ATP Inhibition Research Articles

Abstract 4294: Copper is required for oncogenic BRAF signaling and tumorigenesis.

Abstract The BRAF serine/threonine kinase is mutated, typically at V600, to remain in the active oncogenic state in a large fraction of melanoma, thyroid cancers, and hairy cell leukemia, and to a lesser extent in a wide spectrum of other cancers, thereby activating the kinases MEK1 and MEK2 to stimulate the MAPK pathway and promote cancer. Excitingly, ATP inhibitors of oncogenic BRAF and MEK provide a survival advantage in metastatic melanoma and early clinical studies suggest that coupling BRAF and MEK kinase inhibitors may be even more effective. Thus, the combination of multiple approaches to inhibit MAPK signaling holds great promise for the treatment of BRAF mutation-positive cancers, especially in terms of overcoming resistance. In this regard, we previously found that copper (Cu) influx enhanced MEK1 phosphorylation of its substrates ERK1/2 through a Cu-MEK1 interaction. We show here that genetic loss of the high affinity Cu transporter Ctr1 or mutations in MEK1 that disrupt Cu binding reduced MAPK signaling and oncogenic BRAFV600E-mediated tumorigenesis, which could be rescued by expressing activated ERK2. Importantly, a Cu chelator used in the treatment of Wilson's disease reduced tumor growth of not only BRAFV600E-transformed cells, but also cells resistant to a BRAF inhibitor. Taken together, these results suggest that Cu-chelation therapy could be repurposed for the treatment of BRAFV600E mutation-positive cancers. Citation Format: Donita C. Brady, Matthew S. Crowe, Michelle L. Turski, Dennis J. Thiele, Christopher M. Counter. Copper is required for oncogenic BRAF signaling and tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4294. doi:10.1158/1538-7445.AM2013-4294

A new simple fluorimetric method to assay cytosolic ATP content: application to durum wheat seedlings to assess modulation of mitochondrial potassium channel and uncoupling protein activity under hyperosmotic stress

AbstractThe assays commonly used to determine ATP content in biological samples generally measure total cellular ATP content, but not the different subcellular pools. In this study a new simple method for measuring ATP content in a cytosol-enriched fraction (CEF) was developed, based on a rapid cytosolic ATP extraction (by an isotonic grinding medium that preserves organelle integrity) and its detection monitoring the NADPH fluorescence generated via hexokinase/glucose-6-phosphate dehydrogenase coupled reactions. Four protocols, differing for timing of NADPH generation and for either the presence or absence of some inhibitors of ATP and NADPH metabolism, were compared by determining CEF-ATP, as well as total ATP, in durum wheat (Triticum durum Desf.) etiolated seedlings. The best protocol was the one adopting both simultaneous NADPH generation and use of inhibitors during tissue homogenization. This protocol also showed higher performance than the classical trichloroacetic acid extraction. Using the new method, CEF-ATP content was assessed in control, salt- and osmotic-stressed seedlings, resulting 2.68 ± 0.04, 1.69 ± 0.12 (−40%) and 1.35 ± 0.16 (−50%) μmol/g dry weight, respectively. Finally, the effects of this stress-dependent decrease of cytosolic ATP were evaluated with respect to a possible modulation of two mitochondrial energy-dissipating systems, the uncoupling protein (PUCP) and the K+ channel (PmitoKATP), both inhibited by cytosolic ATP. Experiments carried out at different physiological ATP concentrations suggest that the decreased cytosolic ATP content occurring under hyperosmotic stress may contribute to attenuate inhibition of PmitoKATP, thus promoting its activity (up to about 90%), but not of PUCP, that appears to lose ATP sensitivity under stress condition.

ATP Inhibition Couples Guanylyl Cyclase A and B to Cellular Energy Status

Homodimeric and heterodimeric guanylyl cyclases (GCs) regulate numerous biologic functions by catalyzing the synthesis of cGMP in response to peptide or gaseous ligands, respectively. Atrial natriuretic peptide (NP) and C‐type NP reduce the Km for GC‐A and GC‐B, respectively, by a process that requires ATP binding to a high affinity allosteric activation site in the catalytic domain with an EC50 of ~0.1 mM. Here, we show that higher physiologic concentrations of ATP inhibit GC‐A and GC‐B by binding a low affinity site with an IC50 of ~2 mM in the catalytic domain. Unlike activation, inhibition neither required the 2'ribosyl OH group of ATP nor CNP. The inhibition was mixed type and was observed with ATP and ADP but not AMP. Inhibition was competitive with the product, pyrophosphate but not the substrate, GTP. We suggest that changes in cellular ATP concentrations couple GC activity to the energy status of the cell by binding the pyrophosphate site.

