Transcriptome profiling of the fungus Aspergillus nidulans exposed to a commercial glyphosate-based herbicide under conditions of apparent herbicide tolerance.

Glyphosate-based herbicides, such as Roundup®, are the most widely used non-selective, broad-spectrum herbicides. The release of these compounds in large amounts into the environment is susceptible to affect soil quality and health, especially because of the non-target effects on a large range of organisms including soil microorganisms. The soil filamentous fungus Aspergillus nidulans, a well-characterized experimental model organism that can be used as a bio-indicator for agricultural soil health, has been previously shown to be highly affected by Roundup GT Plus (R450: 450 g/L of glyphosate) at concentrations far below recommended agricultural application rate, including at a dose that does not cause any macroscopic effect. In this study, we determined alterations in the transcriptome of A. nidulans when exposed to R450 at a dose corresponding to the no-observed-adverse-effect level (NOAEL) for macroscopic parameters. A total of 1816 distinct genes had their expression altered. The most affected biological functions were protein synthesis, amino acids and secondary metabolisms, stress response, as well as detoxification pathways through cytochromes P450, glutathione-S-transferases, and ABC transporters. These results partly explain the molecular mechanisms underlying alterations in growth parameters detected at higher concentrations for this ascomycete fungus. In conclusion, our results highlight molecular disturbances in a soil fungus under conditions of apparent tolerance to the herbicide, and thus confirm the need to question the principle of "substantial equivalence" when applied to plants made tolerant to herbicides.

Glycogen synthase kinase 3-mediated voltage-dependent anion channel phosphorylation controls outer mitochondrial membrane permeability during lipid accumulation.

UNLABELLED: Nonalcoholic steatosis is a liver pathology characterized by fat accumulation and severe metabolic alterations involving early mitochondrial impairment and late hepatocyte cell death. However, mitochondrial dysfunction mechanisms remain elusive. Using four models of nonalcoholic steatosis, i.e., livers from patients with fatty liver disease, ob/ob mice, mice fed a high-fat diet, and in vitro models of lipotoxicity, we show that outer mitochondrial membrane permeability is altered and identified a posttranslational modification of voltage-dependent anion channel (VDAC), a membrane channel and NADH oxidase, as a cause of early mitochondrial dysfunction. Thus, in nonalcoholic steatosis VDAC exhibits reduced threonine phosphorylation, which increases the influx of water and calcium into mitochondria, sensitizes the organelle to matrix swelling, depolarization, and cytochrome c release without inducing cell death. This also amplifies VDAC enzymatic and channel activities regulation by calcium and modifies its interaction with proteic partners. Moreover, lipid accumulation triggers a rapid lack of VDAC phosphorylation by glycogen synthase kinase 3 (GSK3). Pharmacological and genetic manipulations proved GSK3 to be responsible for VDAC phosphorylation in normal cells. Notably, VDAC phosphorylation level correlated with steatosis severity in patients. CONCLUSION: VDAC acts as an early sensor of lipid toxicity and its GSK3-mediated phosphorylation status controls outer mitochondrial membrane permeabilization in hepatosteatosis.

Identification of periplakin as a new target for autoreactivity in idiopathic pulmonary fibrosis.

RATIONALE: Injury to alveolar epithelial cells is central to the pathophysiology of idiopathic pulmonary fibrosis (IPF). An abnormal autoimmune response directed against antigens of the alveolar epithelium may contribute to the disease. OBJECTIVES: To detect circulating autoantibodies (autoAbs) directed against epithelial structures. METHODS: We performed immunoblot by separating human placental amnion extract or alveolar epithelial cell (A549 cell line) proteins on polyacrylamide gels, blotting on nitrocellulose membranes, and incubating with serum from patients with IPF (n = 40) or healthy subjects (n = 40). Proteomic analysis and mass spectrometry characterized the target protein. Inhibition experiments performed with the correspondent recombinant protein confirmed our results. MEASUREMENTS AND MAIN RESULTS: We identified IgG autoAbs recognizing a 200-kD protein in the serum of patients with IPF. Proteomic analysis identified this protein as human periplakin (PPL), a component of desmosomes. Anti-PPL Abs were found by immunoblot in both serum and bronchoalveolar lavage in patients with IPF: 16/40 (40%) of them were positive versus none of the control subjects. Immunohistochemistry revealed that PPL was strongly expressed in bronchial and alveolar epithelium, but that PPL exhibited changes in intracellular localization among normal and fibrotic alveolar epithelium. In an alveolar epithelial wound repair assay, an anti-PPL IgG decreased cell migration. Recombinant PPL induced bronchoalveolar lavage T lymphocyte proliferation. Patients with IPF with anti-PPL Abs had a more severe respiratory disease, despite no difference in survival. CONCLUSIONS: We found a new circulating autoAb directed against PPL in patients with IPF, associated with a more severe disease.

