Illuminating the life of GPCRs

The investigation of biological systems highly depends on the possibilities that allow scientists to visualize and quantify biomolecules and their related activities in real-time and non-invasively. G-protein coupled receptors represent a family of very dynamic and highly regulated transmembrane proteins that are involved in various important physiological processes. Since their localization is not confined to the cell surface they have been a very attractive "moving target" and the understanding of their intracellular pathways as well as the identified protein-protein-interactions has had implications for therapeutic interventions. Recent and ongoing advances in both the establishment of a variety of labeling methods and the improvement of measuring and analyzing instrumentation, have made fluorescence techniques to an indispensable tool for GPCR imaging. The illumination of their complex life cycle, which includes receptor biosynthesis, membrane targeting, ligand binding, signaling, internalization, recycling and degradation, will provide new insights into the relationship between spatial receptor distribution and function. This review covers the existing technologies to track GPCRs in living cells. Fluorescent ligands, antibodies, auto-fluorescent proteins as well as the evolving technologies for chemical labeling with peptide- and protein-tags are described and their major applications concerning the GPCR life cycle are presented

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Deciphering a new role for the transcriptional coregulator RIP140 in the regulation of glucose metabolism and differentiation of cancer cells

La reprogrammation métabolique est une caractéristique des cellules cancéreuses qui sont capables, entre autres, de passer d’un métabolisme aérobie (phosphorylation oxydative) à un métabolisme anaérobie (glycolyse) en fonction de leurs besoins. Ceci leur permet de survivre dans des environnements pauvres en nutriments et de résister à certains traitements anti-cancéreux. En effet, les rechutes fréquentes des leucémies aiguës myéloïdes (LAM) peuvent être dues par une reprogrammation métabolique favorisant la phosphorylation oxydative. Expliquer les mécanismes responsables de la reprogrammation métabolique des cellules cancéreuses pourrait conduire au développement de nouvelles thérapies contre le cancer. Mon projet s’intéresse au rôle du corégulateur transcriptionnel RIP140 dans la flexibilité métabolique et la différenciation des cellules cancéreuses. RIP140 (Receptor Interacting Protein of 140 Kda) est un corégulateur transcriptionnel qui participe à la régulation de l’expression de gènes cibles de nombreux facteurs de transcription (récepteurs nucléaires, facteurs AP-1, SP1, NF-KB et E2F1, etc.). Grace à son large interactome, RIP140 est impliqué dans la régulation de nombreux processus physiologiques et physiopathologiques comme le métabolisme ou le cancer respectivement. Nous nous sommes donc intéressés au rôle potentiel de RIP140 dans le contrôle du métabolisme dans un contexte cancéreux. Nous avons démontré que la perte d’expression de RIP140 dans des fibroblastes embryonnaires murins transformés (MEF RIPKO) induit une augmentation de la prolifération cellulaire, de la croissance tumorale ainsi qu’une augmentation de la glycolyse. De plus, l’avantage prolifératif des MEF RIPKO est abrogé en absence de glucose ou en présence d’inhibiteurs de la glycolyse in vitro et in vivo dans des expériences de xénogreffes. Enfin, l’expression des gènes de la glycolyse GLUT3 et G6PD est augmentée dans les MEF RIPKO et ses gènes sont nécessaires à la prolifération des cellules RIPKO. Nous avons confirmé l’ensemble de ces résultats dans différentes lignées cancéreuses mammaires, montrant un effet général de RIP140 sur le métabolisme glucidique des cellules cancéreuses. Des expériences d’immunoprécipitation de la chromatine montrent la présence de RIP140 au promoteur de GLUT3. Nous avons décrypté le mécanisme moléculaire responsable des effets de RIP140 qui implique le gène suppresseur de tumeur p53 et le facteur HIF-2α (Hypoxia Inducible Factor 2α). Nos données établissent pour la première fois RIP140 comme un modulateur critique de l’interférence entre les facteurs HIF et p53 dans le contrôle du métabolisme