Pharmadynamic (PD) & pharmacokinetic (PK) properties of AF‐219: first in class, selective, clinical P2X3 antagonist in development for chronic pain and related conditions

P2X3 receptors are ATP‐gated channels with expression somewhat limited to primary afferent populations suspected of chronic sensitization in pain‐related disorders & offering therapeutic potential for antagonists (Ford, 2012, Purinergic Signal. 8:3–26). We describe preclinical PD & PK of AF‐219 (MW 353), a selective P2X3 antagonist optimized from an HTS lead.AF‐219 blocked ATP activation of rat & human P2X3 & P2X2/3 trimers in calcium flux & voltage‐clamp assays: IC50 values 25–200 nM; unlike A‐317491 & TNP‐ATP, allosteric inhibition of ATP was displayed. In homogenate binding studies, AF‐219, but not ATP or TNP‐ATP, displaced specific binding of [3H]AF‐353. At 10 μM no activity was seen at other P2X channels & in a battery of receptors, enzymes & channels.Favorable PK properties were observed for AF‐219 in rat, rabbit, dog & primate: high metabolic stability; oral availability (38–82 %F), elimination half‐life (t½ 1.5–7.6 h) & plasma free‐fraction (55–67% protein‐bound); predominant renal elimination (parent). AF‐219 was dose‐dependently efficacious (1–60 mg/kg, p.o. or s.c.; total plasma range 100–3000 ng/ml) in rodent models of hyperalgesia (neuropathic & inflammatory) or visceral hypersensitivity. The favorable PD, PK, & in vivo activity confirm AF‐219 as an excellent medicinal candidate to establish the role of P2X3 receptors in chronic pain and related conditions.

Purinergic modulation of smooth muscle intracellular Ca2+ concentration

Basal, steady‐state Ca2+ levels are determined by the balance between Ca2+ release/influx and uptake/extrusion mechanisms. Extrinsic factors can alter this balance to affect smooth muscle excitability. Extracellular ATP acts as an inhibitory neurotransmitter in gastrointestinal and vascular (portal vein) smooth muscle. This study examined the effect of extracellular ATP on the steady‐state intracellular Ca2+ concentration ([Ca2+]c). Extracellular ATP (1 mM; applied via picopritzer) reversibly decreased steady‐state [Ca2+]c (measured using fluo 4). To assess whether ATP functioned by altering influx or extrusion [Ca2+]c and inward currents were measured simultaneously in voltage‐clamped myocytes during a depolarizing pulse. ATP decreased the peak amplitude without altering the voltage‐dependence of the inward current. The P2 purinoceptor inhibitor, suramin, and the P2Y1 specific inhibitor, MRS2179, each prevented ATP inhibition of steady‐state [Ca2+]c and inward current. Interestingly, suramin and MRS2179 each also increased the magnitude of the inward current per se, indicating tonic ATP inhibition of Ca2+ channels by endogenous ATP. ATP did not alter Ca2+ uptake/extrusion mechanisms. ATP appears to influence Ca2+ influx mechanisms in smooth muscle via P2Y receptors to exert a negative regulation of excitability.

Loss of pH Control in Plasmodium falciparum Parasites Subjected to Oxidative Stress

The intraerythrocytic malaria parasite is susceptible to oxidative stress and this may play a role in the mechanism of action of some antimalarial agents. Here we show that exposure of the intraerythrocytic malaria parasite to the oxidising agent hydrogen peroxide results in a fall in the intracellular ATP level and inhibition of the parasite's V-type H+-ATPase, causing a loss of pH control in both the parasite cytosol and the internal digestive vacuole. In contrast to the V-type H+-ATPase, the parasite's digestive vacuole H+-pyrophosphatase is insensitive to hydrogen peroxide-induced oxidative stress. This work provides insights into the effects of oxidative stress on the intraerythrocytic parasite, as well as providing an alternative possible explanation for a previous report that light-induced oxidative stress causes selective lysis of the parasite's digestive vacuole.

