The RanBP2/RanGAP1-SUMO complex gates β-arrestin2 nuclear entry to regulate the Mdm2-p53 signalling axis

Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein β-arrestin2 (β-arr2) regulates tumor suppressor p53 by counteracting Mdm2. β-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. β-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. β-arr2 can be SUMOylated, but no information is available on how SUMO may regulate β-arr2 nucleocytoplasmic shuttling. While we found β-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and β-arr2, via a SUMO interaction motif (SIM), that is required for β-arr2 cytonuclear trafficking. This SIM promotes association of β-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective β-arr2 nuclear entry. Mutation of the SIM inhibits β-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a β-arr2 SIM nuclear entry checkpoint, coupled with active β-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis

Functions of Tumour Suppressor PTEN : Regulation through Beta-arrestins and intramolecular interaction

La protéine suppresseur de tumeurs PTEN (Phosphatase and tensin deleted on chromosome 10) est une phosphatase lipidique. En déphosphorylant le phosphatidylinositol (3,4,5) trisphosphate (PIP3) en PI(4,5) P2, PTEN contre-régule la voie PI3K/Akt et inhibe la prolifération. D’autres fonctions de PTEN peuvent être indépendantes de son activité phosphatase lipidique, notamment l’inhibition de la migration. Bien que PTEN soit, après p53, le suppresseur de tumeurs le plus muté dans un large panel de cancers (gliomes, prostate, sein, endomètre…), les mécanismes par lesquels ses fonctions sont régulées ne sont pas entièrement élucidés. Par une approche de double-hybride, notre équipe a identifié que les β-arrestines (β-arrs), des protéines d’échafaudage, interagissent avec PTEN. Nos travaux mettent en évidence que l’interaction entre PTEN et les β-arrs permet de moduler ses deux activités dépendantes ou non de son activité phosphatase lipidique. D’une part, les β-arrs augmentent l’activité phosphatase lipidique de PTEN in vitro. La GTPase RhoA et sa kinase d’aval ROCK activent PTEN, et ceci se fait par l’intermédiaire des β-arrs. La stimulation du récepteur à l’acide lysophosphatidique (LPA), qui active la voie RhoA/ROCK, augmente la formation du complexe PTEN/β-arrs et permet le recrutement du complexe à la membrane. Par l’effet positif sur l’activité phosphatase lipidique de PTEN, les β-arrs participent à l’inhibition d’Akt et de la prolifération dans les fibroblastes embryonnaires de souris (MEF). A l’inverse dans les gliomes U373, les β-arrs lèvent l’inhibition de la migration exercée par le domaine C2 de PTEN, indépendamment de son activité phosphatase lipidique. En aval de l’activation de RhoA induite par blessure du tapis cellulaire, les β-arrs interagissent davantage avec PTEN et rétablissent la migration des gliomes. De ce fait, les β-arrs régulent différentiellement les fonctions de PTEN importantes pour le contrôle de la prolifération cellulaire et la migration. Enfin, l’activité et la localisation de PTEN sont modulées par des interactions intramoléculaires entre ses domaines catalytiques, C2 et sa queue C-terminale régulatrice. Ces interactions régulent le passage d’une conformation fermée vers une conformation ouverte et active de PTEN. Grâce au développement d’un biosenseur de PTEN basé sur le transfert d’énergie par résonnance (RET), nous pouvons suivre pour la première fois les changements conformationnels de PTEN dans les cellules vivantes. En utilisant ce biosenseur nous montrons que la mutation des résidus impliqués dans les interactions intramoléculaires entraine des changements de conformation détectés par des variations de RET. De plus, l’activation de voies de signalisation