Modulation of septin and molecular motor recruitment in the microtubule environment of the Taxol-resistant human breast cancer cell line MDA-MB-231.

Cell resistance to low doses of paclitaxel (Taxol) involves a modulation of microtubule (MT) dynamics. We applied a proteomic approach based on 2-DE coupled with MS to identify changes in the MT environment of Taxol-resistant breast cancer cells. Having established a proteomic pattern of the microtubular proteins extracted from MDA-MB-231 cells, we verified by Western blotting that in resistant cells, α- and ß-tubulins (more specifically the ßIII and ßIV isotypes) increased. Interestingly, four septins (SEPT2, 8, 9 and 11), which are GTPases involved in cytokinesis and in MT/actin cytoskeleton organization, were overexpressed and enriched in the MT environment of Taxol-resistant cells compared to their sensitive counterpart. Changes in the MT proteome of resistant cells also comprised increased kinesin-1 heavy chain expression and recruitment on MTs while dynein light chain-1 was downregulated. Modulation of motor protein recruitment around MTs might reflect their important role in controlling MT dynamics via the organization of signaling pathways. The identification of proteins previously unknown to be linked to taxane-resistance could also be valuable to identify new biological markers of resistance.

Highly cytotoxic and neurotoxic acetogenins of the Annonaceae: new putative biological targets of squamocin detected by activity-based protein profiling.

Acetogenins of the Annonaceae are strong inhibitors of mitochondrial complex I but discrepancies in the structure/activity relationships pled the search for other targets within the whole cell proteome. Combining hemisynthetic work, Cu-catalyzed Huisgen cycloaddition and proteomic techniques we have identified new putative protein targets of squamocin ruling out the previously accepted 'complex I dogma'. These results give new insights into the mechanism of action of these potent neurotoxic molecules.

Proteomic analysis of the soil filamentous fungus Aspergillus nidulans exposed to a Roundup formulation at a dose causing no macroscopic effect: a functional study.

Roundup® is a glyphosate-based herbicide (GBH) used worldwide both in agriculture and private gardens. Thus, it constitutes a substantial source of environmental contaminations, especially for water and soil, and may impact a number of non-target organisms essential for ecosystem balance. The soil filamentous fungus Aspergillus nidulans has been shown to be highly affected by a commercial formulation of Roundup® (R450), containing 450 g/L of glyphosate (GLY), at doses far below recommended agricultural application rate. In the present study, we used two-dimensional gel electrophoresis combined to mass spectrometry to analyze proteomic pattern changes in A. nidulans exposed to R450 at a dose corresponding to the no-observed-adverse-effect level (NOAEL) for macroscopic parameters (31.5 mg/L GLY among adjuvants). Comparative analysis revealed a total of 82 differentially expressed proteins between control and R450-treated samples, and 85% of them (70) were unambiguously identified. Their molecular functions were mainly assigned to cell detoxification and stress response (16%), protein synthesis (14%), amino acid metabolism (13%), glycolysis/gluconeogenesis/glycerol metabolism/pentose phosphate pathway (13%) and Krebs TCA cycle/acetyl-CoA synthesis/ATP metabolism (10%). These results bring new insights into the understanding of the toxicity induced by higher doses of this herbicide in the soil model organism A. nidulans. To our knowledge, this study represents the first evidence of protein expression modulation and, thus, possible metabolic disturbance, in response to an herbicide treatment at a dose that does not cause any visible effect. These data are likely to challenge the concept of "substantial equivalence" when applied to herbicide-tolerant plants.

Search for biomarkers of neurosarcoidosis by proteomic analysis of cerebrospinal fluid.

