Enhanced mild-temperature photothermal therapy by pyroptosis-boosted ATP deprivation with biodegradable nanoformulation.

BACKGROUND: Mild-temperature photothermal therapy (mild PTT) is a safe and promising tumor therapeutic modality by alleviating the damage of healthy tissues around the tumor due to high temperature. However, its therapeutic efficiency is easily restricted by heat shock proteins (HSPs). Thus, exploitation of innovative approaches of inhibiting HSPs to enhance mild PTT efficiency is crucial for the clinical application of PTT. RESULTS: Herein, an innovative strategy is reported: pyroptosis-boosted mild PTT based on a Mn-gallate nanoformulation. The nanoformulation was constructed via the coordination of gallic acid (GA) and Mn2+. It shows an acid-activated degradation and releases the Mn2+ and GA for up-regulation of reactive oxygen species (ROS), mitochondrial dysfunction and pyroptosis, which can result in cellular ATP deprivation via both the inhibiton of ATP generation and incresed ATP efflux. The reduction of ATP and accumulation of ROS provide a powerful approach for inhibiting the expression of HSPs, which enables the nanoformulation-mediated mild PTT. CONCLUSIONS: Our in-vitro and in-vivo results demonstrate that this strategy of pyroptosis-assited PTT can achieve efficient mild PTT efficiency for osteosarcoma therapy.

Nucleolar translocation of human DNA topoisomerase II by ATP depletion and its disruption by the RNA polymerase I inhibitor BMH-21.

DNA topoisomerase II (TOP2) is a nuclear protein that resolves DNA topological problems and plays critical roles in multiple nuclear processes. Human cells have two TOP2 proteins, TOP2A and TOP2B, that are localized in both the nucleoplasm and nucleolus. Previously, ATP depletion was shown to augment the nucleolar localization of TOP2B, but the molecular details of subnuclear distributions, particularly of TOP2A, remained to be fully elucidated in relation to the status of cellular ATP. Here, we analyzed the nuclear dynamics of human TOP2A and TOP2B in ATP-depleted cells. Both proteins rapidly translocated from the nucleoplasm to the nucleolus in response to ATP depletion. FRAP analysis demonstrated that they were highly mobile in the nucleoplasm and nucleolus. The nucleolar retention of both proteins was sensitive to the RNA polymerase I inhibitor BMH-21, and the TOP2 proteins in the nucleolus were immediately dispersed into the nucleoplasm by BMH-21. Under ATP-depleted conditions, the TOP2 poison etoposide was less effective, indicating the therapeutic relevance of TOP2 subnuclear distributions. These results give novel insights into the subnuclear dynamics of TOP2 in relation to cellular ATP levels and also provide discussions about its possible mechanisms and biological significance.

Lung metabolomics after ischemic acute kidney injury reveals increased oxidative stress, altered energy production, and ATP depletion.

Acute kidney injury (AKI) is a complex disease associated with increased mortality that may be due to deleterious distant organ effects. AKI associated with respiratory complications, in particular, has a poor outcome. In murine models, AKI is characterized by increased circulating cytokines, lung chemokine upregulation, and neutrophilic infiltration, similar to other causes of indirect acute lung injury (ALI; e.g., sepsis). Many causes of lung inflammation are associated with a lung metabolic profile characterized by increased oxidative stress, a shift toward the use of other forms of energy production, and/or a depleted energy state. To our knowledge, there are no studies that have evaluated pulmonary energy production and metabolism after AKI. We hypothesized that based on the parallels between inflammatory acute lung injury and AKI-mediated lung injury, a similar metabolic profile would be observed. Lung metabolomics and ATP levels were assessed 4 h, 24 h, and 7 days after ischemic AKI in mice. Numerous novel findings regarding the effect of AKI on the lung were observed including 1) increased oxidative stress, 2) a shift toward alternate methods of energy production, and 3) depleted levels of ATP. The findings in this report bring to light novel characteristics of AKI-mediated lung injury and provide new leads into the mechanisms by which AKI in patients predisposes to pulmonary complications.

Smooth muscle 22α deficiency impairs oxytocin-induced uterine contractility in mice at full-term pregnancy.