connues pour activer PTEN, entrainent des changements conformationnels qui corrèlent avec l’augmentation de l’activité phosphatase lipidique de PTEN. Nos données montrent que le biosenseur peut être utilisé comme outil pour détecter les changements d’activité de PTEN dans les cellules vivantes. L’axe suppresseur de tumeurs/oncogène PTEN/PI3K/Akt joue un rôle essentiel dans la progression tumorale et constitue une cible thérapeutique pour le cancer. L’ensemble de nos travaux permet d’ajouter un degré de compréhension dans la régulation de PTEN, tant par les β-arrs que par l’interaction intramoléculaire et les changements conformationnels.The Tumour Suppressor protein PTEN (Phosphatase and tensin deleted on chromosome 10) is a lipid phosphatase. By converting phosphatidylinositol (3,4,5) trisphosphate (PIP3) to PI(4,5)P2, PTEN inhibits the PI3K/Akt signalling pathway and cell proliferation. Other functions attributed to PTEN, including the inhibition of cell migration, can occur independently of its lipid phosphatase activity. Although PTEN function is dysregulated in a broad range of cancers (gliomas, prostate, breast, endometrium…), the mechanisms by which it is regulated are far from being completely elucidated. Using a two-hybrid approach, our team identified that the molecular scaffolds, β-arrestins (β-arrs), interact with PTEN.Our studies demonstrate that β-arrs modulate distinct functional outputs of PTEN that in turn are dependent or independent on its lipid phosphatase activity. β-arrs increase the lipid phosphatase activity of PTEN in vitro. The small GTPase RhoA and its downstream effector ROCK activate PTEN and this effect requires β-arrs. The stimulation of the lysophosphatidic acid receptor 1 (LPA1-R) receptor, that activates the RhoA/ROCK pathway, was found to increase the association of β-arrs with PTEN and induced plasma membrane translocation of the complex. Through their stimulatory effect on the lipid phosphatase activity of PTEN, β-arrs inhibit the PI3K/Akt pathway and proliferation of mouse embryonic fibroblasts. In contrast, in U373 glioma cells, βarrs release the brake on cell migration, which is mediated by the C2 domain of PTEN independently of its lipid phosphatase activity. Following wounding of a cell monolayer, and RhoA activation, β-arrs show increased association with PTEN, and rescue glioma cell migration. β-arrs therefore differentially regulate functions of PTEN important in the control of cell proliferation and migration.The activity and localization of PTEN are under tight control of intramolecular interactions between its regulatory C-terminal tail, and catalytic and C2 domains. These intramolecular interactions regulate a switch between a closed form of PTEN, and an open and active form that is targeted to the membrane. We have developed a resonance energy transfer (RET)-based biosensor that permits the monitoring of PTEN conformational change in live cells. Using the biosensor we demonstrate that mutation of residues implicated in the intramolecular switch produce conformational rearrangement of PTEN, detected by changes in RET. Furthermore, activation of signalling pathways known to activate PTEN, elicit conformational changes that parallel increased PTEN lipid phosphatase activity in living cells. Combined, these data demonstrate that the biosensor can be used as a tool to detect changes in PTEN tumour suppressor activity in live cells.The tumour suppressor/oncogene PTEN/PI3K/Akt axis plays a key role in tumour progression and represents a major therapeutic target in the treatment of cancer. Our studies help to further our understanding of how tumour suppressor PTEN is controlled by inter- and intramolecular interactions and provide a biosensor that can report changes in PTEN activity

