Regulation Of Mitochondrial Activity Research Articles

Stattic V, a STAT3 inhibitor, affects human spermatozoa through regulation of mitochondrial activity

We have recently shown that many mediators of the JAK/STAT signaling pathway are present in ejaculated human spermatozoa. Among them, STAT3 is detected mainly in membranes and flagellar cytoskeletal fractions. In order to determine the importance of STAT3-mediated signaling, sperm were incubated with Stattic V, a specific inhibitor. Effects on motility were evaluated by CASA, sperm acrosomal integrity was evaluated by FITC conjugated lectin (PSA or PNA) staining, and protein phosphotyrosine content was assessed by Western blot using a monoclonal anti-phosphotyrosine antibody. INDO1-AM and JC-1 were used to measure sperm intracellular calcium and mitochondrial membrane potential, respectively, by flow cytometry, and reactive oxygen species (ROS) production was investigated by luminol-based assay. Percentages of motility and motility parameters were significantly affected by Stattic V. This later also significantly increased intracellular Ca(2+) levels, progesterone- and calcium ionophore (A23187)-induced acrosome reaction. On the other hand, a significant decrease in ATP content was measured when sperm were treated with Stattic V, associated with depolarization of mitochondrial membrane and elevated ROS production. These results suggest that STAT3 is involved in sperm functions, at least through regulation of mitochondrial activity. This further emphasizes that STAT3 mediates cellular activities in a manner different than strictly the activation of gene transcription.

Translational Control of Mitochondrial Energy Production Mediates Neuron Morphogenesis

Mitochondrial energy production is a tightly regulated process involving the coordinated transcription of several genes, catalysis of a plethora of posttranslational modifications, and the formation of very large molecular supercomplexes. The regulation of mitochondrial activity is particularly important for the brain, which is a high-energy-consuming organ that depends on oxidative phosphorylation to generate ATP. Here we show that brain mitochondrial ATP production is controlled by the cytoplasmic polyadenylation-induced translation of an mRNA encoding NDUFV2, a key mitochondrial protein. Knockout mice lacking the Cytoplasmic Polyadenylation Element Binding protein 1 (CPEB1) have brain-specific dysfunctional mitochondria and reduced ATP levels, which is due to defective polyadenylation-induced translation of electron transport chain complex I protein NDUFV2 mRNA. This reduced ATP results in defective dendrite morphogenesis of hippocampal neurons both invitro and invivo. These and other results demonstrate that CPEB1 control of mitochondrial activity is essential for normal brain development.

A PGC-1 Tale: Healthier Intestinal Stem Cells, Longer Life

PGC-1 regulates energy homeostasis and mitochondrial activity. In this issue, Rera etal. (2011) show that dPGC-1 overexpression in Drosophila intestinal stem cells and their immediate progeny extends organismal life span and protects against age-related loss of intestinal homeostasis and integrity, thereby establishing a link between mitochondria, tissue stem cells, and aging.

In situ kinetic trapping reveals a fingerprint of reversible protein thiol oxidation in the mitochondrial matrix

Reactive oxygen species (ROS) are released at the mitochondrial inner membrane by the electron transport chain (ETC). Increasing evidence suggests that mitochondrial H2O2 acts as a signaling molecule and participates in the (feedback) regulation of mitochondrial activity and turnover. It seems likely that key mitochondrial components contain redox-sensitive thiols that help to adapt protein function to changes in electron flow. However, the identity of most redox-regulated mitochondrial proteins remains to be defined. Thioredoxin 2 (Trx2) is the major protein-thiol-reducing oxidoreductase in the mitochondrial matrix. We used in situ mechanism-based kinetic trapping to identify disulfide-exchange interactions of Trx2 within functional mitochondria of intact cells. Mass spectrometry successfully identified known and suspected Trx2 target proteins and, in addition, revealed a set of new candidate target proteins. Our results suggest that the mitochondrial protein biosynthesis machinery is a major target of ETC-derived ROS. In particular, we identified mitochondrial methionyl-tRNA synthetase (mtMetRS) as one of the most prominent Trx2 target proteins. We show that an increase in ETC-derived oxidants leads to an increase in mtMetRS oxidation in intact cells. In conclusion, we find that in situ kinetic trapping provides starting points for future functional studies of intramitochondrial redox regulation.

Mitochondrial sirtuins in the regulation of mitochondrial activity and metabolic adaptation.