Smooth muscle 22α (SM22α, namely Transgelin), as an actin-binding protein, regulates the contractility of vascular smooth muscle cells (VSMCs) by modulation of the stress fiber formation. However, little is known about the roles of SM22α in the regulation of uterine contraction during parturition. Here, we showed that contraction in response to oxytocin (OT) was significantly decreased in the uterine muscle strips from SM22α knockout (Sm22α-KO) mice, especially at full-term pregnancy, which may be resulted from impaired formation of stress fibers. Furthermore, serious mitochondrial damage such as the mitochondrial swelling, cristae disruption and even disappearance were observed in the myometrium of Sm22α-KO mice at full-term pregnancy, eventually resulting in the collapse of mitochondrial membrane potential and impairment in ATP synthesis. Our data indicate that SM22α is necessary to maintain uterine contractility at delivery in mice, and acts as a novel target for preventive or therapeutic manipulation of uterine atony during parturition.

Early detection of skeletal muscle bioenergetic deficit by magnetic resonance spectroscopy in cigarette smoke-exposed mice.

Skeletal muscle dysfunction is a common complication and an important prognostic factor in patients with chronic obstructive pulmonary disease (COPD). It is associated with intrinsic muscular abnormalities of the lower extremities, but it is not known whether there is an easy way to predict its presence. Using a mouse model of chronic cigarette smoke exposure, we tested the hypothesis that magnetic resonance spectroscopy allows us to detect muscle bioenergetic deficit in early stages of lung disease. We employed this technique to evaluate the synthesis rate of adenosine triphosphate (ATP) and characterize concomitant mitochondrial dynamics patterns in the gastrocnemius muscle of emphysematous mice. The fibers type composition and citrate synthase (CtS) and cytochrome c oxidase subunit IV (COX4) enzymatic activities were evaluated. We found that the rate of ATP synthesis was reduced in the distal skeletal muscle of mice exposed to cigarette smoke. Emphysematous mice showed a significant reduction in body weight gain, in the cross-sectional area of the total fiber and in the COX4 to CtS activity ratio, due to a significant increase in CtS activity of the gastrocnemius muscle. Taken together, these data support the hypothesis that in the early stage of lung disease, we can detect a decrease in ATP synthesis in skeletal muscle, partly caused by high oxidative mitochondrial enzyme activity. These findings may be relevant to predict the presence of skeletal bioenergetic deficit in the early stage of lung disease besides placing the mitochondria as a potential therapeutic target for the treatment of COPD comorbidities.

miR-214 represses mitofusin-2 to promote renal tubular apoptosis in ischemic acute kidney injury.

Disruption of mitochondrial dynamics is an important pathogenic event in both acute and chronic kidney diseases, but the underlying mechanism remains poorly understood. Here, we report the regulation of mitofusin-2 (Mfn2; a key mitochondrial fusion protein) by microRNA-214 (miR-214) in renal ischemia-reperfusion that contributes to mitochondrial fragmentation, renal tubular cell death, and ischemic acute kidney injury (AKI). miR-214 was induced, whereas Mfn2 expression was decreased, in mouse ischemic AKI and cultured rat kidney proximal tubular cells (RPTCs) following ATP depletion treatment. Overexpression of miR-214 decreased Mfn2. Conversely, inhibition of miR-214 with anti-miR-214 prevented Mfn2 downregulation in RPTCs following ATP depletion. Anti-miR-214 further ameliorated mitochondrial fragmentation and apoptosis, whereas overexpression of miR-214 increased apoptosis, in ATP-depleted RPTCs. To test regulation in vivo, we established a mouse model with miR-214 specifically deleted from kidney proximal tubular cells (PT-miR-214-/-). Compared with wild-type mice, PT-miR-214-/- mice had less severe tissue damage, fewer apoptotic cells, and better renal function after ischemic AKI. miR-214 induction in ischemic AKI was suppressed in PT-miR-214-/- mice, accompanied by partial preservation of Mfn2 in kidneys. These results unveil the miR-214/Mfn2 axis that contributes to the disruption of mitochondrial dynamics and tubular cell death in ischemic AKI, offering new therapeutic targets.

Interacciones neuroinmunológicas en el ictus / Neuroimmunological interactions in stroke