Fonctions de la protéine suppresseur de tumeurs PTEN (régulation par les b-arrestines et par l'interaction intramoléculaire)

La protéine suppresseur de tumeurs PTEN (Phosphatase and tensin deleted on chromosome 10) est une phosphatase lipidique. En déphosphorylant le phosphatidylinositol (3,4,5) trisphosphate (PIP3) en PI(4,5) P2, PTEN contre-régule la voie PI3K/Akt et inhibe la prolifération. D autres fonctions de PTEN peuvent être indépendantes de son activité phosphatase lipidique, notamment l inhibition de la migration. Bien que PTEN soit, après p53, le suppresseur de tumeurs le plus muté dans un large panel de cancers (gliomes, prostate, sein, endomètre ), les mécanismes par lesquels ses fonctions sont régulées ne sont pas entièrement élucidés. Par une approche de double-hybride, notre équipe a identifié que les -arrestines ( -arrs), des protéines d échafaudage, interagissent avec PTEN. Nos travaux mettent en évidence que l interaction entre PTEN et les -arrs permet de moduler ses deux activités dépendantes ou non de son activité phosphatase lipidique. D une part, les -arrs augmentent l activité phosphatase lipidique de PTEN in vitro. La GTPase RhoA et sa kinase d aval ROCK activent PTEN, et ceci se fait par l intermédiaire des -arrs. La stimulation du récepteur à l acide lysophosphatidique (LPA), qui active la voie RhoA/ROCK, augmente la formation du complexe PTEN/ -arrs et permet le recrutement du complexe à la membrane. Par l effet positif sur l activité phosphatase lipidique de PTEN, les -arrs participent à l inhibition d Akt et de la prolifération dans les fibroblastes embryonnaires de souris (MEF). A l inverse dans les gliomes U373, les -arrs lèvent l inhibition de la migration exercée par le domaine C2 de PTEN, indépendamment de son activité phosphatase lipidique. En aval de l activation de RhoA induite par blessure du tapis cellulaire, les -arrs interagissent davantage avec PTEN et rétablissent la migration des gliomes. De ce fait, les -arrs régulent différentiellement les fonctions de PTEN importantes pour le contrôle de la prolifération cellulaire et la migration. Enfin, l activité et la localisation de PTEN sont modulées par des interactions intramoléculaires entre ses domaines catalytiques, C2 et sa queue C-terminale régulatrice. Ces interactions régulent le passage d une conformation fermée vers une conformation ouverte et active de PTEN. Grâce au développement d un biosenseur de PTEN basé sur le transfert d énergie par résonnance (RET), nous pouvons suivre pour la première fois les changements conformationnels de PTEN dans les cellules vivantes. En utilisant ce biosenseur nous montrons que la mutation des résidus impliqués dans les interactions intramoléculaires entraine des changements de conformation détectés par des variations de RET. De plus, l activation de voies de signalisation connues pour activer PTEN, entrainent des changements conformationnels qui corrèlent avec l augmentation de l activité phosphatase lipidique de PTEN. Nos données montrent que le biosenseur peut être utilisé comme outil pour détecter les changements d activité de PTEN dans les cellules vivantes. L axe suppresseur de tumeurs/oncogène PTEN/PI3K/Akt joue un rôle essentiel dans la progression tumorale et constitue une cible thérapeutique pour le cancer. L ensemble de nos travaux permet d ajouter un degré de compréhension dans la régulation de PTEN, tant par les -arrs que par l interaction intramoléculaire et les changements conformationnels.The Tumour Suppressor protein PTEN (Phosphatase and tensin deleted on chromosome 10) is a lipid phosphatase. By converting phosphatidylinositol (3,4,5) trisphosphate (PIP3) to PI(4,5)P2, PTEN inhibits the PI3K/Akt signalling pathway and cell proliferation. Other functions attributed to PTEN, including the inhibition of cell migration, can occur independently of its lipid phosphatase activity. Although PTEN function is dysregulated in a broad range of cancers (gliomas, prostate, breast, endometrium ), the mechanisms by which it is regulated are far from being completely elucidated. Using a two-hybrid approach, our team identified that the molecular scaffolds, -arrestins ( -arrs), interact with PTEN.Our studies demonstrate that -arrs modulate distinct functional outputs of PTEN that in turn are dependent or independent on its lipid phosphatase activity. -arrs increase the lipid phosphatase activity of PTEN in vitro. The small GTPase RhoA and its downstream effector ROCK activate PTEN and this effect requires -arrs. The stimulation of the lysophosphatidic acid receptor 1 (LPA1-R) receptor, that activates the RhoA/ROCK pathway, was found to increase the association of -arrs with PTEN and induced plasma membrane translocation of the complex. Through their stimulatory effect on the lipid phosphatase activity of PTEN, -arrs inhibit the PI3K/Akt pathway and proliferation of mouse embryonic fibroblasts. In contrast, in U373 glioma cells, -arrs release the brake on cell migration, which is mediated by the C2 domain of PTEN independently of its lipid phosphatase activity. Following wounding of a cell monolayer, and RhoA activation, -arrs show increased association with PTEN, and rescue glioma cell migration. -arrs therefore differentially regulate functions of PTEN important in the control of cell proliferation and migration.The activity and localization of PTEN are under tight control of intramolecular interactions between its regulatory C-terminal tail, and catalytic and C2 domains. These intramolecular interactions regulate a switch between a closed form of PTEN, and an open and active form that is targeted to the membrane. We have developed a resonance energy transfer (RET)-based biosensor that permits the monitoring of PTEN conformational change in live cells. Using the biosensor we demonstrate that mutation of residues implicated in the intramolecular switch produce conformational rearrangement of PTEN, detected by changes in RET. Furthermore, activation of signalling pathways known to activate PTEN, elicit conformational changes that parallel increased PTEN lipid phosphatase activity in living cells. Combined, these data demonstrate that the biosensor can be used as a tool to detect changes in PTEN tumour suppressor activity in live cells.The tumour suppressor/oncogene PTEN/PI3K/Akt axis plays a key role in tumour progression and represents a major therapeutic target in the treatment of cancer. Our studies help to further our understanding of how tumour suppressor PTEN is controlled by inter- and intramolecular interactions and provide a biosensor that can report changes in PTEN activity.PARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