In eukaryotes, mitochondria carry out numerous functions that are central to cellular and organismal health. How mitochondrial activities are regulated in response to differing environmental conditions, such as variations in diet, remains an important unsolved question in biology. Here, we review emerging evidence suggesting that reversible acetylation of mitochondrial proteins on lysine residues represents a key mechanism by which mitochondrial functions are adjusted to meet environmental demands. In mammals, three members of the sirtuin class of NAD(+)-dependent deacetylases - SIRT3, SIRT4, and SIRT5 - localize to mitochondria and regulate targets involved in a diverse array of biochemical pathways. The importance of this activity is highlighted by recent studies of SIRT3 indicating that this protein suppresses the emergence of diverse age-related pathologies: hearing loss, cardiac fibrosis, and malignancy. Together, these findings argue that mitochondrial protein acetylation represents a central means by which mammals regulate mitochondrial functions to maintain cellular and organismal homeostasis.

Essential Regulation of Cell Bioenergetics by Constitutive InsP3 Receptor Ca2+ Transfer to Mitochondria

Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of mitochondrial activity is incompletely understood. Here we identified an unexpected and essential role for constitutive InsP(3)R-mediated Ca(2+) release in maintaining cellular bioenergetics. Macroautophagy provides eukaryotes with an adaptive response to nutrient deprivation that prolongs survival. Constitutive InsP(3)R Ca(2+) signaling is required for macroautophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to diminished mitochondrial Ca(2+) uptake. Mitochondrial uptake of InsP(3)R-released Ca(2+) is fundamentally required to provide optimal bioenergetics by providing sufficient reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer results in enhanced phosphorylation of pyruvate dehydrogenase and activation of AMPK, which activates prosurvival macroautophagy. Thus, constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration and maintenance of normal cell bioenergetics.

Nuclear Receptor SHP, a Death Receptor That Targets Mitochondria, Induces Apoptosis and Inhibits Tumor Growth

Small heterodimer partner (SHP) is an epigenetically regulated nuclear transcriptional repressor that suppresses the development of liver cancer by inhibiting cellular growth. Here we report a novel cytoplasmic function of SHP through its regulation of mitochondrial activity. SHP is a pivotal cell death receptor that targets mitochondria, where it binds with Bcl-2, disrupts Bcl-2/Bid interaction, and induces cytochrome c release. The apoptosis inducer AHPN {retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid} acts by regulating SHP gene expression and promotes the translocation of SHP from the nucleus to the mitochondria. Induction of apoptosis by SHP activation inhibits peritoneal pancreatic tumor growth. Our findings provide for the first time a mechanism by which SHP regulates cell survival, namely, by controlling mitochondrial function via modulating the activity of Bcl-2 through AHPN-mediated or AHPN-independent action. Thus, SHP regulates a mechanism by which apoptotic signals can mediate local control of mitochondrial function and apoptosis, which in turn may limit tumorigenesis.

Nutrition Controls Mitochondrial Biogenesis in the Drosophila Adipose Tissue through Delg and Cyclin D/Cdk4

Mitochondria are cellular organelles that perform critical metabolic functions: they generate energy from nutrients but also provide metabolites for de novo synthesis of fatty acids and several amino acids. Thus mitochondrial mass and activity must be coordinated with nutrient availability, yet this remains poorly understood. Here, we demonstrate that Drosophila larvae grown in low yeast food have strong defects in mitochondrial abundance and respiration activity in the larval fat body. This correlates with reduced expression of genes encoding mitochondrial proteins, particularly genes involved in oxidative phosphorylation. Second, genes involved in glutamine metabolism are also expressed in a nutrient-dependent manner, suggesting a coordination of amino acid synthesis with mitochondrial abundance and activity. Moreover, we show that Delg (CG6338), the Drosophila homologue to the alpha subunit of mammalian transcription factor NRF-2/GABP, is required for proper expression of most genes encoding mitochondrial proteins. Our data demonstrate that Delg is critical to adjust mitochondrial abundance in respect to Cyclin D/Cdk4, a growth-promoting complex and glutamine metabolism according to nutrient availability. However, in contrast to nutrients, Delg is not involved in the regulation of mitochondrial activity in the fat body. These findings are the first genetic evidence that the regulation of mitochondrial mass can be uncoupled from mitochondrial activity.

