Regulation of the autophagic PI3KC3 complex by laforin/malin E3-ubiquitin ligase, two proteins involved in Lafora disease.

Lafora progressive myoclonus epilepsy is a fatal rare neurodegenerative disorder characterized by the accumulation of insoluble abnormal glycogen deposits in the brain and peripheral tissues. Mutations in at least two genes are responsible for the disease: EPM2A, encoding the glucan phosphatase laforin, and EPM2B, encoding the RING-type E3-ubiquitin ligase malin. Both laforin and malin form a functional complex in which laforin recruits the substrates to be ubiquitinated by malin. We and others have described that, in cellular and animal models of this disease, there is an autophagy impairment which leads to the accumulation of dysfunctional mitochondria. In addition, we established that the autophagic defect occurred at the initial steps of autophagosome formation. In this work, we present evidence that in cellular models of the disease there is a decrease in the amount of phosphatidylinositol-3P. This is probably due to defective regulation of the autophagic PI3KC3 complex, in the absence of a functional laforin/malin complex. In fact, we demonstrate that the laforin/malin complex interacts physically and co-localizes intracellularly with core components of the PI3KC3 complex (Beclin1, Vps34 and Vps15), and that this interaction is specific and results in the polyubiquitination of these proteins. In addition, the laforin/malin complex is also able to polyubiquitinate ATG14L and UVRAG. Finally, we show that overexpression of the laforin/malin complex increases PI3KC3 activity. All these results suggest a new role of the laforin/malin complex in the activation of autophagy via regulation of the PI3KC3 complex and explain the defect in autophagy described in Lafora disease.

Hydrogen Sulfide Improves Cardiomyocyte Function in a Cardiac Arrest Model.

BACKGROUND Cardioplegic arrest is a common procedure for many types of cardiac surgery, and different formulations have been proposed to enhance its cardio-protective effect. Hydrogen sulfide is an important signaling molecule that has cardio-protective properties. We therefore studied the cardio-protective effect of hydrogen sulfide in cardiac cell culture and its potential therapeutic use in combination with cardioplegia formulations. MATERIAL AND METHODS We added hydrogen sulfide donor GYY4137 to HL-1 cells to study its protective effect in nutrient starved conditions. In addition, we tested the potential use of GYY4137 when it is added into two different cardioplegia formulations: Cardi-Braun® solution and del Nido solution in an ex vivo Langendorff perfused rat hearts model. RESULTS We observed that eight-hour pre-treatment with GYY4137 significantly suppressed apoptosis in nutrient-starved HL-1 cells (28% less compared to untreated cells; p<0.05), maintained ATP content, and reduced protein synthesis. In ex vivo experiments, Cardi-Braun® and del Nido cardioplegia solutions supplemented with GYY4137 significantly reduced the pro-apoptotic protein caspase-3 content and preserved ATP content. Furthermore, GYY4137 supplemented cardioplegia solutions decreased the S-(5-adenosyl)-L-methionine/S-(adenosyl)-L-homocysteine ratio, reducing the oxidative stress in cardiac tissue. Finally, heart beating analysis revealed the preservation of the inter-beat interval and the heart rate in del Nido cardioplegia solution supplemented with GYY4137. CONCLUSIONS GYY4137 preconditioning preserved energetic state during starved conditions, attenuating the cardiomyocytes apoptosis in vitro. The addition of GYY4137 to cardioplegia solutions prevented apoptosis, ATP consumption, and oxidative stress in perfused rat hearts, restoring its electrophysiological status after cardiac arrest. These findings suggested that GYY4137 sulfide donor may improve the cardioplegia solution performance during cardiac surgery.

Defective Expression of the Mitochondrial-tRNA Modifying Enzyme GTPBP3 Triggers AMPK-Mediated Adaptive Responses Involving Complex I Assembly Factors, Uncoupling Protein 2, and the Mitochondrial Pyruvate Carrier.

