Na+ controls hypoxic signalling by the mitochondrial respiratory chain.

All metazoans depend on the consumption of O2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1-4, a phenomenon that occurs in hypoxia4-8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production of reactive oxygen species by modulating the fluidity of the inner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification of the matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition of Na+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism.

HIF1α Suppresses Tumor Cell Proliferation through Inhibition of Aspartate Biosynthesis.

Cellular aspartate drives cancer cell proliferation, but signaling pathways that rewire aspartate biosynthesis to control cell growth remain largely unknown. Hypoxia-inducible factor-1α (HIF1α) can suppress tumor cell proliferation. Here, we discovered that HIF1α acts as a direct repressor of aspartate biosynthesis involving the suppression of several key aspartate-producing proteins, including cytosolic glutamic-oxaloacetic transaminase-1 (GOT1) and mitochondrial GOT2. Accordingly, HIF1α suppresses aspartate production from both glutamine oxidation as well as the glutamine reductive pathway. Strikingly, the addition of aspartate to the culture medium is sufficient to relieve HIF1α-dependent repression of tumor cell proliferation. Furthermore, these key aspartate-producing players are specifically repressed in VHL-deficient human renal carcinomas, a paradigmatic tumor type in which HIF1α acts as a tumor suppressor, highlighting the in vivo relevance of these findings. In conclusion, we show that HIF1α inhibits cytosolic and mitochondrial aspartate biosynthesis and that this mechanism is the molecular basis for HIF1α tumor suppressor activity.

Identification of S-Nitrosylated and Reversibly Oxidized Proteins by Fluorescence Switch and Complementary Techniques.

S-nitrosylation and other reversible oxidative posttranslational modifications of proteins are part of the nonclassical mechanisms of nitric oxide signaling. The biotin switch technique for specifically labeling S-nitrosylated proteins opened the way to proteomic identification of these modifications. Since then, several variations and adaptations of the original method have been applied.We describe here the protocols of several techniques that can be used for the proteomic identification of these modifications, as well as for the detailed characterization of the modification of individual proteins. The fluorescence switch technique allows the proteomic identification of S-nitrosylated proteins based on their fluorescent labeling coupled to electrophoretic separation, as well as the comparison of the overall modification in different samples. The redox fluorescence switch is an adaptation to detect all the proteins reversibly oxidized in cysteine residues. We also describe the protocols of complementary techniques that allow comparing the extent of modification of individual proteins in several conditions by biotin switch, and the identification of modified residues by differential labeling adapted for mass spectrometry identification.

Fusogenic properties of the Ectodomain of HCV E2 envelope protein.

The steps leading from hepatitis C virus (HCV) attachment to the hepatocytes to the fusion of viral and cellular membranes remain uncharacterized. In this regard, we have studied the mechanism underlying the HCV fusion process using liposomes and a truncated form of E2 protein lacking the transmembrane region, E2661 (amino acids 384-661). E2661 has been previously obtained by using the baculovirus expression system and shown to behave as an independent folding domain (M. Rodriguez-Rodriguez, D. Tello, B. Yelamos, J. Gomez-Gutierrez, B. Pacheco, S. Ortega, A.G. Serrano, D.L. Peterson, F. Gavilanes, Structural properties of the ectodomain of hepatitis C virus E2 envelope protein, Virus Res. 139 (2009) 91-99). This form has been used in lipid-protein interaction studies with different model vesicles, at different pHs and by employing a variety of fluorescent assays. The obtained results indicate that E2661 induces vesicle aggregation, lipid mixing and liposome leakage, reaching higher values in the presence of negatively charged phospholipids and cholesterol at acidic pH. Therefore, the results of these studies would be indicative of an HCV infection process through receptor mediated endocytosis. Accordingly, E2 might be important in the HCV initial infective steps, interacting with the target membranes and giving rise to their subsequent destabilization.

Índice C en médicos de dos centros hospitalarios chilenos según el test de competencia moral de Lind / Assessment of moral competence of physicians

Background: Moral competence (MC) in physicians is fundamental, given the increasing complexity of medicine. The "Moral Competence Test" (MCT © Lind) evaluates this feature and its indicator is the C Index (CI). Aim: To explore moral competence and its associated factors among physicians working in Chile. Material and Methods: The MCT was answered by 236 physicians from two medical centers who voluntarily participated in the study. Besides the test, participants completed an encrypted form giving information about gender, years in practice and post-graduate studies. Results: The average CI value of the participants was 20,9. Post-graduate studies had a significant positive influence on CI. There was a significant decrease in CI, between 16 and 20 years of professional exercise. Gender and the area of post-graduate studies did not have a significant influence. Conclusions: The studied physicians showed a wide range of CI which was positively affected by the postgraduate studies performed. The years of professional practice had a negative influence. Expanding training opportunities during professional practice could have a positive effect on CM as measured by CI.

