Taiane Schneider

Antimicrobial activity of Amazonian oils against Paenibacillus species

The Gram-positive, spore-forming bacterium Paenibacillus larvae is the primary bacterial pathogen of honeybee brood and the causative agent of American foulbrood disease (AFB). One of the feasible alternative treatments being used for their control of this disease is essential oils. In this study in vitro antimicrobial activity of Andiroba and Copaíba essential oils against Paenibacillus species, including P. larvae was evaluated. Minimal inhibitory concentration (MIC) in Mueller-Hinton broth by the microdilution method was assessed. Andiroba registered MIC values of 1.56-25%, while the MICs values obtained for Copaíba oil were of 1.56-12.5%. In order to determine the time-response effect of essential oils on P. larvae, this microorganism was exposed to the oils for up to 48 h. After 24 h treatment with Andiroba oil and after 48 h treatment with Copaíba oil no viable cells of P. larvae ATCC 9545 were observed. The possible toxic effect of essential oils were assessed by the spraying application method of the same concentrations of MICs. Bee mortality was evident only in treatment with Andiroba oil and the Copaíba oil shows no toxic effects after 10 days of observation. Taking together ours results showed for the first time that these oils presented a high activity against Paenibacillus species showing that Copaíba oil may be a candidate for the treatment or prevention of AFB.

Contribution of S6K1/MAPK Signaling Pathways in the Response to Oxidative Stress: Activation of RSK and MSK by Hydrogen Peroxide

Cells respond to different kind of stress through the coordinated activation of signaling pathways such as MAPK or p53. To find which molecular mechanisms are involved, we need to understand their cell adaptation. The ribosomal protein, S6 kinase 1 (S6K1), is a common downstream target of signaling by hormonal or nutritional stress. Here, we investigated the initial contribution of S6K1/MAPK signaling pathways in the cell response to oxidative stress produced by hydrogen peroxide (H2O2). To analyze S6K1 activation, we used the commercial anti-phospho-Thr389-S6K1 antibody most frequently mentioned in the bibliography. We found that this antibody detected an 80-90 kDa protein that was rapidly phosphorylated in response to H2O2 in several human cells. Unexpectedly, this phosphorylation was insensitive to both mTOR and PI3K inhibitors, and knock-down experiments showed that this protein was not S6K1. RSK and MSK proteins were candidate targets of this phosphorylation. We demonstrated that H2O2 stimulated phosphorylation of RSK and MSK kinases at residues that are homologous to Thr389 in S6K1. This phosphorylation required the activity of either p38 or ERK MAP kinases. Kinase assays showed activation of RSK and MSK by H2O2. Experiments with mouse embryonic fibroblasts from p38 animals’ knockout confirmed these observations. Altogether, these findings show that the S6K1 signaling pathway is not activated under these conditions, clarify previous observations probably misinterpreted by non-specific detection of proteins RSK and MSK by the anti-phospho-Thr389-S6K1 antibody, and demonstrate the specific activation of MAPK signaling pathways through ERK/p38/RSK/MSK by H2O2.

A nonsense variant in HERC1 is associated with intellectual disability, megalencephaly, thick corpus callosum and cerebellar atrophy

Megalencephaly is a congenital condition characterized by severe overdeveloped brain size. This phenotype is often caused by mutations affecting the RTK/PI3K/mTOR (receptor tyrosine kinase-phosphatidylinositol-3-kinase-AKT) signaling and its downstream pathway of mammalian target of rapamycin (mTOR). Here, using a whole-exome sequencing in a Moroccan consanguineous family, we show that a novel autosomal-recessive neurological condition characterized by megalencephaly, thick corpus callosum and severe intellectual disability is caused by a homozygous nonsense variant in the HERC1 gene. Assessment of the primary skin fibroblast from the proband revealed complete absence of the HERC1 protein. HERC1 is an ubiquitin ligase that interacts with tuberous sclerosis complex 2, an upstream negative regulator of the mTOR pathway. Our data further emphasize the role of the mTOR pathway in the regulation of brain development and the power of next-generation sequencing technique in elucidating the genetic etiology of autosomal-recessive disorders and suggest that HERC1 defect might be a novel cause of autosomal-recessive syndromic megalencephaly.