GTPBP3 is an evolutionary conserved protein presumably involved in mitochondrial tRNA (mt-tRNA) modification. In humans, GTPBP3 mutations cause hypertrophic cardiomyopathy with lactic acidosis, and have been associated with a defect in mitochondrial translation, yet the pathomechanism remains unclear. Here we use a GTPBP3 stable-silencing model (shGTPBP3 cells) for a further characterization of the phenotype conferred by the GTPBP3 defect. We experimentally show for the first time that GTPBP3 depletion is associated with an mt-tRNA hypomodification status, as mt-tRNAs from shGTPBP3 cells were more sensitive to digestion by angiogenin than tRNAs from control cells. Despite the effect of stable silencing of GTPBP3 on global mitochondrial translation being rather mild, the steady-state levels and activity of Complex I, and cellular ATP levels were 50% of those found in the controls. Notably, the ATPase activity of Complex V increased by about 40% in GTPBP3 depleted cells suggesting that mitochondria consume ATP to maintain the membrane potential. Moreover, shGTPBP3 cells exhibited enhanced antioxidant capacity and a nearly 2-fold increase in the uncoupling protein UCP2 levels. Our data indicate that stable silencing of GTPBP3 triggers an AMPK-dependent retrograde signaling pathway that down-regulates the expression of the NDUFAF3 and NDUFAF4 Complex I assembly factors and the mitochondrial pyruvate carrier (MPC), while up-regulating the expression of UCP2. We also found that genes involved in glycolysis and oxidation of fatty acids are up-regulated. These data are compatible with a model in which high UCP2 levels, together with a reduction in pyruvate transport due to the down-regulation of MPC, promote a shift from pyruvate to fatty acid oxidation, and to an uncoupling of glycolysis and oxidative phosphorylation. These metabolic alterations, and the low ATP levels, may negatively affect heart function.

CERKL, a retinal disease gene, encodes an mRNA-binding protein that localizes in compact and untranslated mRNPs associated with microtubules.

The function of CERKL (CERamide Kinase Like), a causative gene of retinitis pigmentosa and cone-rod dystrophy, still awaits characterization. To approach its cellular role we have investigated the subcellular localization and interaction partners of the full length CERKL isoform, CERKLa of 532 amino acids, in different cell lines, including a photoreceptor-derived cell line. We demonstrate that CERKLa is a main component of compact and untranslated mRNPs and that associates with other RNP complexes such as stress granules, P-bodies and polysomes. CERKLa is a protein that binds through its N-terminus to mRNAs and interacts with other mRNA-binding proteins like eIF3B, PABP, HSP70 and RPS3. Except for eIF3B, these interactions depend on the integrity of mRNAs but not of ribosomes. Interestingly, the C125W CERKLa pathological mutant does not interact with eIF3B and is absent from these complexes. Compact mRNPs containing CERKLa also associate with microtubules and are found in neurites of neural differentiated cells. These localizations had not been reported previously for any member of the retinal disorders gene family and should be considered when investigating the pathogenic mechanisms and therapeutical approaches in these diseases.

PTEN increases autophagy and inhibits the ubiquitin-proteasome pathway in glioma cells independently of its lipid phosphatase activity.

Two major mechanisms of intracellular protein degradation, autophagy and the ubiquitin-proteasome pathway, operate in mammalian cells. PTEN, which is frequently mutated in glioblastomas, is a tumor suppressor gene that encodes a dual specificity phosphatase that antagonizes the phosphatidylinositol 3-kinase class I/AKT/mTOR pathway, which is a key regulator of autophagy. Here, we investigated in U87MG human glioma cells the role of PTEN in the regulation of autophagy and the ubiquitin-proteasome pathway, because both are functionally linked and are relevant in cancer progression. Since U87MG glioma cells lack a functional PTEN, we used stable clones that express, under the control of a tetracycline-inducible system (Tet-on), wild-type PTEN and two of its mutants, G129E-PTEN and C124S-PTEN, which, respectively, lack the lipid phosphatase activity only and both the lipid and the protein phosphatase activities of this protein. Expression of PTEN in U87MG glioma cells decreased proteasome activity and also reduced protein ubiquitination. On the contrary, expression of PTEN increased the autophagic flux and the lysosomal mass. Interestingly, and although PTEN negatively regulates the phosphatidylinositol 3-kinase class I/AKT/mTOR signaling pathway by its lipid phosphatase activity, both effects in U87MG cells were independent of this activity. These results suggest a new mTOR-independent signaling pathway by which PTEN can regulate in opposite directions the main mechanisms of intracellular protein degradation.

Glucose induces autophagy under starvation conditions by a p38 MAPK-dependent pathway.

Autophagy is a natural process of 'self-eating' that occurs within cells and can be either pro-survival or can cause cell death. As a pro-survival mechanism, autophagy obtains energy by recycling cellular components such as macromolecules or organelles. In response to nutrient deprivation, e.g. depletion of amino acids or serum, autophagy is induced and most of these signals converge on the kinase mTOR (mammalian target of rapamycin). It is commonly accepted that glucose inhibits autophagy, since its deprivation from cells cultured in full medium induces autophagy by a mechanism involving AMPK (AMP-activated protein kinase), mTOR and Ulk1. However, we show in the present study that under starvation conditions addition of glucose produces the opposite effect. Specifically, the results of the present study demonstrate that the presence of glucose induces an increase in the levels of LC3 (microtubule-associated protein 1 light chain)-II, in the number and volume density of autophagic vacuoles and in protein degradation by autophagy. Addition of glucose also increases intracellular ATP, which is in turn necessary for the induction of autophagy because the glycolysis inhibitor oxamate inhibits it, and there is also a good correlation between LC3-II and ATP levels. Moreover, we also show that, surprisingly, the induction of autophagy by glucose is independent of AMPK and mTOR and mainly relies on p38 MAPK (mitogen-activated protein kinase).