du glucose dans les cellules cancéreuses mammaires. En parallèle, je me suis intéressé au rôle de RIP140 dans la chimiorésistance des leucémies aiguës myéloïdes (LAM). Nous montrons qu’à l’inverse des tumeurs solides, une forte expression de RIP140 est associée à un mauvais pronostic dans les LAM. La modulation de l’expression de RIP140 grâce à l’utilisation de shRNA dans des lignées de LAM induit une modification de la prolifération par induction de l’apoptose. Des données de RNAseq établies dans une lignée d’AML dont l’expression de RIP140 a été inhibée par rapport au contrôle ont montrées que RIP140 est impliqué dans la régulation de l’apoptose, du cycle cellulaire, de la phosphorylation oxydative et du métabolisme de l’acide rétinoïque. Nous nous sommes donc intéressés à la sensibilité des cellules de LAM à l’all trans retinoic acid (ATRA), une molécule induisant la différenciation, en fonction de l’expression de RIP140. Nous avons démontré que l’inhibition de l’expression de RIP140 potentialise les effets de l’ATRA tant au niveau prolifératif qu’au niveau des marqueurs de différentiation. A terme, ce facteur pourrait être proposé comme marqueur prédictif de la réponse aux agents différentiant pour cette pathologie.Metabolic reprogramming is a hallmark of cancer cells that are capable, among other things, of going from an aerobic metabolism (oxidative phosphorylation) to an anaerobic metabolism (glycolysis) according to their needs. This allows them to survive in nutrient-poor environments and to resist to certain anti-cancer treatments. Explaining the mechanisms responsible for the metabolic reprogramming of cancer cells could lead to the development of new cancer therapies. My thesis project aims to decipher the role of the transcriptional coregulator RIP140 in cancer cell metabolic flexibility and chemoresistance. RIP140 (Receptor Interacting Protein of 140 Kda) is a trancriptionnal coregulator that interact with various transcription factors (nuclear receptor, factors such as AP-1, SP1, NF-KB et E2F1, etc.) in order to regulate gene expression. Thanks to its large interactome, it is implicated in the regulation of various physiological and pathophysiological processes such as metabolism or cancer, respectively. In this context we were interested in the potential role of RIP140 control in cancer metabolism. We demonstrate that the loss of RIP140 expression in transformed mouse embryonic fibroblasts (MEF RIPKO) increase cell proliferation, tumor growth and glycolysis. Moreover, this proliferative advantage is abrogated by glucose deprivation or glycolysis inhibition with pharmacological agent in vitro and in vivo in xenografted mice. We also show that glycolysis gene such as GLUT3 or G6PD are upregulated in RIPKO MEF and that cell proliferation is dependent of their expression. We confirmed those results in cancer cell lines showing a general effect of RIP140 on cancer cells metabolism. Chromatin immunoprecipitation experiment show that RIP140 is present at GLUT3 gene promotor. Our data establish for the first time RIP140 as a critical modulator of the transcriptional interplay between HIF2α (Hypoxia Inducible Factor 2α) and p53 in the control of glucose metabolism in cancer cells. In parallel, I was interested in the role of RIP140 in the chemoresistance of acute myeloid leukemia (AML) whose frequent relapses can be due to a metabolic reprogramming enhancing oxidative phosphorylation metabolism. We show that, in contrast to solid tumors, high expression of RIP140 is associated with a poor prognosis in AML. Modulation of RIP140 expression in AML cell lines using shRNA induces a decrease of proliferation via apoptosis control. RNAseq data established on OCI-AML2 where RIP140 expression was reduce compared to control show that RIP140 is implicated in the regulation of apoptosis, cell cycle, oxidative phosphorylation and retinoic acid metabolism. The next step was to test AML cells sensitivity to all trans retinoic acid (ATRA), a differentiating agent, when RIP140 expression was depressed. We show that RIP140 inhibition potentiates ATRA effects on AML cells. The overall results will determine the involvement of RIP140 in AML chemoresistance. At the end, this factor could be proposed as a predictive marker of response to treatments for this pathology