Guaraná, a Highly Caffeinated Food, Presents in vitro Antitumor Activity in Colorectal and Breast Cancer Cell Lines by Inhibiting AKT/mTOR/S6K and MAPKs Pathways

ABSTRACT The mammalian target of rapamycin (mTOR) and mitogen-activated protein kinases (MAPKs) pathways are frequently upregulated in cancer. Some authors have reported that some antioxidant molecules could be potential inhibitors of these pathways. Therefore, we investigated the in vitro antitumor effect of guaraná by inhibiting the AKT/mTOR/S6K and MAPKs pathways. Colorectal and breast cancer cell lineages, HT-29 and MCF-7 cells, respectively, were exposed to different guaraná concentrations (0.1, 1, 10, and 100 µg/mL) as well as its main bioactive molecule, caffeine, in proportional concentrations to those found in the extract. Western blot, clonogenic assay, and growth curve were performed. Moreover, we investigated the potential cytotoxic effect of guaraná in normal cells. The results revealed that guaraná and caffeine inhibited some MAPKs proteins (p-p38 and p-HSP27) in MCF-7 cells. However, they did not affect this pathway in HT-29 cells. Furthermore, guaraná inhibited mTORC1 (p-S6K) and mTORC2 (p-AKT) in MCF-7 cells, but only mTORC1 in HT-29 cells. Caffeine only inhibited the mTOR pathway in MCF-7 cells. Guaraná decreased the colony formation and cell growth in MCF-7 and HT-29 cells. Guaraná did not affect normal cells. In conclusion, guaraná could be an important agent in antitumor pharmacologic therapies by inhibiting the mTOR and MAPKs pathways.

NEURL4 regulates the transcriptional activity of tumor suppressor protein p53 by modulating its oligomerization

p53 is a transcription factor that regulates important cellular processes related to tumor suppression, including induction of senescence, apoptosis, and DNA repair as well as the inhibition of angiogenesis and cell migration. Therefore, it is critical to understand the molecular mechanism that regulates it. p53 tetramerization is a key step in its activation process and the regulation of this oligomerization, an important control point. The E3 ubiquitin ligase HERC2 controls the p53 transcriptional activity by regulation of its oligomerization state. HERC2-interacting proteins such as the adaptor-like protein with six neuralized domains NEURL4 are also candidates to regulate p53 activity. Here, we demonstrate the existence of an interaction network between NEURL4, HERC2 and p53 proteins. We report a functional interaction between NEURL4 and p53, involving the C-terminal region of p53 and the neuralized domains 3 and 4 of NEURL4. Through this interaction, NEURL4 regulates the transcriptional activity of p53. Thus, NEURL4 depletion reduced the transcriptional activity whereas NEURL4 overexpression increased it. In both cases, p53 stability was not affected. Although NEURL4 may interact with p53 independently of the E3 ubiquitin ligase HERC2, we observed that both proteins are needed to regulate the transcriptional activity of p53. Clonogenic assays confirmed the functional relevance of this interaction observing a decrease in cell growth by NEURL4 overexpression correlated to the increase of cellular cycle inhibitor p21 by p53 activation. Under these conditions, NEURL4 activated p53 oligomerization. All these findings identify NEURL4 as a novel regulator of the p53s signaling.p53 is a transcription factor that regulates important cellular processes related to tumor suppression, including induction of senescence, apoptosis, and DNA repair as well as the inhibition of angiogenesis and cell migration. Therefore, it is critical to understand the molecular mechanism that regulates it. p53 tetramerization is a key step in its activation process and the regulation of this oligomerization, an important control point. The E3 ubiquitin ligase HERC2 controls the p53 transcriptional activity by regulation of its oligomerization state. HERC2-interacting proteins such as the adaptor-like protein with six neuralized domains NEURL4 are also candidates to regulate p53 activity. Here, we demonstrate the existence of an interaction network between NEURL4, HERC2 and p53 proteins. We report a functional interaction between NEURL4 and p53, involving the C-terminal region of p53 and the neuralized domains 3 and 4 of NEURL4. Through this interaction, NEURL4 regulates the transcriptional activity of p53. Thus, NEURL4 depletion reduced the transcriptional activity whereas NEURL4 overexpression increased it. In both cases, p53 stability was not affected. Although NEURL4 may interact with p53 independently of the E3 ubiquitin ligase HERC2, we observed that both proteins are needed to regulate the transcriptional activity of p53. Clonogenic assays confirmed the functional relevance of this interaction observing a decrease in cell growth by NEURL4 overexpression correlated to the increase of cellular cycle inhibitor p21 by p53 activation. Under these conditions, NEURL4 activated p53 oligomerization. All these findings identify NEURL4 as a novel regulator of the p53’s signaling.