Withdrawal of essential amino acids increases autophagy by a pathway involving Ca2+/calmodulin-dependent kinase kinase-ß (CaMKK-ß).

Autophagy is the main lysosomal catabolic process that becomes activated under stress conditions, such as amino acid starvation and cytosolic Ca(2+) upload. However, the molecular details on how both conditions control autophagy are still not fully understood. Here we link essential amino acid starvation and Ca(2+) in a signaling pathway to activate autophagy. We show that withdrawal of essential amino acids leads to an increase in cytosolic Ca(2+), arising from both extracellular medium and intracellular stores, which induces the activation of adenosine monophosphate-activated protein kinase (AMPK) via Ca(2+)/calmodulin-dependent kinase kinase-ß (CaMKK-ß). Furthermore, we show that autophagy induced by amino acid starvation requires AMPK, as this induction is attenuated in its absence. Subsequently, AMPK activates UNC-51-like kinase (ULK1), a mammalian autophagy-initiating kinase, through phosphorylation at Ser-555 in a process that requires CaMKK-ß. Finally, the mammalian target of rapamycin complex C1 (mTORC1), a negative regulator of autophagy downstream of AMPK, is inhibited by amino acid starvation in a Ca(2+)-sensitive manner, and CaMKK-ß appears to be important for mTORC1 inactivation, especially in the absence of extracellular Ca(2+). All these results highlight that amino acid starvation regulates autophagy in part through an increase in cellular Ca(2+) that activates a CaMKK-ß-AMPK pathway and inhibits mTORC1, which results in ULK1 stimulation.

Regulation of autophagy by glucose in Mammalian cells.

Autophagy is an evolutionarily conserved process that contributes to maintain cell homeostasis. Although it is strongly regulated by many extracellular factors, induction of autophagy is mainly produced by starvation of nutrients. In mammalian cells, the regulation of autophagy by amino acids, and also by the hormone insulin, has been extensively investigated, but knowledge about the effects of other autophagy regulators, including another nutrient, glucose, is more limited. Here we will focus on the signalling pathways by which environmental glucose directly, i.e., independently of insulin and glucagon, regulates autophagy in mammalian cells, but we will also briefly mention some data in yeast. Although glucose deprivation mainly induces autophagy via AMPK activation and the subsequent inhibition of mTORC1, we will also comment other signalling pathways, as well as evidences indicating that, under certain conditions, autophagy can be activated by glucose. A better understanding on how glucose regulates autophagy not only will expand our basic knowledge of this important cell process, but it will be also relevant to understand common human disorders, such as cancer and diabetes, in which glucose levels play an important role.

Mechanisms of autophagy and apoptosis: Recent developments in breast cancer cells.

Autophagy, the pathway whereby cell components are degraded by lysosomes, is involved in the cell response to environmental stresses, such as nutrient deprivation, hypoxia or exposition to chemotherapeutic agents. Under these conditions, which are reminiscent of certain phases of tumor development, autophagy either promotes cell survival or induces cell death. This strengthens the possibility that autophagy could be an important target in cancer therapy, as has been proposed. Here, we describe the regulation of survival and death by autophagy and apoptosis, especially in cultured breast cancer cells. In particular, we discuss whether autophagy represents an apoptosis-independent process and/or if they share common pathways. We believe that understanding in detail the molecular mechanisms that underlie the relationships between autophagy and apoptosis in breast cancer cells could improve the available treatments for this disease.

BRCA1 negatively regulates formation of autophagic vacuoles in MCF-7 breast cancer cells.