Role Of Hif2α Oxygen Sensing Pathway In Bronchial Epithelial Club Cell Proliferation.

Oxygen-sensing pathways executed by the hypoxia-inducible factors (HIFs) induce a cellular adaptive program when oxygen supply becomes limited. However, the role of the HIF oxygen-sensing pathway in the airway response to hypoxic stress in adulthood remains poorly understood. Here we found that in vivo exposure to hypoxia led to a profound increase in bronchial epithelial cell proliferation mainly confined to Club (Clara) cells. Interestingly, this response was executed by hypoxia-inducible factor 2α (HIF2α), which controls the expression of FoxM1, a recognized proliferative factor of Club cells. Furthermore, HIF2α induced the expression of the resistin-like molecules α and ß (RELMα and ß), previously considered bronchial epithelial growth factors. Importantly, despite the central role of HIF2α, this proliferative response was not initiated by in vivo Vhl gene inactivation or pharmacological inhibition of prolyl hydroxylase oxygen sensors, indicating the molecular complexity of this response and the possible participation of other oxygen-sensing pathways. Club cells are principally involved in protection and maintenance of bronchial epithelium. Thus, our findings identify a novel molecular link between HIF2α and Club cell biology that can be regarded as a new HIF2α-dependent mechanism involved in bronchial epithelium adaptation to oxygen fluctuations.

High-yield production of a chimeric glycoprotein based on permuted E1 and E2 HCV envelope ectodomains.

In this report it is described for the first time the expression and purification of large quantities of a soluble and correctly folded chimeric recombinant protein, E2661E1340, containing the permuted Hepatitis C virus (HCV) glycoprotein ectodomains E1 (amino acids 192-340) and E2 (amino acids 384-661). Using the baculovirus/insect cell expression system, 8mg of secreted protein were purified from 1L of culture media, a yield 4 times higher than the described for its counterpart E1341E2661. This permuted chimeric protein is glycosylated and possesses a high tendency to self-associate. The fluorescence emission spectrum indicates that Trp residues occupy a relatively low hydrophobic environment. The secondary structure was determined by deconvolution of the far-UV circular dichroism spectrum yielding 13% α-helix structure, 49% extended structure and 38% non-ordered structure. E2661E1340 binds to antibodies present in human sera from HCV-positive patients, a binding that is blocked at different levels by a rabbit anti-E2661 antibody. All these structural and antigenic features of E2661E1340 are very similar to those described for E1340E2661, Thus, this high-yield isolated chimeric protein may be a valuable tool to study the first steps of the HCV infection.

Fusogenic properties of the ectodomains of hepatitis C virus envelope proteins.

We have used an isolated chimeric protein E1340 E2661 that includes the ectodomains of the envelope proteins of hepatitis C virus to study its interaction with model membranes. E1340 E2661 has some of the membrane destabilization properties, vesicle aggregation, lipid mixing and the release of internal aqueous content, which have previously been ascribed to fusion proteins. The effects are preferentially produced on vesicles of acidic phospholipids which would indicate the importance of the electrostatic interactions. In fact, an increase of the ionic strength of the buffer induced a considerable decrease of the destabilizing properties. Moreover, fluorescence polarization studies show that the recombinant protein reduces the amplitude of the thermal transition of dimyristoylphosphatidylglycerol vesicles and increases the transition temperature at pH 5.0 in a dose-dependent manner, indicating its insertion into the bilayer. Furthermore, a decrease of the pH induces a conformational change in the protein structure as evidenced by fluorescence of tryptophan residues and 4,4'-bis(1-anilinonaphthalene-8-sulfonate). A model for the fusion of hepatitis C virus with the host cell membrane can be postulated. The dissociation of E1E2 dimers would uncover the fusion peptides which can then interact with the polar lipid heads of the outer leaflet of the lipid bilayer and next insert into the hydrophobic moiety producing the destabilization of the bilayer which finally leads to fusion.

Fungal extracellular ribotoxins as insecticidal agents.

Fungal ribotoxins were discovered almost 50 years ago as extracellular ribonucleases (RNases) with antitumoral properties. However, the biological function of these toxic proteins has remained elusive. The discovery of the ribotoxin HtA, produced by the invertebrates pathogen Hirsutella thompsonii, revived the old proposal that insecticidal activity would be their long searched function. Unfortunately, HtA is rather singular among all ribotoxins known in terms of sequence and structure similarities. Thus, it was intriguing to answer the question of whether HtA is just an exception or, on the contrary, the paradigmatic example of the ribotoxins function. The work presented uses HtA and α-sarcin, the most representative member of the ribotoxins family, to show their strong toxic action against insect larvae and cells.

Differential redox proteomics allows identification of proteins reversibly oxidized at cysteine residues in endothelial cells in response to acute hypoxia.

