The ubiquitin ligase HERC1 regulates cell migration via RAF-dependent regulation of MKK3/p38 signaling

Protein modifications by phosphorylation or ubiquitylation have been selected throughout evolution as efficient regulatory mechanisms of cellular processes. Cell migration is a complex, highly coordinated process where these mechanisms must participate in an integrated manner to transmit signaling during migration. In this study, we show that the ubiquitin ligase HERC1 regulates the p38 signaling pathway, and that this regulation is mediated by the MAPK kinase MKK3. Moreover, we demonstrate a crosstalk between RAF and MKK3/p38 pathways where RAF acts upstream of MKK3. Mechanistically, HERC1 regulates the protein levels of C-RAF and MKK3. Thus, HERC1 ubiquitylates C-RAF, targeting it for proteasomal degradation, and RAF proteins regulate MKK3 mRNA levels. Accordingly, HERC1 knockdown induces C-RAF stabilization and activation of RAF proteins; in turn, this activation increases MKK3, which phosphorylates and activates p38. The importance of these observations is demonstrated by HERC1 regulation of cell migration through regulation of p38 signaling via a RAF-dependent mechanism. Thus, HERC1 plays an essential role as a regulator of crosstalk between RAF/MKK3/p38 signaling pathways during cell migration

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

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 p53's signaling

Large HERCs function as tumor suppressors

Ubiquitin ligases regulate numerous cellular processes, including tissue homeostasis, cellular metabolism, and cell cycle progression. These enzymes recognize, interact with and ubiquitylate specific substrates. Homologous to the E6-AP carboxyl terminus (HECT) and regulator of chromosome condensation 1 (RCC1)-like domain-containing proteins (HERCs) belong to the family of HECT ubiquitin ligases. There are six human HERCs which can be divided into two subgroups: large HERCs (HERC1-2) and small HERCs (HERC3-6). Alterations in the function of large HERCs are associated with serious pathologies such as neurological disorders. Mutations in human HERC1 have been associated with overgrowth, intellectual disability and some autistic features; while mutations in HERC2 have been identified as the cause of a neurodevelopmental disorder with similarities to Angelman syndrome and autism

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

Contribution of S6K1/MAPK signaling pathways in the response to oxidative stress: activation of RSK and MSK by hydrogen peroxide

Trobareu correccions de l'article a: http://dx.doi.org/10.1371/annotation/0b485bd9-b1b2-4c60-ab22-3ac5d271dc59Cells 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

Iniciação Científica na segunda metade dos anos finais do Ensino Fundamental: percursos da Equipe de Professores do Projeto PIXEL do Colégio de Aplicação da UFRGS

Este relato de experiência tem como objetivo apresentar como foi constituído e como estrutura-se atualmente o componente curricular Iniciação Científica (IC) no Projeto PIXEL, que abrange a segunda metade dos anos finais do Ensino Fundamental no Colégio de Aplicação (CAp) da UFRGS. O trabalho na IC organiza-se a partir de um planejamento semanal da equipe de professores, o que possibilitou uma série de reflexões e práticas neste componente curricular. O trabalho com a IC oportuniza o desenvolvimento de competências gerais em ambos os anos (8° e 9°) de seu desenvolvimento. São elas: a argumentação, a autonomia, a autoria, a cooperação e a colaboração e a resolução de problemas. Essas competências são essenciais à IC e servem de referência para criar os critérios de avaliação, não somente desse componente curricular, mas de todos os demais componentes do Projeto de Ensino PIXEL. A IC ocorre em quatro períodos semanais e seus projetos são desenvolvidos ao longo de um semestre. Eles culminam em produções escritas estruturadas – em formatos de reportagem científica para os 8ºs anos, e com formatos diversos, que incluem desde relatórios até manuais para os 9ºs. Ambos os anos se envolvem em Mostras de Trabalhos, nas quais os estudantes apresentam seus processos de pesquisa utilizando-se dos mais diversos recursos, fazendo a devolutiva da investigação para a comunidade. O trabalho da IC na escola não se trata, portanto, de uma aula que propõe situações-problema hipotéticas na expectativa de que os estudantes encontrem a resposta “correta”, mas sim, de criar propostas em que a complexidade e a diversidade do mundo se evidenciem, sejam parte do problema e de suas possíveis soluções. Os resultados que temos observado a partir do que é desenvolvido na IC do PIXEL, mostram o desenvolvimento de aprendizagens que permitem ao estudante a busca de informações e verificação de fontes, que façam comparações, que defendam seu ponto de vista e que elaborem estratégias e conclusões. A IC do PIXEL é uma proposta de ensino e de aprendizagem que ressignifica o papel do professor, aproximando-o dos estudantes, pois coloca-o na posição de orientador – aquele que tem experiência de pesquisa – e também de pesquisador – aquele que aprende sobre os temas ao longo do trabalho com cada estudante

Increasing power plant efficiency with clustering methods and Variable Importance Index assessment

Power plant performance can decrease along with its life span, and move away from the design and commissioning targets. Maintenance issues, operational practices, market restrictions, and financial objectives may lead to that behavior, and the knowledge of appropriate actions could support the system to retake its original operational performance. This paper applies unsupervised machine learning techniques to identify operating patterns based on the power plant’s historical data which leads to the identification of appropriate steam generator efficiency conditions. The selected operational variables are evaluated in respect to their impact on the system performance, quantified by the Variable Importance Index. That metric is proposed to identify the variables among a much wide set of monitored data whose variation impacts the overall power plant operation, and should be controlled with more attention. Principal Component Analysis (PCA) and k-means++ clustering techniques are used to identify suitable operational conditions from a one-year-long data set with 27 recorded variables from a steam generator of a 360MW thermal power plant. The adequate number of clusters is identified by the average Silhouette coefficient and the Variable Importance Index sorts nine variables as the most relevant ones, to finally group recommended settings to achieve the target conditions. Results show performance gains in respect to the average historical values of 73.5% and the lowest efficiency condition records of 68%, to the target steam generator efficiency of 76%

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 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