Introducción: El ictus es una de las principales causas de mortalidad en el mundo y debido al incremento en la expectativa de vida su incidencia va en aumento; sin embargo, el desarrollo de nuevos medicamentos con utilidad clínica ha sido prácticamente nulo, por lo que hasta la fecha el tratamiento de estos pacientes es muy limitado. Desarrollo: La evidencia básica y clínica en el área señala que tras un infarto cerebral se producen una serie de cambios neuroquímicos, entre los que se encuentran: la depleción energética, la producción de radicales libres, la acumulación de calcio, la desregulación de neurotransmisores, la excitotoxicidad, y de manera tardía, la activación del sistema inmune caracterizada como inflamación. Esta respuesta del sistema inmunológico ha mostrado ser un evento central en la progresión de la patología, en el que destaca la participación de las citocinas proinflamatorias como TNF, que aumentan el daño por excitotoxicidad y por acumulación de calcio, favorecen la formación de radicales libres y en general promueven la muerte celular. Por otro lado, algunas citocinas antiinflamatorias como IL-10 e IL-4 han mostrado tener efectos neuroprotectores e incluso favorecen la recuperación de sinapsis y la neurogénesis, haciendo de la modulación de la respuesta inmunológica un área con mucho potencial terapéutico. Conclusiones: El entendimiento de las relaciones entre el sistema inmunológico y el sistema nervioso no solo nos permite entender con mayor profundidad el fenómeno del ictus, sino que también nos ofrece un nuevo arsenal de estrategias diagnósticas, pronósticas y terapéuticas que podrían mejorar la calidad de vida de las personas aquejadas por esta terrible enfermedad

Introduction: Stroke is one of the leading causes of death in the world; its incidence is increasing due to increased life expectancy. However, treatment options for these patients are limited since no clinically effective drugs have been developed to date. Development: According to clinical evidence, a number of neurochemical changes take place after stroke, including energy depletion, increased free radical synthesis, calcium accumulation, neurotransmitter imbalance, excitotoxicity, and, at a later stage, immune system activation leading to inflammation. Immune response has been shown to be a major factor in disease progression. The release of proinflammatory cytokines such as TNF increase brain damage secondary to excitotoxicity and calcium accumulation, and promote free radical synthesis and cell death through various mechanisms. On the other hand, certain anti-inflammatory cytokines, such as IL-10 and IL-4, have been shown to have a neuroprotective effect and even promote neurogenesis and synapse remodeling, which makes immune modulation a promising treatment approach. Conclusions: Understanding the relationship between the immune system and the nervous system not only deepens our knowledge of stroke but also provides new diagnostic, prognostic, and therapeutic strategies that may increase the quality of life of stroke patients

Deficiency of TPPP2, a factor linked to oligoasthenozoospermia, causes subfertility in male mice.

Oligoasthenozoospermia is a major cause of male infertility; however, its etiology and pathogenesis are unclear and may be associated with specific gene abnormalities. This study focused on Tppp2 (tubulin polymerization promoting protein family member 2), whose encoded protein localizes in elongating spermatids at stages IV-VIII of the seminiferous epithelial cycle in testis and in mature sperm in the epididymis. In human and mouse sperm, in vitro inhibition of TPPP2 caused significantly decreased motility and ATP content. Studies on Tppp2 knockout (KO) mice demonstrated that deletion of TPPP2 resulted in male subfertility with a significantly decreased sperm count and motility. In Tppp2-/- mice, increased irregular mitochondria lacking lamellar cristae, abnormal expression of electron transfer chain molecules, lower ATP levels, decreased mitochondrial membrane potential and increased apoptotic index were observed in sperm, which could be the potential causes for its oligoasthenozoospermia phenotype. Moreover, we identified a potential TPPP2-interactive protein, eEf1b (eukaryotic translation elongation factor 1 beta), which plays an important role in protein translation extension. Thus, TPPP2 is probably a potential pathogenic factor in oligoasthenozoospermia. Deficiency of TPPP2 might affect the translation of specific proteins, altering the structure and function of sperm mitochondria, and resulting in decreased sperm count, motility and fertility.

Bioenergetic dysfunction in a zebrafish model of acute hyperammonemic decompensation.

Acute hyperammonemic encephalopathy is a life-threatening manifestation of individuals with urea cycle disorders, which is associated with high mortality rates and severe neurological sequelae in survivors. Cerebral bioenergetic failure has been proposed as one of the key mechanisms underlying hyperammonemia-induced brain damage, but data supporting this hypothesis remain inconclusive and partially contradictory. Using a previously established zebrafish model of acute hyperammonemic decompensation, we unraveled that acute hyperammonemia leads to a transamination-dependent withdrawal of 2-oxoglutarate (alpha-ketoglutarate) from the tricarboxylic acid (TCA) cycle with consecutive TCA cycle dysfunction, ultimately causing impaired oxidative phosphorylation with ATP shortage, decreased ATP/ADP-ratio and elevated lactate concentrations. Thus, our study supports and extends the hypothesis that cerebral bioenergetic dysfunction is an important pathophysiological hallmark of hyperammonemia-induced neurotoxicity.