Adaptation to decreased oxygen availability (hypoxia) is crucial for proper cell function and survival. In metazoans, this is partly achieved through gene transcriptional responses mediated by hypoxia-inducible factors (HIFs). There is abundant evidence that production of reactive oxygen species (ROS) increases during hypoxia, which contributes to the activation of the HIF pathway. In addition to altering the cellular redox balance, leading to oxidative stress, ROS can transduce signals by reversibly modifying the redox state of cysteine residues in certain proteins. Using the "redox fluorescence switch" (RFS), a thiol redox proteomic technique that fluorescently labels reversibly oxidized cysteines, we analyzed endothelial cells subjected to acute hypoxia and subsequent reoxygenation. We observed a general increase in cysteine oxidation during hypoxia, which was reversed by reoxygenation, and two-dimensional electrophoresis revealed the differential oxidation of specific proteins. Using complementary derivatization techniques, we confirmed the modification of individual target proteins and identified specific cysteine residues that were oxidized in hypoxic conditions, thereby overcoming several limitations associated with fluorescence derivatization. These findings provide an important basis for future studies of the role of these modifications in HIF activation and in other acute adaptive responses to hypoxia.

Expression and structural properties of a chimeric protein based on the ectodomains of E1 and E2 hepatitis C virus envelope glycoproteins.

Hepatitis C virus encodes two enveloped glycoproteins, E1 and E2, which are involved in viral attachment and entry into target cells. We have obtained in insect cells infected by recombinant baculovirus a chimeric secreted recombinant protein, E1(341)E2(661,) containing the ectodomains of E1 and E2. The described procedure allows the purification of approximately 2mg of protein from 1L of culture media. Sedimentation velocity experiments and SDS-PAGE in the absence of reducing agents indicate that the protein has a high tendency to self-associate, the dimer being the main species observed. All the oligomeric forms observed maintain a conformation which is recognized by the conformation-dependent monoclonal antibody H53 directed against the E2 ectodomain. The spectroscopic properties of E1(341)E2(661) are those of a three-dimensionally structured protein. Moreover, the chimeric protein is able to bind to human antibodies present in HCV-positive human sera. Accordingly, this chimeric soluble polypeptide chain may be a valuable tool to study the structure-function relationship of HCV envelope proteins.

A "fluorescence switch" technique increases the sensitivity of proteomic detection and identification of S-nitrosylated proteins.

Protein S-nitrosylation is a reversible post-translational modification of protein cysteines that is increasingly being considered as a signal transduction mechanism. The "biotin switch" technique marked the beginning of the study of the S-nitrosoproteome, based on the specific replacement of the labile S-nitrosylation by a more stable biotinylation that allowed further detection and purification. However, its application for proteomic studies is limited by its relatively low sensitivity. Thus, typical proteomic experiments require high quantities of protein extracts, which precludes the use of this method in a number of biological settings. We have developed a "fluorescence switch" technique that, when coupled to 2-DE proteomic methodologies, allows the detection and identification of S-nitrosylated proteins by using limited amounts of starting material, thus significantly improving the sensitivity. We have applied this methodology to detect proteins that become S-nitrosylated in endothelial cells when exposed to S-nitroso-L-cysteine, a physiological S-nitrosothiol, identifying already known S-nitrosylation targets, as well as proteins that are novel targets. This "fluorescence switch" approach also allowed us to identify several proteins that are denitrosylated by thioredoxin in cytokine-activated RAW264.7 (murine macrophage) cells. We believe that this method represents an improvement in order to approach the identification of S-nitrosylated proteins in physiological conditions.

Structural properties of the ectodomain of hepatitis C virus E2 envelope protein.

We describe the structural and antigenic properties of a soluble form of hepatitis C virus E2 envelope protein ectodomain ending at residue 661 (E2(661)) which is obtained in large quantities in a baculovirus/insect cell system. The protein is secreted to the cellular medium by virus-infected cells. E2(661) is glycosylated and possesses a high tendency to self-associate. In fact, analytical ultracentrifugation and size exclusion chromatography studies show that the purified protein is mainly composed of dimers, trimers and tetramers being the dimer the smallest species present in solution. The secondary structure was determined by deconvolution of the far-UV circular dichroism spectrum yielding 8% alpha-helix structure, 47% extended structure and 45% non-ordered structure. The near-UV CD spectrum is indicative of a folded structure. The fluorescence emission spectrum indicates that Trp residues occupy a relatively low hydrophobic environment. Finally, E2(661) binds to a monoclonal conformation specific antibody and to antibodies present in human sera from HCV-positive patients. All these features suggest that the secreted protein possesses a native-like conformation. The use of this independent folding domain may contribute to shed light on the biology of HCV and could also be used as a vaccine in the prevention of HCV infection.