A estimulação russa no fortalecimento da musculatura abdominal Russian stimulation in strengthening abdominal muscle

INTRODUÇÃO - A flacidez muscular surge com maior frequência nas mulheres, o que causa fator ruim para a estética corporal. OBJETIVO - Analisar os resultados da corrente russa no fortalecimento da musculatura abdominal. MÉTODOS - Revisão bibliográfica com base nas publicações acessíveis pelas seguintes bases de dados: Medline/Pubmed, Scielo, Lilacs com cruzamento dos descritores corrente russa, flacidez, abdômen. O uso da eletroestimulação age tanto sobre as fibras brancas, que respondem pela velocidade, como também sobre as fibras vermelhas dado à sua sustentação, e ainda sobre as fibras intermediárias. CONCLUSÃO - Os dados publicados mostram a satisfação e êxito do tratamento, enfatizando que a corrente russa favorece o aumento da hipertrofia e força muscular.<br>INTRODUCTION - Muscle weakness appears most often in women, the factor that causes bad esthetics. OBJECTIVE - To analyze the results of the Russian current strengthening the abdominal muscles. METHODS - Literature review based on publications available in the following databases: Medline / Pubmed, Scielo, Lilacs with crossing headings Russian current, sagging, abdomen. The use of electrical stimulation acts both on the white fibers, which account for the speed, but also on the red fibers given their support, and on intermediate fibers. CONCLUSION - The data published show the satisfaction and success of treatment, emphasizing that the Russian current promotes increase of muscle strength and hypertrophy

PROSPECÇÃO TECNOLÓGICA DO JABORANDI (Pilocarpus microphyllus): ESPÉCIE ECONOMICAMENTE IMPORTANTE NO NORTE E NORDESTE DO BRASIL

A espécie Pilocarpus microphyllus Stapf ex Wardlew é uma planta nativa do Norte e Nordeste do Brasil de ocorrência no leste do Estado do Pará, oeste e norte do Maranhão e ao norte do Piauí. É uma das espécies medicinais brasileiras de maior destaque constando em farmacopéias de todo o mundo, devido ao uso de seu princípio ativo pilocarpina. Ao utilizar a base de periódicos Web of Science com o gênero Pilocarpus, o resultado foi de 56 artigos e para a espécie Pilocarpus microphyllus o resultado encontrado foi de 17 artigos. A busca por patentes foi utilizando a base de patentes Derwent Innovation Index, INPI, EPO e USPTO obtendo um resultado contrastante com o número de artigos, considerando o Japão em destaque na proteção da pesquisa envolvendo Pilocarpus. O gênero Pilocarpus com ênfase na espécie Pilcarpus microphyllus por meio dos artigos publicados e das patentes concedidas tanto no Brasil como em outros países destaca a relevante importância econômica e industrial da espécie bem como estabelece perspectivas futuras na descoberta de novas aplicações farmacológicas e biotecnológicas dos outros alcalóides que até o momento permanecem desconhecidas.</em