Force Spectrum Microscopy Using Mitochondrial Fluctuations of Control and ATP-Depleted Cells.

A single-cell assay of active and passive intracellular mechanical properties of mammalian cells could give significant insight into cellular processes. Force spectrum microscopy (FSM) is one such technique, which combines the spontaneous motion of probe particles and the mechanical properties of the cytoskeleton measured by active microrheology using optical tweezers to determine the force spectrum of the cytoskeleton. A simpler and noninvasive method to perform FSM would be very useful, enabling its widespread adoption. Here, we develop an alternative method of FSM using measurement of the fluctuating motion of mitochondria. Mitochondria of the C3H-10T1/2 cell line were labeled and tracked using confocal microscopy. Mitochondrial probes were selected based on morphological characteristics, and their mean-square displacement, creep compliance, and distributions of directional change were measured. We found that the creep compliance of mitochondria resembles that of particles in viscoelastic media. However, comparisons of creep compliance between controls and cells treated with pharmacological agents showed that perturbations to the actomysoin network had surprisingly small effects on mitochondrial fluctuations, whereas microtubule disruption and ATP depletion led to a significantly decreased creep compliance. We used properties of the distribution of directional change to identify a regime of thermally dominated fluctuations in ATP-depleted cells, allowing us to estimate the viscoelastic parameters for a range of timescales. We then determined the force spectrum by combining these viscoelastic properties with measurements of spontaneous fluctuations tracked in control cells. Comparisons with previous measurements made using FSM revealed an excellent match.

Muscle wasting and branched-chain amino acid, alpha-ketoglutarate, and ATP depletion in a rat model of liver cirrhosis.

The aim of the study was to examine whether a rat model of liver cirrhosis induced by carbon tetrachloride (CCl4) is a suitable model of muscle wasting and alterations in amino acid metabolism in cirrhotic humans. Rats were treated by intragastric gavage of CCl4 or vehicle for 45 days. Blood plasma and different muscle types-tibialis anterior (mostly white fibres), soleus (red muscle) and extensor digitorum longus (white muscle) - were analysed at the end of the study. Characteristic biomarkers of impaired hepatic function were found in the plasma of cirrhotic animals. The weights and protein contents of all muscles of CCl4-treated animals were lower when compared with controls. Increased concentrations of glutamine (GLN) and aromatic amino acids (phenylalanine and tyrosine) and decreased concentrations of branched-chain amino acids (BCAA), glutamate (GLU), alanine and aspartate were found in plasma and muscles. In the soleus muscle, GLN increased more and GLU and BCAA decreased less than in the extensor digitorum and tibialis muscles. Increased chymotrypsin-like activity (indicating enhanced proteolysis) and decreased α-ketoglutarate and ATP levels were found in muscles of cirrhotic animals. ATP concentration also decreased in blood plasma. It is concluded that a rat model of CCl4-induced cirrhosis is a valid model for the investigation of hepatic cachexia that exhibits alterations in line with a theory of role of ammonia in pathogenesis of BCAA depletion, citric cycle and mitochondria dysfunction, and muscle wasting in cirrhotic subjects. The findings indicate more effective ammonia detoxification to GLN in red than in white muscles.

Memantine attenuates cell apoptosis by suppressing the calpain-caspase-3 pathway in an experimental model of ischemic stroke.

Ischemic stroke, the second leading cause of death worldwide, leads to excessive glutamate release, over-activation of N-methyl-D-aspartate receptor (NMDAR), and massive influx of calcium (Ca2+), which may activate calpain and caspase-3, resulting in cellular damage and death. Memantine is an uncompetitive NMDAR antagonist with low-affinity/fast off-rate. We investigated the potential mechanisms through which memantine protects against ischemic stroke in vitro and in vivo. Middle cerebral artery occlusion-reperfusion (MCAO) was performed to establish an experimental model of ischemic stroke. The neuroprotective effects of memantine on ischemic rats were evaluated by neurological deficit scores and infarct volumes. The activities of calpain and caspase-3, and expression levels of microtubule-associated protein-2 (MAP2) and postsynaptic density-95 (PSD95) were determined by Western blotting. Additionally, Nissl staining and immunostaining were performed to examine brain damage, cell apoptosis, and neuronal loss induced by ischemia. Our results show that memantine could significantly prevent ischemic stroke-induced neurological deficits and brain infarct, and reduce ATP depletion-induced neuronal death. Moreover, memantine markedly suppressed the activation of the calpain-caspase-3 pathway and cell apoptosis, and consequently, attenuated brain damage and neuronal loss in MCAO rats. These results provide a molecular basis for the role of memantine in reducing neuronal apoptosis and preventing neuronal damage, suggesting that memantine may be a promising therapy for stroke patients.