Etude du rôle du corégulateur transcriptionnel RIP140 dans le métabolisme glucidique et la différenciation des cellules cancéreuses

Metabolic reprogramming is a hallmark of cancer cells that are capable, among other things, of going from an aerobic metabolism (oxidative phosphorylation) to an anaerobic metabolism (glycolysis) according to their needs. This allows them to survive in nutrient-poor environments and to resist to certain anti-cancer treatments. Explaining the mechanisms responsible for the metabolic reprogramming of cancer cells could lead to the development of new cancer therapies. My thesis project aims to decipher the role of the transcriptional coregulator RIP140 in cancer cell metabolic flexibility and chemoresistance. RIP140 (Receptor Interacting Protein of 140 Kda) is a trancriptionnal coregulator that interact with various transcription factors (nuclear receptor, factors such as AP-1, SP1, NF-KB et E2F1, etc.) in order to regulate gene expression. Thanks to its large interactome, it is implicated in the regulation of various physiological and pathophysiological processes such as metabolism or cancer, respectively. In this context we were interested in the potential role of RIP140 control in cancer metabolism. We demonstrate that the loss of RIP140 expression in transformed mouse embryonic fibroblasts (MEF RIPKO) increase cell proliferation, tumor growth and glycolysis. Moreover, this proliferative advantage is abrogated by glucose deprivation or glycolysis inhibition with pharmacological agent in vitro and in vivo in xenografted mice. We also show that glycolysis gene such as GLUT3 or G6PD are upregulated in RIPKO MEF and that cell proliferation is dependent of their expression. We confirmed those results in cancer cell lines showing a general effect of RIP140 on cancer cells metabolism. Chromatin immunoprecipitation experiment show that RIP140 is present at GLUT3 gene promotor. Our data establish for the first time RIP140 as a critical modulator of the transcriptional interplay between HIF2α (Hypoxia Inducible Factor 2α) and p53 in the control of glucose metabolism in cancer cells. In parallel, I was interested in the role of RIP140 in the chemoresistance of acute myeloid leukemia (AML) whose frequent relapses can be due to a metabolic reprogramming enhancing oxidative phosphorylation metabolism. We show that, in contrast to solid tumors, high expression of RIP140 is associated with a poor prognosis in AML. Modulation of RIP140 expression in AML cell lines using shRNA induces a decrease of proliferation via apoptosis control. RNAseq data established on OCI-AML2 where RIP140 expression was reduce compared to control show that RIP140 is implicated in the regulation of apoptosis, cell cycle, oxidative phosphorylation and retinoic acid metabolism. The next step was to test AML cells sensitivity to all trans retinoic acid (ATRA), a differentiating agent, when RIP140 expression was depressed. We show that RIP140 inhibition potentiates ATRA effects on AML cells. The overall results will determine the involvement of RIP140 in AML chemoresistance. At the end, this factor could be proposed as a predictive marker of response to treatments for this pathology.La reprogrammation métabolique est une caractéristique des cellules cancéreuses qui sont capables, entre autres, de passer d’un métabolisme aérobie (phosphorylation oxydative) à un métabolisme anaérobie (glycolyse) en fonction de leurs besoins. Ceci leur permet de survivre dans des environnements pauvres en nutriments et de