Sarcoidosis is a systemic granulomatous disease, which mostly affects lung. Central nervous system can be affected causing a neurosarcoidosis in 5 to 15% of all sarcoidosis patients. The definitive diagnosis is established on histological examination of brain granulomas. Angiotensin converting enzyme is currently the most relevant biomarker to confirm a probable diagnosis; however, it lacks sensitivity and specificity. We aim to find novel biomarkers of neurosarcoidosis in cerebrospinal fluid (CSF) by proteomic analysis, combining two-dimension electrophoresis (2-DE) and mass spectrometry. We performed CSF proteomic profile of both patients (group S) and control subjects (group H). The statistical analysis of 2-D gels highlighted 42 spots significantly different between the two groups. Twenty-five spots were subjected to tryptic digestion; the peptides were analyzed by MALDI-TOF and MALDI-TOF-TOF, giving rise to 10 identifications. Among the identified proteins, low-molecular-mass-kininogen and vitamin-D-binding-protein were increased, while transthyretin was decreased. These proteins have probably an intrathecal source and could be interesting candidates. This study led to the identification of several proteins which can be used for the diagnosis and/or monitoring of neurosarcoidosis. These putative biomarkers have to be confirmed on a larger cohort and assessed for their sensitivity and specificity.

Differential Peripheral Proteomic Biosignature of Fluoxetine Response in a Mouse Model of Anxiety/Depression.

The incorporation of peripheral biomarkers in the treatment of major depressive disorders (MDD) could improve the efficiency of treatments and increase remission rate. Peripheral blood mononuclear cells (PBMCs) represent an attractive biological substrate allowing the identification of a drug response signature. Using a proteomic approach with high-resolution mass spectrometry, the present study aimed to identify a biosignature of antidepressant response (fluoxetine, a Selective Serotonin Reuptake Inhibitor) in PBMCs in a mouse model of anxiety/depression. Following determination of an emotionality score, using complementary behavioral analysis of anxiety/depression across three different tests (Elevated Plus Maze, Novelty Suppressed Feeding, Splash Test), we showed that a 4-week corticosterone treatment (35 µg/ml, CORT model) in C57BL/6NTac male mice induced an anxiety/depressive-like behavior. Then, chronic fluoxetine treatment (18 mg/kg/day for 28 days in the drinking water) reduced corticosterone-induced increase in emotional behavior. However, among 46 fluoxetine-treated mice, only 30 of them presented a 50% decrease in emotionality score, defining fluoxetine responders (CORT/Flx-R). To determine a peripheral biological signature of fluoxetine response, proteomic analysis was performed from PBMCs isolated from the "most" affected corticosterone/vehicle (CORT/V), corticosterone/fluoxetine responders and non-responders (CORT/Flx-NR) animals. In comparison to CORT/V, a total of 263 proteins were differently expressed after fluoxetine exposure. Expression profile of these proteins showed a strong similarity between CORT/Flx-R and CORT/Flx-NR (R = 0.827, p < 1e-7). Direct comparison of CORT/Flx-R and CORT/Flx-NR groups revealed 100 differently expressed proteins, representing a combination of markers associated either with the maintenance of animals in a refractory state, or associated with behavioral improvement. Finally, 19 proteins showed a differential direction of expression between CORT/Flx-R and CORT/Flx-NR that drove them away from the CORT-treated profile. Among them, eight upregulated proteins (RPN2, HSPA9, NPTN, AP2B1, UQCRC2, RACK-1, TOLLIP) and one downregulated protein, TLN2, were previously associated with MDD or antidepressant drug response in the literature. Future preclinical studies will be required to validate whether proteomic changes observed in PBMCs from CORT/Flx-R mice mirror biological changes in brain tissues.

Native metabotropic glutamate receptor 4 depresses synaptic transmission through an unusual Gαq transduction pathway.

In cerebellar cortex, mGlu4 receptors located on parallel fibers play an essential role in normal motor function, but the molecular mechanisms involved are not yet completely understood. Using a strategy combining biochemical and electrophysiological approaches in the rodent cerebellum, we demonstrate that presynaptic mGlu4 receptors control synaptic transmission through an atypical activation of Gαq proteins. First, the Gαq subunit, PLC and PKC signaling proteins present in cerebellar extracts are retained on affinity chromatography columns grafted with different sequences of the cytoplasmic domain of mGlu4 receptor. The i2 loop and the C terminal domain were used as baits, two domains that are known to play a pivotal role in coupling selectivity and efficacy. Second, in situ proximity ligation assays show that native mGlu4 receptors and Gαq subunits are in close physical proximity in cerebellar cortical slices. Finally, electrophysiological experiments demonstrate that the molecular mechanisms underlying mGlu4 receptor-mediated inhibition of transmitter release at cerebellar Parallel Fiber (PF) - Molecular Layer Interneuron (MLI) synapses involves the Gαq-PLC signaling pathway. Taken together, our results provide compelling evidence that, in the rodent cerebellar cortex, mGlu4 receptors act by coupling to the Gαq protein and PLC effector system to reduce glutamate synaptic transmission.