In recent years, the function of different tumour suppressors in the regulation of macroautophagy has been studied. We show here that BRCA1, unlike other tumour suppressors, negatively regulates formation of autophagosomes and lysosomal mass under conditions of both basal and enhanced autophagy. In MCF-7 breast cancer cells, increased formation of autophagic vacuoles after inactivation of BRCA1 by siRNAs is associated with an increase in reactive oxygen species, such as superoxide anion and hydrogen peroxide. This allows one to propose an antioxidant function for BRCA1 and suggests that dysfunctional mitochondria and the generated reactive oxygen species excess could explain the increased macroautophagy observed in the absence of BRCA1. In addition, a quick decrease in BRCA1 levels occurs when MCF-7 cells are switched to a nutrient-poor environment that stimulates macroautophagy and that is also reminiscent of certain phases of tumour growth. Inhibition of BRCA1 synthesis has an important role in this reduction, while there are almost no changes in BRCA1 degradation by lysosomes and proteasomes. Therefore, BRCA1 produces macroautophagy inhibition by reducing the formation of autophagic vacuoles, and this, together with the other results presented here, shows new functional aspects of BRCA1 that could help to clarify the role of autophagy in cancer development.

Regulation of glycogen synthesis by the laforin-malin complex is modulated by the AMP-activated protein kinase pathway.

Lafora progressive myoclonus epilepsy (LD) is a fatal autosomal recessive neurodegenerative disorder characterized by the presence of glycogen-like intracellular inclusions called Lafora bodies. LD is caused by mutations in two genes, EPM2A and EPM2B, encoding respectively laforin, a dual-specificity protein phosphatase, and malin, an E3 ubiquitin ligase. Previously, we and others have suggested that the interactions between laforin and PTG (a regulatory subunit of type 1 protein phosphatase) and between laforin and malin are critical in the pathogenesis of LD. Here, we show that the laforin-malin complex downregulates PTG-induced glycogen synthesis in FTO2B hepatoma cells through a mechanism involving ubiquitination and degradation of PTG. Furthermore, we demonstrate that the interaction between laforin and malin is a regulated process that is modulated by the AMP-activated protein kinase (AMPK). These findings provide further insights into the critical role of the laforin-malin complex in the control of glycogen metabolism and unravel a novel link between the energy sensor AMPK and glycogen metabolism. These data advance our understanding of the functional role of laforin and malin, which hopefully will facilitate the development of appropriate LD therapies.

Tissue-specific autophagy alterations and increased tumorigenesis in mice deficient in Atg4C/autophagin-3.

Atg4C/autophagin-3 is a member of a family of cysteine proteinases proposed to be involved in the processing and delipidation of the mammalian orthologues of yeast Atg8, an essential component of an ubiquitin-like modification system required for execution of autophagy. To date, the in vivo role of the different members of this family of proteinases remains unclear. To gain further insights into the functional relevance of Atg4 orthologues, we have generated mutant mice deficient in Atg4C/autophagin-3. These mice are viable and fertile and do not display any obvious abnormalities, indicating that they are able to develop the autophagic response required during the early neonatal period. However, Atg4C-/--starved mice show a decreased autophagic activity in the diaphragm as assessed by immunoblotting studies and by fluorescence microscopic analysis of samples from Atg4C-/- GFP-LC3 transgenic mice. In addition, animals deficient in Atg4C show an increased susceptibility to develop fibrosarcomas induced by chemical carcinogens. Based on these results, we propose that Atg4C is not essential for autophagy development under normal conditions but is required for a proper autophagic response under stressful conditions such as prolonged starvation. We also propose that this enzyme could play an in vivo role in events associated with tumor progression.

A single point mutation in the low-density lipoprotein receptor switches the degradation of its mature protein from the proteasome to the lysosome.

Pathogenic mutations in the low-density lipoprotein receptor prevent cholesterol uptake and cause familial hypercholesterolemia. In comparison to the biogenesis and endocytic trafficking of this receptor and some of its mutants, their degradation mechanisms are not well understood. Therefore, to gain some insights into this aspect, we analyzed the effects of proteasomal and lysosomal inhibitors on the levels of the wild type low-density lipoprotein receptor and a mutant form, C358Y, which was prevalent in a sample of Spanish familial hypercholesterolemia patients. In transfected cells, the mutant C358Y exhibited lower activity than the wild type receptor, as well as retarded post-translational processing of its precursor to the mature form. Interestingly, about 30% of the mutant precursor was degraded by a lysosomal pathway. Moreover, its mature form was more rapidly degraded than the wild type receptor (half lives of 5.3 and 10.9 h, respectively) and its degradation was exclusively dependent on a lysosomal pathway. In contrast, the mature form of the wild type receptor was mainly degraded by proteasomes and, to a minor extent (30%), by lysosomes. We conclude that a single mutation in the low-density lipoprotein receptor switches the degradation of the mature receptor from a proteasomal to a lysosomal pathway which degrades the protein at a faster rate. This suggests cooperation of proteasomes and lysosomes in the degradation of the low-density lipoprotein receptor and adds an intriguing new aspect to our understanding of receptor-mediated endocytosis.