Targeting Spare CC Chemokine Receptor 5 (CCR5) as a Principle to Inhibit HIV-1 Entry.

International audience: CCR5 binds the chemokines CCL3, CCL4, and CCL5 and is the major coreceptor for HIV-1 entry into target cells. Chemokines are supposed to form a natural barrier against human immunodeficiency virus, type 1 (HIV-1) infection. However, we showed that their antiviral activity is limited by CCR5 adopting low-chemokine affinity conformations at the cell surface. Here, we investigated whether a pool of CCR5 that is not stabilized by chemokines could represent a target for inhibiting HIV infection. We exploited the characteristics of the chemokine analog PSC-RANTES (N-α-(n-nonanoyl)-des-Ser(1)-[l-thioprolyl(2), l-cyclohexylglycyl(3)]-RANTES(4-68)), which displays potent anti-HIV-1 activity. We show that native chemokines fail to prevent high-affinity binding of PSC-RANTES, analog-mediated calcium release (in desensitization assays), and analog-mediated CCR5 internalization. These results indicate that a pool of spare CCR5 may bind PSC-RANTES but not native chemokines. Improved recognition of CCR5 by PSC-RANTES may explain why the analog promotes higher amounts of β-arrestin 2*CCR5 complexes, thereby increasing CCR5 down-regulation and HIV-1 inhibition. Together, these results highlight that spare CCR5, which might permit HIV-1 to escape from chemokines, should be targeted for efficient viral blockade

Administration of Hypoxia-Activated Prodrug Evofosfamide after Conventional Adjuvant Therapy Enhances Therapeutic Outcome and Targets Cancer-Initiating Cells in Preclinical Models of Colorectal Cancer.

Purpose: Cancer-initiating cells (C-IC) have been described in multiple cancer types, including colorectal cancer. C-ICs are defined by their capacity to self-renew, thereby driving tumor growth. C-ICs were initially thought to be static entities; however, recent studies have determined these cells to be dynamic and influenced by microenvironmental cues such as hypoxia. If hypoxia drives the formation of C-ICs, then therapeutic targeting of hypoxia could represent a novel means to target C-ICs. Experimental Design: Patient-derived colorectal cancer xenografts were treated with evofosfamide, a hypoxia-activated prodrug (HAP), in combination with 5-fluorouracil (5-FU) or chemoradiotherapy (5-FU and radiation; CRT). Treatment groups included both concurrent and sequential dosing regimens. Effects on the colorectal cancer-initiating cell (CC-IC) fraction were assessed by serial passage in vivo limiting dilution assays. FAZA-PET imaging was utilized as a noninvasive method to assess intratumoral hypoxia. Results: Hypoxia was sufficient to drive the formation of CC-ICs and colorectal cancer cells surviving conventional therapy were more hypoxic and C-IC-like. Using a novel approach to combination therapy, we show that sequential treatment with 5-FU or CRT followed by evofosfamide not only inhibits tumor growth of xenografts compared with 5-FU or CRT alone, but also significantly decreases the CC-IC fraction. Furthermore, noninvasive FAZA-PET hypoxia imaging was predictive of a tumor's response to evofosfamide. Conclusions: Our data demonstrate a novel means to target the CC-IC fraction by adding a HAP sequentially after conventional adjuvant therapy, as well as the use of FAZA-PET as a biomarker for hypoxia to identify tumors that will benefit most from this approach. (C) 2018 AACR