Effects of opioids on proximal renal tubular cells undergoing ATP depletion.

This study investigated the effect of morphine, fentanyl, butorphanol and buprenorphine on viability and caspase-3 activity in renal proximal tubular cells exposed to opioids for 2 h before or 12 h after chemical anoxia. Cell viability decreased regardless the treatment although intracellular ATP content was elevated in morphine and fentanyl pre-treated cells at 12 h. Anoxia increased caspase activity but this effect was significantly reduced in cells treated before or after with morphine, fentanyl and in cell treated with butorphanol for 12 h. No influence of buprenorphine was detected. Morphine, fentanyl and butorphanol might have protective effects during kidney ischemia.

Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification.

ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane.

Evaluation of energy metabolism and calcium homeostasis in cells affected by Shwachman-Diamond syndrome.

Isomorphic mutation of the SBDS gene causes Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have ribosome biogenesis and their protein synthesis altered, which are two high-energy consuming cellular processes. The reported changes in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest an energy production defect in SDS cells. In our work, we have demonstrated that SDS cells display a Complex IV activity impairment, which causes an oxidative phosphorylation metabolism defect, with a consequent decrease in ATP production. These data were confirmed by an increased glycolytic rate, which compensated for the energetic stress. Moreover, the signalling pathways involved in glycolysis activation also appeared more activated; i.e. we reported AMP-activated protein kinase hyper-phosphorylation. Notably, we also observed an increase in a mammalian target of rapamycin phosphorylation and high intracellular calcium concentration levels ([Ca(2+)]i), which probably represent new biochemical equilibrium modulation in SDS cells. Finally, the SDS cell response to leucine (Leu) was investigated, suggesting its possible use as a therapeutic adjuvant to be tested in clinical trials.

[AGGREGATION OF METABOLICALLY DEPLETED HUMAN ERYTHROCYTES].

An aggregation of erythrocytes in autologous plasma after blood storage for 14 days at 4 °C was studied using photometry and light microscopy. The decrease of ATP content, the formation of echinocytes and spheroechinocytes, the decrease of rouleaux form of erythrocyte aggregation were observed during the storage. On the other hand the aggregates of echinocytes were formed in the stored blood. The addition of plasma from the fresh blood didn't restore the normal discocytic shape and aggregation of erythrocytes in the stored blood. The possible mechanisms of erythrocytes and echinocytes aggregation are discussed.

Coenzyme Q biosynthesis in health and disease.

Coenzyme Q (CoQ, or ubiquinone) is a remarkable lipid that plays an essential role in mitochondria as an electron shuttle between complexes I and II of the respiratory chain, and complex III. It is also a cofactor of other dehydrogenases, a modulator of the permeability transition pore and an essential antioxidant. CoQ is synthesized in mitochondria by a set of at least 12 proteins that form a multiprotein complex. The exact composition of this complex is still unclear. Most of the genes involved in CoQ biosynthesis (COQ genes) have been studied in yeast and have mammalian orthologues. Some of them encode enzymes involved in the modification of the quinone ring of CoQ, but for others the precise function is unknown. Two genes appear to have a regulatory role: COQ8 (and its human counterparts ADCK3 and ADCK4) encodes a putative kinase, while PTC7 encodes a phosphatase required for the activation of Coq7. Mutations in human COQ genes cause primary CoQ(10) deficiency, a clinically heterogeneous mitochondrial disorder with onset from birth to the seventh decade, and with clinical manifestation ranging from fatal multisystem disorders, to isolated encephalopathy or nephropathy. The pathogenesis of CoQ(10) deficiency involves deficient ATP production and excessive ROS formation, but possibly other aspects of CoQ(10) function are implicated. CoQ(10) deficiency is unique among mitochondrial disorders since an effective treatment is available. Many patients respond to oral CoQ(10) supplementation. Nevertheless, treatment is still problematic because of the low bioavailability of the compound, and novel pharmacological approaches are currently being investigated. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Thiamine Deprivation Produces a Liver ATP Deficit and Metabolic and Genomic Effects in Mice: Findings Are Parallel to Those of Biotin Deficiency and Have Implications for Energy Disorders.