Breathing lessons: Tor tackles the mitochondria

Breathing lessons: Tor tackles the mitochondria

Proteomic profiling of endothelin-1-stimulated hypertrophic cardiomyocytes reveals the increase of four different desmin species and α-B-crystallin

We performed a proteomic investigation on primary cultures of neonatal rat cardiomyocytes after treatment with 10 nM endothelin-1 (ET1) for 48 h, an in vitro model for cardiac hypertrophy. Two-dimensional gel electrophoresis profiles of cell lysates were compared after colloidal Coomassie Blue staining. 12 protein spots that significantly changed in density due to ET1 stimulation were selected for in-gel digestion and identified through mass spectrometry. Of these, 8 spots were increased and 4 were decreased. Four of the increased proteins were identified as desmin, the cardiac component of intermediate filaments and one as α-B-crystallin, a molecular chaperone that binds desmin. All the desmins increased 2- to 5-fold, and α-B-crystallin increased 2-fold after ET1 treatment. Desmin cytoskeleton has been implicated in the regulation of mitochondrial activity and distribution, as well as in the formation of amyloid bodies. Mitochondria-specific fluorescent probe MitoTracker indicated mitochondrial redistribution in hypertrophic cells. An increase of amyloid aggregates containing desmin upon treatment with ET1 was detected by filter assay. Of the four proteins that showed decreased abundance after ET1 treatment, the chaperones hsp60 and grp75 were decreased 13- and 9-fold, respectively. In conclusion, proteomic profiling of ET1-stimulated rat neonatal cardiomyocytes reveals specific changes in cardiac molecular phenotype mainly involving intermediate filament and molecular chaperone proteins.

The role of mitochondria in status epilepticus

Reflecting their central roles in cellular metabolism,including energy production and calcium homeostasis, mi-tochondria have been proposed to act as the “stress sensor”of the cell, and in extreme circumstances (e.g., excitotoxi-city) the executioner. A large body of work has establishedmitochondrial dysfunction as the “lowest common denom-inator” in a range of inherited (Kwong et al., 2006) and ac-quired (Cock, 2005) neurodegenerative disease processes.In addition to bioenergetic failure and increased cytosoliccalcium, consequences of impaired mitochondrial func-tion (summarized in Fig. 1) include oxidative stress (ex-cessive free radical production and impaired synthesis ofantioxidants, especially glutathione), and the release ofkey proteins into the cytosol (mitochondrial permeabilitytransition) triggering cell death pathways (caspases). How-ever, it is also increasingly clear that many of the “patho-logical” processes also have key physiological roles inmetabolic control and cell signaling, in which context pro-teins such as uncoupling proteins have attracted particu-lar interest, but are not yet fully understood (Cock, 2005).On this background, interest in the role of mitochondriain seizure-related brain damage has rapidly developed overthe last decade; though only work specifically relating tostatus epilepticus (as opposed to chronic epilepsy) will bediscussed here.

Hypertrophic growth in cardiac myocytes is mediated by Myc through a Cyclin D2-dependent pathway

c-Myc (Myc) is highly expressed in developing embryos where it regulates body size by controlling proliferation but not cell size. However, Myc is also induced in many postmitotic tissues, including adult myocardium, in response to stress where the predominant form of growth is an increase in cell size (hypertrophy) and not number. The function of Myc induction in this setting is unproven. Therefore, to explore Myc's role in hypertrophic growth, we created mice where Myc can be inducibly inactivated, specifically in adult myocardium. Myc-deficient hearts demonstrated attenuated stress-induced hypertrophic growth, secondary to a reduction in cell growth of individual myocytes. To explore the dependence of Myc-induced cell growth on CycD2, we created bigenic mice where Myc can be selectively activated in CycD2-null adult myocardium. Myc-dependent hypertrophic growth and cell cycle reentry is blocked in CycD2-deficient hearts. However, in contrast to Myc-induced DNA synthesis, hypertrophic growth is independent of CycD2-induced Cdk2 activity. These data suggest that Myc is required for a normal hypertrophic response and that its growth-promoting effects are also mediated through a CycD2-dependent pathway.