SIRT1 protects the heart from ER stress-induced cell death through eIF2α deacetylation.

Over the past decade, endoplasmic reticulum (ER) stress has emerged as an important mechanism involved in the pathogenesis of cardiovascular diseases including heart failure. Cardiac therapy based on ER stress modulation is viewed as a promising avenue toward effective therapies for the diseased heart. Here, we tested whether sirtuin-1 (SIRT1), a NAD+-dependent deacetylase, participates in modulating ER stress response in the heart. Using cardiomyocytes and adult-inducible SIRT1 knockout mice, we demonstrate that SIRT1 inhibition or deficiency increases ER stress-induced cardiac injury, whereas activation of SIRT1 by the SIRT1-activating compound STAC-3 is protective. Analysis of the expression of markers of the three main branches of the unfolded protein response (i.e., PERK/eIF2α, ATF6 and IRE1) showed that SIRT1 protects cardiomyocytes from ER stress-induced apoptosis by attenuating PERK/eIF2α pathway activation. We also present evidence that SIRT1 physically interacts with and deacetylates eIF2α. Mass spectrometry analysis identified lysines K141 and K143 as the acetylation sites on eIF2α targeted by SIRT1. Furthermore, mutation of K143 to arginine to mimic eIF2α deacetylation confers protection against ER stress-induced apoptosis. Collectively, our findings indicate that eIF2α deacetylation on lysine K143 by SIRT1 is a novel regulatory mechanism for protecting cardiac cells from ER stress and suggest that activation of SIRT1 has potential as a therapeutic approach to protect the heart against ER stress-induced injury.

MALDI-TOF MS applied to apoC-III glycoforms of patients with congenital disorders affecting O-glycosylation. Comparison with two-dimensional electrophoresis.

PURPOSE: The O-glycan abnormalities accompanying some congenital disorders of glycosylation, namely conserved oligomeric Golgi-congenital disorders of glycosylation (COG-CDGs) and ATP6V0A2-CDGs, are mainly detected using electrophoresis methods applied to circulating apolipoprotein C-III. The objective of this study was to evaluate the reliability of MALDI-TOF MS of apoC-III for the detection and characterization of CDG-associated O-glycan defects. EXPERIMENTAL DESIGN: plasmas from CDG-negative, COG-CDG, and ATP6V0A2-CDG patients were analyzed and results were compared to those obtained using 2DE followed by Western blot. RESULTS: MALDI-TOF of apoC-III allowed to detect various significant O-glycan abnormalities in CDG-patients with emphasis to COG-CDG. Furthermore, in CDG samples, comparison study between 2DE and MALDI-TOF showed a particular behavior of monosialylated apoC-III in the mass spectrometer that could be related to an abnormal O-glycan structure. CONCLUSIONS AND CLINICAL RELEVANCE: MALDI-TOF MS appears as a powerful technique for the analysis of apoC-III glycoforms for potential routine screening of COG- and ATP6V0A2-CDGs.

Immunization using GroEL decreases Clostridium difficile intestinal colonization.

Clostridium difficile is a pathogen which is responsible for diarrhea and colitis, particularly after treatment with antibiotics. Clinical signs are mainly due to two toxins, TcdA and TcdB. However, the first step of pathogenesis is the colonization process. We evaluated C. difficile surface proteins as vaccine antigens in the hamster model to prevent intestinal colonization. This vaccination induced a partial protection of hamsters against death after a C. difficile challenge. A proteomic analysis of animal sera allowed us to identify proteins which could be responsible for the protection observed. Among these proteins, we identified the GroEL heat shock protein. To confirm the role of the specific GroEL antibodies in the delayed C. difficile colonization of hamsters, we performed an immunization assay in a mouse model. After intranasal immunization with the recombinant protein GroEL, we observed a lower C. difficile intestinal colonization in the immunized group as compared to the control group.