reticulum via the SERCA pump and in the mitochondria by the electrophoretic uniporter. The NCX has also been found at the inner membrane of the nuclear envelope (NE) and has been proposed to mediate Ca²⁺ flux between the nucleoplasm and the NE (Xie et al. 2002), and then to the ER (Wu et al. 2009) in neuronal and certain other cell types. Ca²⁺ binding proteins also contributed to Ca²⁺ buffering: In this review, we will not cover them, as we will only discuss the systems that extrude Ca²⁺ out of the cell.Open in a separate windowFigure 1.A schematic representation of the structures involved in cellular Ca²⁺ homeostasis. The model shows a cell with its Ca²⁺-transporting systems: Ca²⁺-ATPases (plasma membrane and sarco/endoplasmic reticulum, PMCA and SERCA), plasma membrane (PM) Ca²⁺ channels, Na⁺/Ca²⁺ exchangers (NCX and NCLX), 1,4,5-triphosphate receptor (IP₃R) and ryanodine receptor (RyR), the electrophoretic mitochondrial uptake uniporter (U). Mitochondria are drawn as yellow ellipses, nucleus as orange circle and endoplasmic reticulum is colored in red. The different Ca²⁺-transporting systems cooperate to maintain the Ca²⁺ concentration gradient between the extracellular and the intracellular ambient.The PMCA pump is a minor component of the total protein of the plasma membrane (less than 0.1% of it). Quantitatively, it is overshadowed by the more powerful NCX in excitable tissue like heart; however, even cells in which the NCX predominates, the PMCA pump is likely to be the fine tuner of cytosolic Ca²⁺, as it can operate in a concentration range in which the low affinity NCX is relatively very inefficient.The PMCA was discovered in erythrocytes (Schatzmann 1966), and was then described and characterized in numerous other cell types. It was purified in 1979 using a calmodulin affinity column (Niggli et al. 1979), and cloned about 10 years later (Shull and Greeb 1988; Verma et al. 1988). It shows the same essential membrane topology properties of the SERCA pump. Molecular modeling work using the structure of the SERCA pump as a template (Toyoshima et al. 2000) predicts the same general features of the latter, with 10 transmembrane domains and the large cytosolic headpiece divided into the three main cytosolic A, N, and P domains. The Na⁺/Ca²⁺ cotransport process was discovered at about the same time as PMCA by two independent groups working on heart (Reuter and Seitz 1968) and on the squid giant axon (Baker et al. 1969). The exchanger was cloned in 1990 (Nicoll et al. 1990). The sequence was initially predicted to correspond to a protein with 11 transmembrane domains and one large cytosolic loop linking transmembrane domain five and six but a revised model predicting only nine transmembrane domains is now generally accepted.     

Mitochondrial Function in Human Neuroblastoma Cells Is Up-Regulated and Protected by NQO1, a Plasma Membrane Redox Enzyme

Background

Recent findings suggest that NADH-dependent enzymes of the plasma membrane redox system (PMRS) play roles in the maintenance of cell bioenergetics and oxidative state. Neurons and tumor cells exhibit differential vulnerability to oxidative and metabolic stress, with important implications for the development of therapeutic interventions that promote either cell survival (neurons) or death (cancer cells).

Methods and Findings

Here we used human neuroblastoma cells with low or high levels of the PMRS enzyme NADH-quinone oxidoreductase 1 (NQO1) to investigate how the PMRS modulates mitochondrial functions and cell survival. Cells with elevated NQO1 levels exhibited higher levels of oxygen consumption and ATP production, and lower production of reactive oxygen species. Cells overexpressing NQO1 were more resistant to being damaged by the mitochondrial toxins rotenone and antimycin A, and exhibited less oxidative/nitrative damage and less apoptotic cell death. Cells with basal levels of NQO1 resulted in increased oxidative damage to proteins and cellular vulnerability to mitochondrial toxins. Thus, mitochondrial functions are enhanced and oxidative stress is reduced as a result of elevated PMRS activity, enabling cells to maintain redox homeostasis under conditions of metabolic and energetic stress.

Conclusion

These findings suggest that NQO1 is a potential target for the development of therapeutic agents for either preventing neuronal degeneration or promoting the death of neural tumor cells.     