résister à certains traitements anti-cancéreux. En effet, les rechutes fréquentes des leucémies aiguës myéloïdes (LAM) peuvent être dues par une reprogrammation métabolique favorisant la phosphorylation oxydative. Expliquer les mécanismes responsables de la reprogrammation métabolique des cellules cancéreuses pourrait conduire au développement de nouvelles thérapies contre le cancer. Mon projet s’intéresse au rôle du corégulateur transcriptionnel RIP140 dans la flexibilité métabolique et la différenciation des cellules cancéreuses. RIP140 (Receptor Interacting Protein of 140 Kda) est un corégulateur transcriptionnel qui participe à la régulation de l’expression de gènes cibles de nombreux facteurs de transcription (récepteurs nucléaires, facteurs AP-1, SP1, NF-KB et E2F1, etc.). Grace à son large interactome, RIP140 est impliqué dans la régulation de nombreux processus physiologiques et physiopathologiques comme le métabolisme ou le cancer respectivement. Nous nous sommes donc intéressés au rôle potentiel de RIP140 dans le contrôle du métabolisme dans un contexte cancéreux. Nous avons démontré que la perte d’expression de RIP140 dans des fibroblastes embryonnaires murins transformés (MEF RIPKO) induit une augmentation de la prolifération cellulaire, de la croissance tumorale ainsi qu’une augmentation de la glycolyse. De plus, l’avantage prolifératif des MEF RIPKO est abrogé en absence de glucose ou en présence d’inhibiteurs de la glycolyse in vitro et in vivo dans des expériences de xénogreffes. Enfin, l’expression des gènes de la glycolyse GLUT3 et G6PD est augmentée dans les MEF RIPKO et ses gènes sont nécessaires à la prolifération des cellules RIPKO. Nous avons confirmé l’ensemble de ces résultats dans différentes lignées cancéreuses mammaires, montrant un effet général de RIP140 sur le métabolisme glucidique des cellules cancéreuses. Des expériences d’immunoprécipitation de la chromatine montrent la présence de RIP140 au promoteur de GLUT3. Nous avons décrypté le mécanisme moléculaire responsable des effets de RIP140 qui implique le gène suppresseur de tumeur p53 et le facteur HIF-2α (Hypoxia Inducible Factor 2α). Nos données établissent pour la première fois RIP140 comme un modulateur critique de l’interférence entre les facteurs HIF et p53 dans le contrôle du métabolisme du glucose dans les cellules cancéreuses mammaires. En parallèle, je me suis intéressé au rôle de RIP140 dans la chimiorésistance des leucémies aiguës myéloïdes (LAM). Nous montrons qu’à l’inverse des tumeurs solides, une forte expression de RIP140 est associée à un mauvais pronostic dans les LAM. La modulation de l’expression de RIP140 grâce à l’utilisation de shRNA dans des lignées de LAM induit une modification de la prolifération par induction de l’apoptose. Des données de RNAseq établies dans une lignée d’AML dont l’expression de RIP140 a été inhibée par rapport au contrôle ont montrées que RIP140 est impliqué dans la régulation de l’apoptose, du cycle cellulaire, de la phosphorylation oxydative et du métabolisme de l’acide rétinoïque. Nous nous sommes donc intéressés à la sensibilité des cellules de LAM à l’all trans retinoic acid (ATRA), une molécule induisant la différenciation, en fonction de l’expression de RIP140. Nous avons démontré que l’inhibition de l’expression de RIP140 potentialise les effets de l’ATRA tant au niveau prolifératif qu’au niveau des marqueurs de différentiation. A terme, ce facteur pourrait être proposé comme marqueur prédictif de la réponse aux agents différentiant pour cette pathologie