The E3 ubiquitin ligase HERC1 controls the ERK signaling pathway targeting C-RAF for degradation

The RAF/MEK/ERK cascade is a conserved intracellular signaling pathway that controls fundamental cellular processes including growth, proliferation, differentiation, survival and migration. Aberrant regulation of this signaling pathway has long been associated with human cancers. A major point of regulation of this pathway occurs at the level of the serine/threonine protein kinase C-RAF. Here, we show how the E3 ubiquitin ligase HERC1 regulates ERK signaling. HERC1 knockdown induced cellular proliferation, which is associated with an increase in ERK phosphorylation and in C-RAF protein levels. We demonstrate that overexpression of wild-type C-RAF is sufficient to increase ERK phosphorylation. Experiments with pharmacological inhibitors of RAF activity, or with interference RNA, show that the regulation of ERK phosphorylation by HERC1 is RAF-dependent. Immunoprecipitation, pull-down and confocal fluorescence microscopy experiments demonstrate an interaction between HERC1 and C-RAF proteins. Mechanistically, HERC1 controls C-RAF stability by regulating its polyubiquitylation in a lysine 48-linked chain. In vitro ubiquitylation assays indicate that C-RAF is a substrate of the E3 ubiquitin ligase HERC1. Altogether, we show how HERC1 can regulate cell proliferation through the activation of ERK signaling by a mechanism that affects C-RAF’s stability.

The HERC2 ubiquitin ligase is essential for embryonic development and regulates motor coordination

A mutation in the HERC2 gene has been linked to a severe neurodevelopmental disorder with similarities to the Angelman syndrome. This gene codifies a protein with ubiquitin ligase activity that regulates the activity of tumor protein p53 and is involved in important cellular processes such as DNA repair, cell cycle, cancer, and iron metabolism. Despite the critical role of HERC2 in these physiological and pathological processes, little is known about its relevance in vivo. Here, we described a mouse with targeted inactivation of the Herc2 gene. Homozygous mice were not viable. Distinct from other ubiquitin ligases that interact with p53, such as MDM2 or MDM4, p53 depletion did not rescue the lethality of homozygous mice. The HERC2 protein levels were reduced by approximately one-half in heterozygous mice. Consequently, HERC2 activities, including ubiquitin ligase and stimulation of p53 activity, were lower in heterozygous mice. A decrease in HERC2 activities was also observed in human skin fibroblasts from individuals with an Angelman-like syndrome that express an unstable mutant protein of HERC2. Behavioural analysis of heterozygous mice identified an impaired motor synchronization with normal neuromuscular function. This effect was not observed in p53 knockout mice, indicating that a mechanism independent of p53 activity is involved. Morphological analysis showed the presence of HERC2 in Purkinje cells and a specific loss of these neurons in the cerebella of heterozygous mice. In these animals, an increase of autophagosomes and lysosomes was observed. Our findings establish a crucial role of HERC2 in embryonic development and motor coordination.