Differentiation Between Alterations in Plasma and Mitochondrial Membrane Potentials in Synaptosomes Using a Carbocyanine Dye

The cationic potentiometric fluorescent probe 3,3'-diethylthiadicarbocyanine iodide [DiS-C2(5)] was used in synaptosomes to assess the relative contributions of plasma and mitochondrial membrane potentials (psi p and psi m, respectively) to overall fluorescence. Addition of synaptosomes to media containing 0.5 microM dye caused a decrease in fluorescence intensity due to dye accumulation, which equilibrated usually within 5 min. Depolarization of mitochondria by combined treatment with cyanide and oligomycin increased fluorescence by 42%, indicating significant prior accumulation of dye into intrasynaptosomal mitochondria. psi p was calculated to be -54 mV and was not altered significantly by prior depolarization of psi m with cyanide and oligomycin (hereafter referred to as "poisoned" synaptosomes). Similarly, the linear relationship between dye fluorescence and psi p was not altered by depolarization of psi m. Valinomycin, a K+ ionophore, caused a psi p-dependent increase in fluorescence in control (nonpoisoned) synaptosomes, but did not alter fluorescence of poisoned synaptosomes except when the extracellular concentration of K+ ([K+]e) was 2 mM, in which case valinomycin hyperpolarized psi p by about 5 mV. The pore-forming antibiotic gramicidin depolarized both psi p and psi m maximally. Under these conditions, Triton X-100 further increased fluorescence by 40%, indicating significant dye binding to synaptosomal components. In poisoned synaptosomes depolarized by 75 mM K+, gramicidin caused a decrease in fluorescence intensity (hyperpolarization of psi p). The organic solvent dimethyl sulfoxide, used as a vehicle for the hydrophobic ionophores, had voltage-dependent effects on psi p and psi m.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Synaptic Transmission by Mitochondrial Ion Channels

Mitochondria are abundant within neuronal presynaptic terminals, where they provide energy for sustained neurotransmitter secretion. Injection of Bcl-xL protein into squid giant presynaptic terminal potentiates neurotransmitter release, while a naturally occurring, proteolytic fragment of BCL-xL causes rundown of synaptic function. The cleaved form of BCL-xL generates large, multiconductance ion channel activity in synaptic mitochondrial outer membranes. A rapid onset of synaptic rundown can also be produced by depriving the synapse of oxygen, and hypoxia also induces large channel activity in mitochondrial outer membranes. Channel activity induced by cleaved BCL-xL or by hypoxia is attenuated by NADH, an inhibitor of the voltage-dependent anion channel (VDAC) of mitochondrial outer membranes. Finally, the large conductances elicited by hypoxia are prevented by the addition of a protease inhibitor that prevents cleavage of BCL-xL. The opposing activities of BCL-xL and its proteolytic fragment may regulate the release of ATP from mitochondria during synaptic transmission.     

Deregulation of Mitochondrial Membrane Potential by Mitochondrial Insertion of Granzyme B and Direct Hax-1 Cleavage

The cytoplasm and the nucleus have been identified as activity sites for granzyme B (GrB) following its delivery from cytotoxic lymphocyte granules into target cells. Here we report on the ability of exogenous GrB to insert into and function within a proteinase K-resistant mitochondrial compartment. We identified Hax-1 (HS-1-associated protein X-1), a mitochondrial protein involved in the maintenance of mitochondrial membrane potential, as a GrB substrate within the mitochondrion. GrB cleaves Hax-1 into two major fragments: an N-terminal fragment that localizes to mitochondria and a C-terminal fragment that localizes to the cytosol after being released from GrB-treated mitochondria. The N-terminal Hax-1 fragment major cellular impact is on the regulation of mitochondrial polarization. Overexpression of wild-type Hax-1 or its uncleavable mutant form protects the mitochondria against GrB or valinomycin-mediated depolarization. The N-terminal Hax-1 fragment functions as a dominant negative form of Hax-1, mediating mitochondrial depolarization in a cyclophilin D-dependent manner. Thus, induced expression of the N-terminal Hax-1 fragment results in mitochondrial depolarization and subsequent lysosomal degradation of such altered mitochondria. This study is the first to demonstrate GrB activity within the mitochondrion and to identify Hax-1 cleavage as a novel mechanism for GrB-mediated mitochondrial depolarization.