Thiamine is one of several essential cofactors for ATP generation. Its deficiency, like in beriberi and in the Wernicke-Korsakoff syndrome, has been studied for many decades. However, its mechanism of action is still not completely understood at the cellular and molecular levels. Since it acts as a coenzyme for dehydrogenases of pyruvate, branched-chain keto acids, and ketoglutarate, its nutritional privation is partly a phenocopy of inborn errors of metabolism, among them maple syrup urine disease. In the present paper, we report metabolic and genomic findings in mice deprived of thiamine. They are similar to the ones we have previously found in biotin deficiency, another ATP generation cofactor. Here we show that thiamine deficiency substantially reduced the energy state in the liver and activated the energy sensor AMP-activated kinase. With this vitamin deficiency, several metabolic parameters changed: blood glucose was diminished and serum lactate was increased, but insulin, triglycerides, and cholesterol, as well as liver glycogen, were reduced. These results indicate a severe change in the energy status of the whole organism. Our findings were associated with modified hepatic levels of the mRNAs of several carbon metabolism genes: a reduction of transcripts for liver glucokinase and fatty acid synthase and augmentation of those for carnitine palmitoyl transferase 1 and phosphoenolpyruvate carboxykinase as markers for glycolysis, fatty acid synthesis, beta-oxidation, and gluconeogenesis, respectively. Glucose tolerance was initially increased, suggesting augmented insulin sensitivity, as we had found in biotin deficiency; however, in the case of thiamine, it was diminished from the 3rd week on, when the deficient animals became undernourished, and paralleled the changes in AKT and mTOR, 2 main proteins in the insulin signaling pathway. Since many of the metabolic and gene expression effects on mice deprived of thiamine are similar to those in biotin deficiency, it may be that they result from a more general impairment of oxidative phosphorylation due to a shortage of ATP generation cofactors. These findings may be relevant to energy-related disorders, among them several inborn errors of metabolism, as well as common energy disorders like obesity, diabetes, and neurodegenerative illnesses.

Crystal structures of a polypeptide processing and secretion transporter.

Bacteria secrete peptides and proteins to communicate, to poison competitors, and to manipulate host cells. Among the various protein-translocation machineries, the peptidase-containing ATP-binding cassette transporters (PCATs) are appealingly simple. Each PCAT contains two peptidase domains that cleave the secretion signal from the substrate, two transmembrane domains that form a translocation pathway, and two nucleotide-binding domains that hydrolyse ATP. In Gram-positive bacteria, PCATs function both as maturation proteases and exporters for quorum-sensing or antimicrobial polypeptides. In Gram-negative bacteria, PCATs interact with two other membrane proteins to form the type 1 secretion system. Here we present crystal structures of PCAT1 from Clostridium thermocellum in two different conformations. These structures, accompanied by biochemical data, show that the translocation pathway is a large α-helical barrel sufficient to accommodate small folded proteins. ATP binding alternates access to the transmembrane pathway and also regulates the protease activity, thereby coupling substrate processing to translocation.

Structurally diverse c-Myc inhibitors share a common mechanism of action involving ATP depletion.

The c-Myc (Myc) oncoprotein is deregulated in a large proportion of diverse human cancers. Considerable effort has therefore been directed at identifying pharmacologic inhibitors as potential anti-neoplastic agents. Three such groups of small molecule inhibitors have been described. The first is comprised of so-called "direct" inhibitors, which perturb Myc's ability to form productive DNA-binding heterodimers in association with its partner, Max. The second group is comprised of indirect inhibitors, which largely function by targeting the BET-domain protein BRD4 to prevent the proper formation of transcriptional complexes that assemble in response to Myc-Max DNA binding. Thirdly, synthetic lethal inhibitors cause the selective apoptosis of Myc over-expressing either by promoting mitotic catastrophe or altering Myc protein stability. We report here a common mechanism by which all Myc inhibitors, irrespective of class, lead to eventual cellular demise. This involves the depletion of ATP stores due to mitochondrial dysfunction and the eventual down-regulation of Myc protein. The accompanying metabolic de-regulation causes neutral lipid accumulation, cell cycle arrest, and an attempt to rectify the ATP deficit by up-regulating AMP-activated protein kinase (AMPK). These responses are ultimately futile due to the lack of functional Myc to support the requisite anabolic response. Finally, the effects of Myc depletion on ATP levels, cell cycle arrest, differentiation and AMPK activation can be mimicked by pharmacologic inhibition of the mitochondrial electron transport chain without affecting Myc levels. Thus, all Myc inhibitors promote a global energy collapse that appears to underlie many of their phenotypic consequences.