Mitochondrial JNK phosphorylation as a novel therapeutic target to inhibit neuroinflammation and apoptosis after neonatal ischemic brain damage

Neonatal encephalopathy is associated with high mortality and life-long developmental consequences. Therapeutic options are very limited. We assessed the effects of D-JNKi, a small peptide c-Jun N-terminal kinase (JNK) MAP kinase inhibitor, on neuroinflammation, mitochondrial integrity and neuronal damage in a neonatal rat model of ischemic brain damage.Hypoxic–ischemic (HI) brain injury was induced in postnatal-day 7 rats by unilateral carotid artery occlusion and hypoxia, and was followed by intraperitoneal D-JNKi treatment.We demonstrate here for the first time that a single intraperitoneal injection with D-JNKi directly after HI strongly reduces neonatal brain damage by >85% with a therapeutic window of at least 6h. D-JNKi treatment also restored cognitive and motor function as analyzed at 9weeks post-insult.Neuroprotective D-JNKi treatment inhibited phosphorylation of nuclear c-Jun (P-c-Jun), and consequently reduced activity of the AP-1 transcription factor and production of cerebral cytokines/chemokines as determined at 3 and 24h post-HI. Inhibition of P-c-Jun by D-JNKi is thought to be mediated via inhibition of the upstream phosphorylation of cytosolic and nuclear JNK and/or by preventing the direct interaction of phosphorylated (P-)JNK with c-Jun. Surprisingly, however, HI did not induce a detectable increase in P-JNK in cytosol or nucleus. Notably, we show here for the first time that HI induces P-JNK only in the mitochondrial fraction, which was completely prevented by D-JNKi treatment. The hypothesis that mitochondrial JNK activation is key to HI brain injury was supported by data showing that treatment of rat pups with SabKIM1 peptide, a specific mitochondrial JNK inhibitor, is also neuroprotective. Inhibition of HI-induced mitochondrial JNK activation was associated with preservation of mitochondrial integrity as evidenced by prevention of ATP loss and inhibition of lipid peroxidation. The HI-induced increase in apoptotic markers (cytochrome c release and caspase 3 activation) as analyzed at 24h post-HI were also strongly reduced by D-JNKi and the mitochondrial anti-apoptotic proteins Bcl-2 and Bcl-xL were upregulated. Neuroprotection was lost after repeated 0+3h D-JNKi treatment which was associated with complete inhibition of the second peak of AP-1 activity and disability to upregulate mitochondrial Bcl-2 and Bcl-xL.We show here for the first time that D-JNKi treatment efficiently protects the neonatal brain against ischemic brain damage and subsequent cognitive and motor impairment. We propose that inhibition of phosphorylation of mitochondrial JNK is a pivotal step in preventing early loss of mitochondrial integrity leading to reduced neuroinflammation and inhibition of apoptotic neuronal loss. Moreover we show the crucial role of upregulation of mitochondrial anti-apoptotic proteins to maintain neuroprotection.