Changes in the proteolytic activities of proteasomes and lysosomes in human fibroblasts produced by serum withdrawal, amino-acid deprivation and confluent conditions.

The contribution of the main proteolytic pathways to the degradation of long-lived proteins in human fibroblasts grown under different conditions was investigated. The effects of various commonly used pharmacological inhibitors of protein degradation were first analysed in detail. By choosing specific inhibitors of lysosomes and proteasomes, it was observed that together both pathways accounted for 80% or more of the degradation of cell proteins. With lysosomal inhibitors, it was found that serum withdrawal or amino-acid deprivation strongly stimulated macroautophagy but not other lysosomal pathways, whereas confluent conditions had no effect on macroautophagy and slightly activated other lysosomal pathways. Prolonged (24 h) serum starvation of confluent cultures strongly decreased the macroautophagic pathway, whereas the activity of other lysosomal pathways increased. These changes correlated with electron microscopic observations and morphometric measurements of lysosomes. With proteasomal inhibitors, it was found that, in exponentially growing cells in the absence of serum, activity of the ubiquitin-proteasome pathway increases, whereas under confluent conditions the contribution (in percentage) of proteasomes to degradation decreases, especially in cells deprived of amino acids. Interestingly, in confluent cells, the levels of two components of the 19 S regulatory complex and those of an interchangeable beta-subunit decreased. This was associated with a marked increase in the levels of components of PA28-immunoproteasomes. Thus confluent conditions affect proteasomes in a way that resembles treatment with interferon-gamma. Altogether, these results show that the activity of the various proteolytic pathways depends on the growth conditions of cells and will be useful for investigation of the specific signals that control their activity.

The GTPase activity and C-terminal cysteine of the Escherichia coli MnmE protein are essential for its tRNA modifying function.

The Escherichia coli MnmE protein is a three-domain protein that exhibits a very high intrinsic GTPase activity and low affinity for GTP and GDP. The middle GTPase domain, when isolated, conserves the high intrinsic GTPase activity of the entire protein, and the C-terminal domain contains the only cysteine residue present in the molecule. MnmE is an evolutionarily conserved protein that, in E. coli, has been shown to control the modification of the uridine at the wobble position of certain tRNAs. Here we examine the biochemical and functional consequences of altering amino acid residues within conserved motifs of the GTPase and C-terminal domains of MnmE. Our results indicate that both domains are essential for the MnmE tRNA modifying function, which requires effective hydrolysis of GTP. Thus, it is shown for the first time that a confirmed defect in the GTP hydrolase activity of MnmE results in the lack of its tRNA modifying function. Moreover, the mutational analysis of the GTPase domain indicates that MnmE is closer to classical GTPases than to GTP-specific metabolic enzymes. Therefore, we propose that MnmE uses a conformational change associated with GTP hydrolysis to promote the tRNA modification reaction, in which the C-terminal Cys may function as a catalytic residue. We demonstrate that point mutations abolishing the tRNA modifying function of MnmE confer synthetic lethality, which stresses the importance of this function in the mRNA decoding process.

Role of proteasomes in the degradation of short-lived proteins in human fibroblasts under various growth conditions.

Degradation of proteins in the cells occurs by proteasomes, lysosomes and other cytosolic and organellar proteases. It is believed that proteasomes constitute the major proteolytic pathway under most conditions, especially when degrading abnormal and other short-lived proteins. However, no systematic analysis of their role in the overall degradation of truly short-lived cell proteins has been carried out. Here, the degradation of short-labelled proteins was examined in human fibroblasts by release of trichloroacetic acid-soluble radioactivity. The kinetics of degradation was decomposed into two, corresponding to short- and long-lived proteins, and the effect of proteasomal and lysosomal inhibitors on their degradation, under various growth conditions, was separately investigated. From the degradation kinetics of proteins labelled for various pulse times it can be estimated that about 30% of newly synthesised proteins are degraded with a half-life of approximately 1h. These rapidly degraded proteins should mostly include defective ribosomal products. Deprivation of serum and confluent conditions increased the degradation of the pool of long-lived proteins in fibroblasts without affecting, or affecting to a lesser extent, the degradation of the pool of short-lived proteins. Inhibitors of proteasomes and of lysosomes prevented more than 80% of the degradation of short-lived proteins. It is concluded that, although proteasomes are responsible of about 40-60% of the degradation of short-lived proteins in normal human fibroblasts, lysosomes have also an important participation in the degradation of these proteins. Moreover, in confluent fibroblasts under serum deprivation, lysosomal pathways become even more important than proteasomes in the degradation of short-lived proteins.