Trendsetter for companies and industrial sites: The EU Emissions Trading Scheme

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The Transcription Coregulator RIP140 Inhibits Cancer Cell Proliferation by Targeting the Pentose Phosphate Pathway

International audienceCancer cells switch their metabolism toward glucose metabolism to sustain their uncontrolled proliferation. Consequently, glycolytic intermediates are diverted into the pentose phosphate pathway (PPP) to produce macromolecules necessary for cell growth. The transcription regulator RIP140 controls glucose metabolism in tumor cells, but its role in cancer-associated reprogramming of cell metabolism remains poorly understood. Here, we show that, in human breast cancer cells and mouse embryonic fibroblasts, RIP140 inhibits the expression of the gene-encoding G6PD, the first enzyme of the PPP. RIP140 deficiency increases G6PD activity as well as the level of NADPH, a reducing cofactor essential for macromolecule synthesis. Moreover, G6PD knock-down inhibits the gain of proliferation observed when RIP140 expression is reduced. Importantly, RIP140-deficient cells are more sensitive to G6PD inhibition in cell proliferation assays and tumor growth experiments. Altogether, this study describes a novel role for RIP140 in regulating G6PD levels, which links its effect on breast cancer cell proliferation to metabolic rewiring

Splicing efficiency of minor introns in a mouse model of SMA predominantly depends on their branchpoint sequence and can involve the contribution of major spliceosome components

International audienceSpinal muscular atrophy (SMA) is a devastating neurodegenerative disease caused by reduced amounts of the ubiquitously expressed Survival of Motor Neuron (SMN) protein. In agreement with its crucial role in the biogenesis of spliceosomal snRNPs, SMN-deficiency is correlated to numerous splicing alterations in patient cells and various tissues of SMA mouse models. Among the snRNPs whose assembly is impacted by SMN-deficiency, those involved in the minor spliceosome are particularly affected. Importantly, splicing of several, but not all U12-dependent introns has been shown to be affected in different SMA models. Here, we have investigated the molecular determinants of this differential splicing in spinal cords from SMA mice. We show that the branchpoint sequence (BPS) is a key element controlling splicing efficiency of minor introns. Unexpectedly, splicing of several minor introns with suboptimal BPS is not affected in SMA mice. Using in vitro splicing experiments and oligonucleotides targeting minor or major snRNAs, we show for the first time that splicing of these introns involves both the minor and major machineries. Our results strongly suggest that splicing of a subset of minor introns is not affected in SMA mice because components of the major spliceosome compensate for the loss of minor splicing activity

RIP140 inhibits glycolysis-dependent proliferation of breast cancer cells by regulating GLUT3 expression through transcriptional crosstalk between hypoxia induced factor and p53

International audienceGlycolysis is essential to support cancer cell proliferation, even in the presence of oxygen. The transcriptional co-regulator RIP140 represses the activity of transcription factors that drive cell proliferation and metabolism and plays a role in mammary tumorigenesis. Here we use cell proliferation and metabolic assays to demonstrate that RIP140-deficiency causes a glycolysis-dependent increase in breast tumor growth. We further demonstrate that RIP140 reduces the transcription of the glucose transporter GLUT3 gene, by inhibiting the transcriptional activity of hypoxia inducible factor HIF-2α in cooperation with p53. Interestingly, RIP140 expression was significantly associated with good prognosis only for breast cancer patients with tumors expressing low GLUT3, low HIF-2α and high p53, thus confirming the mechanism of RIP140 anti-tumor activity provided by our experimental data. Overall, our work establishes RIP140 as a critical modulator of the p53/HIF cross-talk to inhibit breast cancer cell glycolysis and proliferation

Monitoring, reporting and verifying emissions in the climate economy

The monitoring, reporting and verification (MRV) of greenhouse-gas emissions is the cornerstone of carbon pricing and management mechanisms. Here we consider peer-reviewed articles and ‘grey literature’ related to existing MRV requirements and their costs. A substantial part of the literature is the regulatory texts of the 15 most important carbon pricing and management mechanisms currently implemented. Based on a comparison of key criteria such as the scope, cost, uncertainty and flexibility of procedures, we conclude that conventional wisdom on MRV is not often promoted in existing carbon pricing mechanisms. Quantification of emissions uncertainty and incentives to reduce this uncertainty are usually only partially applied, if at all. Further, the time and resources spent on small sources of emissions would be expected to be limited. Although provisions aiming at an effort proportionate to the amount of emissions at stake — ‘materiality’ — are widespread, they are largely outweighed by economies of scale: in all schemes, MRV costs per tonne are primarily driven by the size of the source

Synthesis

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