Distribution of Intracellular ADP Diffusion Restriction in Trout Cardiomyocytes

Cardiomyocytes are compartmentalized by intracellular diffusion restrictions. In our previous study we confirmed that isolated permeabilized cardiomyocytes from rainbow trout (Oncorhynchus mykiss) also display intracellular diffusion restriction (Sokolova et al., BMC Cell Biology, 10:90, 2009). This was surprising, because trout cardiomyocytes are much thinner than those of mammals, lack t-tubules, have a lower sarcoplasmic reticulum density and only a single layer of myofilaments surrounding a central core of mitochondria. However, the extent of diffusion restriction is much smaller than in mammalian cardiomyocytes. The aim of the present study was to find the possible distribution of diffusion restrictions in permeabilized trout cardiomyocytes. We measured mitochondrial respiration stimulated by ADP and ATP, and evaluated the rate of inhibition of ATP stimulated respiration by an ADP trapping system, consisting of phosphoenolpyruvate (PEP) activating endogenous and exogenous pyruvate kinase (PK), which competes with mitochondria for ADP. We found a high activity of hexokinase, which stimulates mitochondrial respiration when activated by glucose. To study its role in more detail, all experiments were performed in the absence and presence of 2 mM glucose. Additionally, we performed ADP titrations on cells alone, in the presence of glucose, and in the presence of creatine kinase and creatine. We found that activation of hexokinase by glucose, but not activation of creatine kinase by creatine, lowered the apparent ADP-affinity in permeabilized trout cardiomyocytes. This is in contrast to the situation in permeabilized mammalian cardiomyocytes, where activation of mitochondrial creatine kinase has a profound effect on the apparent ADP-affinity. This suggests that trout cardiomyocytes have a different ADP-feedback system than mammalian cardiomyocytes. The experimental results will be fitted with a mathematical model (Sepp et al., Biophysical Journal, 98:12, 2010) showing more specifically the distribution and extent of compartmentalization of trout cardiomyocytes.

Awakening Loss-of-Function KATP Channel Mutants with an Engineered ‘Forced Gating’ Approach

Regulation of inwardly-rectifying potassium channels by intracellular ligands couples membrane excitability to important signaling cascades and metabolic pathways. A significant barrier to understand coupling mechanisms between ligand binding and gating is that many functionally important channel motifs are highly sensitive to mutagenesis, resulting in a loss of function phenotype. We have developed a ‘forced gating’ approach that rescues function in electrically silent channel mutants, enabling characterization of channel motifs that are otherwise intractable to electrophysiological recording. This approach involves substitution of a glutamate in the hydrophobic Kir channel bundle crossing (F168E mutation in Kir6.2), generating channels that are pH sensitive and open upon alkalization, due to mutual repulsion of introduced negatively charged side chains in the channel gate. We have implemented this ‘forced gating’ approach in mutagenic scans of the Kir channel slide helix and G-loop, two motifs proposed to play a role in ligand dependent gating of Kir channels. Both motifs are also highly sensitive to mutagenesis, with alanine mutations causing nearly complete loss-of-function at 7/20 slide helix positions, and 8/13 G-loop positions. Without exception, expression of silent mutants on the Kir6.2[F168E] background permitted activation of functional channels in alkaline pH, and measurement of kinetics and potency of ATP inhibition. Our results highlight an essential ‘aspartate anchor’ (Kir6.2 residue D58) that bridges the slide helix and multiple interacting residues in the cytoplasmic domain. Disruption of the highly conserved ‘aspartate anchor’ uncouples the transmembrane and cytoplasmic domains, reducing ATP sensitivity of Kir6.2 far more than any other G-loop or slide helix mutants. These findings indicate a central role for the ‘aspartate anchor’ in coupling ligand binding to Kir gating, and also emphasize the potential general utility of this ‘forced gating’ method to study loss-of-function channel mutants.

Lessons from KATP Channels with Diabetogenic Mutations in Sulfonylurea Receptor 1 (SUR1)

Numerous mutations have been identified in SUR1 (ABCC8) subunit of the neuroendocrine type KATP channel in subjects with neonatal diabetes, neonatal diabetes plus epilepsy and/or other neurological features, maturity-onset diabetes of young, and later-onset diabetes. Patch-clamping, single-channel kinetics analysis, affinity photolabeling and molecular modeling were used to clarify how diabetogenic mutations in different parts of SUR1 affect open probability and sulfonylurea inhibition of SUR1/Kir6.2 KATP channel. Essentially all tested diabetogenic mutations in the canonical TMD1-NBD1-TMD2-NBD2 ABC exporter core of SUR1 hyperactivated KATP by stabilizing the stimulatory Mg-nucleotide bound (outward facing) state of SUR1 without affecting the intrinsic gating of KATP channel or its sensitivity to inhibitory nucleotides. Hyperstimulated mutant channels showed attenuated sulfonylurea inhibition in the presence, but not the absence, of stimulatory MgATP/ADP, indicating that KATP with SUR1 in the inward facing state has the lowest Kd for sulfonylureas. Diabetogenic mutations in the non-canonical TMD0-L0 part of SUR1 hyperactivated KATP by destabilizing its long-lived closed state with the highest affinity to inhibitory ATP or by strengthening the functional coupling between the MgATP/ADP-bound SUR1 core and the active (burst) state of the Kir pore. Mutations destabilizing the long-lived closed state compromised sulfonylurea inhibition of KATP in the absence of nucleotides but not the drug-induced release of stimulatory nucleotides. The findings support the mechanistic model (FEBS Letters, 585:3555-9) in which the TMD0-L0 module couples the SUR1 core with the KATP pore, define the most common ABCC8-associated mechanisms of KATP hyperactivity, and largely explain why the majority of diabetic subjects with mutant SUR1 require body-weight normalized doses of sulfonylureas exceeding those recommended by the FDA for treatment of common type 2 diabetes.