A Radical View of Mitochondrial Function

Endogenous nitric oxide (NO) acts on cytochrome c oxidase, the terminal enzyme in the mitochondrial electron transport chain, inhibiting mitochondrial oxygen consumption and promoting the release of free radicals. Quintero et al. investigated the consequences of NO regulation of mitochondrial activity in vascular endothelial cells, which are highly glycolytic and have abundant NO. Analysis of the effects of inhibitors of mitochondrial electron transport or glycolysis at various oxygen concentrations indicated that ATP production in human umbilical vein endothelial cells (HUVECs) depended more on glycolysis than it did on mitochondrial respiration. Exposure to a low concentration of oxygen (1.5%) that promoted the accumulation of hypoxia-inducible factor 1α (HIF-1α) in human smooth muscle and epithelial kidney cells had little effect on HIF-1α abundance in human vascular endothelial cells unless the production of NO was inhibited. The ability of NO to inhibit HIF-1α accumulation in HUVECs depended on mitochondrial respiration. The production of reactive oxygen species (ROS) in HUVECs depended on oxygen concentration, being greater at 3% oxygen than at 0.5% or 21% oxygen, and, at 3% oxygen, depended on the production of NO. Activation of AMP-activated protein kinase (AMPK) also peaked at 3% oxygen, and this did not depend on the ratio of AMP to ATP or on the abundance of AMP. Rather, the activation of AMPK at 3% oxygen was stimulated by ROS and was inhibited by antioxidants. Thus, the authors came to the intriguing conclusion that, in vascular endothelial cells, mitochondria may function primarily as NO-regulated organelles that signal through oxygen or ROS rather than as the "powerhouse" that provides cells with ATP. M. Quintero, S. L. Colombo, A. Godfrey, S. Moncada, Mitochondria as signaling organelles in the vascular endothelium. Proc. Natl. Acad. Sci. U.S.A. 103 , 5379-5384 (2006). [Abstract] [Full Text]

Formation of Membrane-bound Ring Complexes by Prohibitins in Mitochondria

Prohibitins comprise a remarkably conserved protein family in eukaryotic cells with proposed functions in cell cycle progression, senescence, apoptosis, and the regulation of mitochondrial activities. Two prohibitin homologues, Phb1 and Phb2, assemble into a high molecular weight complex of approximately 1.2 MDa in the mitochondrial inner membrane, but a nuclear localization of Phb1 and Phb2 also has been reported. Here, we have analyzed the biogenesis and structure of the prohibitin complex in Saccharomyces cerevisiae. Both Phb1 and Phb2 subunits are targeted to mitochondria by unconventional noncleavable targeting sequences at their amino terminal end. Membrane insertion involves binding of newly imported Phb1 to Tim8/13 complexes in the intermembrane space and is mediated by the TIM23-translocase. Assembly occurs via intermediate-sized complexes of approximately 120 kDa containing both Phb1 and Phb2. Conserved carboxy-terminal coiled-coil regions in both subunits mediate the formation of large assemblies in the inner membrane. Single particle electron microscopy of purified prohibitin complexes identifies diverse ring-shaped structures with outer dimensions of approximately 270 x 200 angstroms. Implications of these findings for proposed cellular activities of prohibitins are discussed.

Bonsaï, a ribosomal protein S15 homolog, involved in gut mitochondrial activity and systemic growth

The regulation of cellular growth is crucial in the control of cell proliferation. While most of the metabolic energy necessary to sustain growth is produced in mitochondria, the regulation of mitochondrial activity and its implications for growth have remained unexplored. Here, a gene named bonsaï is described, which is essential for normal growth in Drosophila. The Bonsaï protein bears strong homology to prokaryotic ribosomal protein S15 and localizes to mitochondria, suggesting a role in mitochondrial protein translation. Accordingly, bonsaï mutants have defective mitochondrial activity, but surprisingly, only the gut appears affected. Consistent with these observations, bonsaï is predominantly expressed in the gut. These results show that bonsaï plays a preponderant role in gut mitochondria. Although gut mitochondrial respiration is altered in bonsaï mutants, the digestive process appears normal, suggesting that a gut function other than digestion is impaired in the mutants. Cytochrome c oxidase, a respiratory chain enzyme partly encoded by the mitochondrial genome, is found to be active in bonsaï mutants. This suggests that mitochondrial translation is not abolished in the mutants. Altogether, these observations suggest that mitochondrial activity is regulated at the tissue-specific level and that this regulation has profound implications for growth and development.