Citations

Citations

Polo-like kinase 1 as target for cancer therapy

Polo-like kinase 1 (Plk1) is an interesting molecule both as a biomarker and as a target for highly specific cancer therapy for several reasons. Firstly, it is over-expressed in many cancers and can serve as a biomarker to monitor treatment efficacy of Plk1 inhibitors. Furthermore, the Plk1 enzyme is expressed only in dividing cells and is a major regulator of the cell cycle. It controls entry into mitosis and regulates the spindle checkpoint. The expression of Plk1 in normal cells is not nearly as strong as that in cancer cells, which makes Plk1 a discriminating tartget for the development of cancer-specific small molecule drugs. RNA interference experiments in vitro and in vivo have indicated that downregulation of Plk1 expression represents an attractive concept for cancer therapy. Over the years, a number of Plk1 inhibitors have been discovered. Many of these inhibitors are substances that compete with ATP for the substrate binding site. The ATP-competitive inhibitor BI 6727 is currently being clinically tested in cancer patients. Another drug in development, poloxin, is the first Polo-box domain inhibitor of Plk1. This compound is a derivative of the natural product, thymoquinone, derived from Nigella sativa. A novel and promising strategy is to synthesize bifunctional inhibitors that combine the high binding affinity of ATP inhibitors with the specificity of competitive inhibitors.

Possible steps of complete disassembly of post-termination complex by yeast eEF3 deduced from inhibition by translocation inhibitors

Ribosomes, after one round of translation, must be recycled so that the next round of translation can occur. Complete disassembly of post-termination ribosomal complex (PoTC) in yeast for the recycling consists of three reactions: release of tRNA, release of mRNA and splitting of ribosomes, catalyzed by eukaryotic elongation factor 3 (eEF3) and ATP. Here, we show that translocation inhibitors cycloheximide and lactimidomycin inhibited all three reactions. Cycloheximide is a non-competitive inhibitor of both eEF3 and ATP. The inhibition was observed regardless of the way PoTC was prepared with either release factors or puromycin. Paromomycin not only inhibited all three reactions but also re-associated yeast ribosomal subunits. On the other hand, sordarin or fusidic acid, when applied together with eEF2/GTP, specifically inhibited ribosome splitting without blocking of tRNA/mRNA release. From these inhibitor studies, we propose that, in accordance with eEF3’s known function in elongation, the release of tRNA via exit site occurs first, then mRNA is released, followed by the splitting of ribosomes during the disassembly of post-termination complexes catalyzed by eEF3 and ATP.

Mitochondrial KATP channels in skeletal muscle: are protein kinases C and G, and nitric oxide synthase involved in the fatigue process?

Correspondence: Rocio Montoya-Perez Coordinacion General de Estudios de Posgrado, Universidad Michoacana de San Nicolas de Hidalgo, Francisco J Mujica s/n Ciudad Universitaria, Edif C5, Col Felicitas del Rio 58030, Morelia, Michoacan, Mexico Tel +52 44 3322 3500 ext 4157 Email biochio@yahoo.com.mx Background: Fatigue in skeletal muscle is defined as a reduction in the physical power needed to execute a function or as an inability to maintain mitochondrial ATP production. The mitochondrial potassium channel (mitoK ATP ) participates in combating fatigue in skeletal muscle. In this work, we evaluated the role of the mitoK ATP channel activator (diazoxide) and inhibitors of the signaling routes (protein kinase C, staurosporine; protein kinase G, KT5823; and nitric oxide synthase, metil N-Nitro-L-arginine ester, L-NAME), on muscle fatigue tension. In addition, we evaluated the main signaling routes used by the nitric oxide synthase protein and protein kinase C and G, in the presence of their specific activators. Methods: We used the anterior latissimus dorsi skeletal muscle of 2–3-week-old chicks. This muscle consists of slow muscle fibers. Tension was achieved by applying repetitive electrical stimulation that induced fatigue in an in vitro model. Results: Diazoxide significantly reduced muscle fatigue (P = 0.0002 in peak tension, P = 0.000002 in maximum tension) by increasing post-fatigue tension, in spite of the fact that 5-hydroxydecanoate, a selective inhibitor of mitoK ATP , did not suppress post-fatigue tension. Conclusion: Our results suggest a lack of direct interaction in inhibition of the signaling routes during fatigue-induced mitoK ATP activation. This effect is possibly due to the type of skeletal muscle fibers (slow), the stimulation protocols (twitch), and the animal (avian) model used in the study.

Discovery of purinergic signalling, the initial resistance and current explosion of interest.

There has been a remarkable growth of papers published about purinergic signalling via ATP since 1972. I am most grateful to the wonderful PhD students and postdoctoral fellows who have worked with me over the years to pursue the purinergic hypothesis despite early opposition and to the many outstanding scientists around the world who are currently extending the story. Recently, therapeutic approaches to pathological disorders include the development of selective P1 and P2 receptor subtype agonists and antagonists, as well as of inhibitors of extracellular ATP breakdown and of ATP transport enhancers and inhibitors. Medicinal chemists are starting to develop small molecule purinergic drugs that are orally bioavailable and stable in vivo.

Shuttling glucose across brain microvessels, with a little help from GLUT1 and AMP kinase. Focus on “AMP kinase regulation of sugar transport in brain capillary endothelial cells during acute metabolic stress”

The Blood-Brain Barrier: a Gatekeeper of Brain “Individuality” The blood-brain barrier (BBB) maintains the individuality of brain fluid, while allowing it to selectively import nutrients and process toxic products. Its major constituent is a single layer of continuous, nonfenestrated endothelium lining the microvessels, whose unique features account in large part for the integrity of the barrier. The endothelial cell monolayer is the major determinant, but other cells in the central nervous system (e.g., pericytes and astrocytes) contribute to the integrity of the BBB. The interendothelial tight junctions of the BBB are one of its signature elements that greatly restrict paracellular permeability to solutes, fluid, and transmigrating cells. This is markedly distinct from the leakier, discontinuous, and fenestrated endothelium of liver microvessels (sinusoids). For example, the tight junctions of the cerebral endothelium (pial vessels) confer an electrical resistance (1,300 ·cm 2 ) far higher than other endothelia (6). Therefore, communication between blood and the brain interstitial fluid requires individually tailored molecular mechanisms. Transcytosis is the movement of molecules through a cellular barrier. Whereas proteins can traverse through vesicular carrier processes that could carry solutes through fluid phase endocytosis, the brain microvasculature is uniquely poor in plasmalemmal and cytoplasmic caveolae (1). Instead, bidirectional passage of small molecules across the BBB is stringently regulated by numerous transporters and receptors. These typically work in tandem across the luminal and abluminal membranes of the endothelial cell.

TGF-β but not BMP signaling induces prechondrogenic condensation through ATP oscillations during chondrogenesis

Although both TGF-β and BMP signaling enhance expression of adhesion molecules during chondrogenesis, TGF-β but not BMP signaling can initiate condensation of uncondensed mesenchymal cells. However, it remains unclear what causes the differential effects between TGF-β and BMP signaling on prechondrogenic condensation. Our previous report demonstrated that ATP oscillations play a critical role in prechondrogenic condensation. Thus, the current study examined whether ATP oscillations are associated with the differential actions of TGF-β and BMP signaling on prechondrogenic condensation. The result revealed that while both TGF-β1 and BMP2 stimulated chondrogenic differentiation, TGF-β1 but not BMP2 induced prechondrogenic condensation. It was also found that TGF-β1 but not BMP2 induced ATP oscillations and inhibition of TGF-β but not BMP signaling prevented insulin-induced ATP oscillations. Moreover, blockage of ATP oscillations inhibited TGF-β1-induced prechondrogenic condensation. In addition, TGF-β1-driven ATP oscillations and prechondrogenic condensation depended on Ca2+ influx via voltage-dependent calcium channels. This study suggests that Ca2+-driven ATP oscillations mediate TGF-β-induced the initiation step of prechondrogenic condensation and determine the differential effects between TGF-β and BMP signaling on chondrogenesis.