Changes in mitochondrial activity evoked by cholecystokinin in isolated mouse pancreatic acinar cells

In the present study, we have employed confocal laser scanning microscopy to investigate the effect that stimulation of mouse pancreatic acinar cells with the secretagogue cholecystokinin (CCK) has on mitochondrial activity. We have monitored changes in cytosolic as well as mitochondrial Ca 2+ concentrations, mitochondrial membrane potential and FAD autofluorescence by loading the cells with fluo-3, rhod-2 or JC-1, respectively. Our results show that stimulation of cells with cholecystokinin led to release of Ca 2+ from intracellular stores that then accumulated into mitochondria. In the presence of the hormone a depolarization of mitochondrial membrane potential was observed, which partially recovered; in addition a transient increase in FAD autofluorescence could be observed. Similarly, treatment of cells with thapsigargin induced increases in mitochondrial Ca 2+ and FAD autofluorescence, and depolarized mitochondria. Pretreament of cells with thapsigargin blocked cholecystokinin-evoked changes. Similar results were obtained when the cells were incubated in the presence of rotenone, which blocks the mitochondrial electron transport chain. Our findings are consistent with changes in mitochondrial activity in response to stimulation of pancreatic acinar cells with cholecystokinin. Following stimulation, mitochondria take up Ca 2+ that could in turn activate the mitochondrial machinery that may match the energy supply necessary for the cell function during secretion, suggesting that Ca 2+ can act as a regulator of mitochondrial activity.

Estrogen receptor ligands affect mitochondrial activity in SH-SY5Y human neuroblastoma cells.

We have studied the pharmacological regulation of mitochondrial activity in a human neuroblastoma cell line. Cyclosporin A was found to directly alter mitochondrial membrane potential and to decrease mitochondrial permeability as measured using calcein. The estrogen receptor ligands tamoxifen, nafoxidine and clomiphene were identified as agents which affect mitochondrial membrane potential in a cyclosporin A-like manner. Also when mitochondrial permeability was measured using calcein, tamoxifen, nafoxidine and clomiphene were effective in inhibiting dye loss from mitochondria. Nafoxidine and cyclosporin A inhibit effects of mastoparan on SH-SY5Y mitochondria. These studies indicate that estrogen receptor ligands appear to affect mitochondria in a cyclosporin A-like manner in human neuroblastoma cells.

Changes in intramitochondrial cardiolipin distribution in apoptosis-resistant HCW-2 cells, derived from the human promyelocytic leukemia HL-60

Using a cytofluorimetric approach, we studied intramitochondrial cardiolipin (CL) distribution in HCW-2 cells, an apoptosis-resistant clone of human HL-60 cells. In HL-60, about 50% of total CL is distributed in the outer leaflet of mitochondrial inner membrane, while in HCW-2 a significantly higher amount of CL (about 65%) is in that site. In basal conditions, HSW-2 cells also show a reduced mitochondrial membrane potential even if they are able to proliferate as the parental line. Taking into account the complex functions that CL plays in the regulation of mitochondrial activity, it is likely that HCW-2 could produce ATP utilizing more glycolytic pathways rather than mitochondrial respiratory chain.

Regulation of Mitochondrial Activity in Cardiac Cells

Regulation of Mitochondrial Activity in Cardiac Cells

Regulation of mitochondrial activity by indoleacetic acid

Abstract Mitochondria of scutella of a maize inbred were allowed to respire in the presence of indoleacetic acid. Greatest enhancement of Q O 2 N , P/N and P/O was observed with indoleacetic acid concentration around 1.07 · 10−10 M. At concentrations above 1.07 · 10−8 M, O2 uptake was greatly inhibited while phosphorylation was inhibited to a lesser degree. It was shown that enhancement of mitochondrial activity is dependent on CoA; without added CoA, effects of indoleacetic acid were slight. The most effective concentration of CoA was shown to be 0.1 mg/2.8 ml reaction mixture. In the presence of 1.07 · 10−10 M indoleacetic acid, rate of O2 uptake measured at 3-min intervals was greatly increased. In addition, with indoleacetic acid in the reaction mixture, mitochondria showed an almost immediate enhancement of activity while the control reaction and the reaction with high concentration of indoleacetic acid (1.07 · 10−5 M) exhibited a lag of 3–4 min. With regard to the relationship of CoA and indoleacetic acid-induced stimulation of rate of mitochondrial O2 uptake, two points were observed: without added CoA, the rate of O2 uptake is sharply reduced and there is a lag of 7 min before mitochondria show any O2 uptake. The results suggest that indoleacetic acid enhances mitochondrial activity by affecting the enzyme(s) of the oxidative phosphorylation pathway. It is suggested that indoleacetic acid accomplishes such enhancement by acting as an allosteric effector.