Complex I and ATP synthase mediate membrane depolarization and matrix acidification by isoflurane in mitochondria

Short application of the volatile anesthetic isoflurane at reperfusion after ischemia exerts strong protection of the heart against injury. Mild depolarization and acidification of the mitochondrial matrix are involved in the protective mechanisms of isoflurane, but the molecular basis for these changes is not clear. In this study, mitochondrial respiration, membrane potential, matrix pH, matrix swelling, ATP synthesis and -hydrolysis, and H2O2 release were assessed in isolated mitochondria. We hypothesized that isoflurane induces mitochondrial depolarization and matrix acidification through direct action on both complex I and ATP synthase. With complex I-linked substrates, isoflurane (0.5mM) inhibited mitochondrial respiration by 28±10%, and slightly, but significantly depolarized membrane potential and decreased matrix pH. With complex II- and complex IV-linked substrates, respiration was not changed, but isoflurane still decreased matrix pH and depolarized mitochondrial membrane potential. Depolarization and matrix acidification were attenuated by inhibition of ATP synthase with oligomycin, but not by inhibition of mitochondrial ATP- and Ca2+-sensitive K+ channels or uncoupling proteins. Isoflurane did not induce matrix swelling and did not affect ATP synthesis and hydrolysis, but decreased H2O2 release in the presence of succinate in an oligomycin- and matrix pH-sensitive manner. Isoflurane modulated H+ flux through ATP synthase in an oligomycin-sensitive manner. Our results indicate that isoflurane-induced mitochondrial depolarization and acidification occur due to inhibition of the electron transport chain at the site of complex I and increased proton flux through ATP synthase. K+ channels and uncoupling proteins appear not to be involved in the direct effects of isoflurane on mitochondria.

Cytochromecoxidase subunit 4 isoform 2‐knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology

Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (-50 and -29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced P(enh) and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (-12%), reduced total oxygen consumption rate (-8%), improved glucose tolerance, and reduced grip force (-14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long-term lung pathology develops in the knockout mice due to impairment of energy-costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma.

PAK4 kinase activity and somatic mutation promote carcinoma cell motility and influence inhibitor sensitivity

Hepatocyte growth factor (HGF) and its receptor (c-Met) are associated with cancer cell motility and invasiveness. p21-activated kinase 4 (PAK4), a potential therapeutic target, is recruited to and activated by c-Met. In response, PAK4 phosphorylates LIM kinase 1 (LIMK1) in an HGF-dependent manner in metastatic prostate carcinoma cells. PAK4 overexpression is known to induce increased cell migration speed but the requirement for kinase activity has not been established. We have used a panel of PAK4 truncations and mutations in a combination of over-expression and RNAi rescue experiments to determine the requirement for PAK4 kinase activity during carcinoma cell motility downstream of HGF. We find that neither the kinase domain alone nor a PAK4 mutant unable to bind Cdc42 is able to fully rescue cell motility in a PAK4-deficient background. Nevertheless, we find that PAK4 kinase activity and associated LIMK1 activity are essential for carcinoma cell motility, highlighting PAK4 as a potential anti-metastatic therapeutic target. We also show here that overexpression of PAK4 harboring a somatic mutation, E329K, increased the HGF-driven motility of metastatic prostate carcinoma cells. E329 lies within the G-loop region of the kinase. Our data suggest E329K mutation leads to a modest increase in kinase activity conferring resistance to competitive ATP inhibitors in addition to promoting cell migration. The existence of such a mutation may have implications for the development of PAK4-specific competitive ATP inhibitors should PAK4 be further explored for clinical inhibition.