Bernard Monsarrat - Academia.edu (original) (raw)

Papers by Bernard Monsarrat

Molecular & Cellular Proteomics, 2012

The proteasome, a proteolytic complex present in all eu-karyotic cells, is part of the ATP-depend... more The proteasome, a proteolytic complex present in all eu-karyotic cells, is part of the ATP-dependent ubiquitin/ proteasome pathway. It plays a critical role in the regula-tion of many physiological processes. The 20 S proteasome, the catalytic core of the 26 S proteasome, is made of four stacked rings of seven subunits each (7777). Here we studied the human 20 S proteasome using proteomics. This led to the establishment of a fine subunit reference map and to the identification of post-translational modifications. We found that the human 20 S proteasome, purified from erythrocytes, exhibited a high degree of structural heterogeneity, characterized by the presence of multiple isoforms for most of the and subunits, including the catalytic ones, resulting in a total of at least 32 visible spots after Coomassie Blue staining.

$Research paper thumbnail of {"__content__"=>"The costimulatory molecule CD226 signals through VAV1 to amplify TCR signals and promote IL-17 production by CD4 T cells.", "sup"=>{"__content__"=>"+"}}$

Science signaling, Jan 10, 2018

The activation of T cells requires the guanine nucleotide exchange factor VAV1. Using mice in whi... more The activation of T cells requires the guanine nucleotide exchange factor VAV1. Using mice in which a tag for affinity purification was attached to endogenous VAV1 molecules, we analyzed by quantitative mass spectrometry the signaling complex that assembles around activated VAV1. Fifty VAV1-binding partners were identified, most of which had not been previously reported to participate in VAV1 signaling. Among these was CD226, a costimulatory molecule of immune cells. Engagement of CD226 induced the tyrosine phosphorylation of VAV1 and synergized with T cell receptor (TCR) signals to specifically enhance the production of interleukin-17 (IL-17) by primary human CD4 T cells. Moreover, co-engagement of the TCR and a risk variant of CD226 that is associated with autoimmunity (rs763361) further enhanced VAV1 activation and IL-17 production. Thus, our study reveals that a VAV1-based, synergistic cross-talk exists between the TCR and CD226 during both physiological and pathological T cell ...

Molecular and cellular biology, Jul 15, 2017

An error was made during the assembly of the figure that resulted in an inadvertent duplication i... more An error was made during the assembly of the figure that resulted in an inadvertent duplication in the second panel. The corrected image is shown below.

Scientific Reports, 2016

IL-33 is a nuclear cytokine from the IL-1 family that plays important roles in health and disease... more IL-33 is a nuclear cytokine from the IL-1 family that plays important roles in health and disease. Extracellular IL-33 activates a growing number of target cells, including group 2 innate lymphoid cells, mast cells and regulatory T cells, but it remains unclear whether intracellular nuclear IL-33 has additional functions in the nucleus. Here, we used a global proteomic approach based on high-resolution mass spectrometry to compare the extracellular and intracellular roles of IL-33 in primary human endothelial cells, a major source of IL-33 protein in human tissues. We found that exogenous extracellular IL-33 cytokine induced expression of a distinct set of proteins associated with inflammatory responses in endothelial cells. In contrast, knockdown of endogenous nuclear IL-33 expression using two independent RNA silencing strategies had no reproducible effect on the endothelial cell proteome. These results suggest that IL-33 acts as a cytokine but not as a nuclear factor regulating gene expression in endothelial cells. Interleukin-33 (IL-33) is a tissue-derived nuclear cytokine from the IL-1 family with critical roles in tissue homeostasis and repair, type 2 immunity, viral infection, inflammation and allergy 1-5. IL-33 binds to the ST2 receptor expressed on cells of the innate and adaptive immune system 1. Tissue-resident cells such as group 2 innate lymphoid cells (ILC2s), mast cells, and certain subsets of regulatory T cells, constitutively express high levels of ST2 and are major targets of IL-33 in vivo 6-14. Other targets of IL-33 include macrophages, dendritic cells, Th2 cells, eosinophils, basophils, NK and iNKT cells, neutrophils, Th1 cells and CD8 + T cells 4. Studies in humans and animal models suggest a critical role of IL-33 in many important diseases 3,4,12,14-20. The genes encoding IL-33 and ST2 have been reproducibly identified as major susceptibility loci for human asthma in several genome-wide association studies 3,15. IL-33 also appears to be important for other allergic diseases (allergic rhinitis, atopic dermatitis, allergic conjunctivitis), adipose tissue metabolism and obesity, and a variety of diseases associated with tissue injury and repair (myocardial infarction, stroke, wounding, microbial infection, hepatic and pulmonary fibrosis, systemic sclerosis, chronic obstructive pulmonary disease, autoimmune diseases and cancer) 3,4,12,16-20. Given these critical roles in health and disease, a good understanding of IL-33 biology and mode of action is crucial. IL-33 is constitutively expressed in the nuclei of producing cells during homeostasis, including epithelial cells from various barrier tissues, endothelial cells from blood vessels, fibroblastic reticular cells of lymphoid organs, and post-mitotic oligodendrocytes in the brain 21-24. Although already high during homeostasis, expression of IL-33 is further upregulated during inflammation, and the protein can be produced by additional cell types 3,4,23,25. Full length IL-33 is biologically active and it can be released from the nucleus of producing cells after cellular damage or necrotic cell death 26,27. It was thus proposed to function as an alarm signal (alarmin) that alerts immune cells of tissue damage 21,26,27. IL-33 cytokine activity is regulated by nuclear compartmentalization or sequestration 28 and proteolytic maturation 3. During apoptosis, IL-33 is inactivated by caspases that cleave the protein within the IL-1-like cytokine domain 26,27. During inflammation, IL-33 is processed in the central activation domain by inflammatory proteases from mast cells and neutrophils, that generate mature forms of the protein with 10 to 30 fold higher biological activity 29,30. Moreover, mature forms of IL-33 are rapidly inactivated (< 2 h) in the extracellular environment by oxidation of critical cysteine residues 31 .

PROTEOMICS, 2016

The ubiquitin-proteasome pathway (UPP) plays a critical role in the degradation of proteins impli... more The ubiquitin-proteasome pathway (UPP) plays a critical role in the degradation of proteins implicated in cell cycle control, signal transduction, DNA damage response, apoptosis and immune response. Proteasome inhibitors can inhibit the growth of a broad spectrum of human cancer cells by altering the balance of intracellular proteins. However, the targets of these compounds in acute myeloid leukemia (AML) cells have not been fully characterized. Herein, we combined large-scale quantitative analysis by SILAC-MS and targeted quantitative proteomic analysis in order to identify proteins regulated upon proteasome inhibition in two AML cell lines displaying different stages of maturation: immature KG1a cells and mature U937 cells. Indepth data analysis enabled accurate quantification of more than 7000 proteins in these two cell lines. Several candidates were validated by selected reaction monitoring (SRM) measurements in a large number of samples. Despite the broad range of proteins known to be affected by proteasome inhibition, such as heat shock (HSP) and cell cycle proteins, our analysis identified new differentially regulated proteins, including IL-32, MORF family mortality factors and apoptosis inducing factor SIVA, a target of p53. It could explain why proteasome inhibitors induce stronger apoptotic responses in immature AML cells.

Cancer Research, 1996

The metabolism of docetaxel by human liver microsomes was investi gated in vitro and compared wit... more The metabolism of docetaxel by human liver microsomes was investi gated in vitro and compared with that of paditaxel. A main docetaxel metabolite was generated by human liver microsomes in the presence of NADPH: retention time in high pressure liquid chromatography and its ion fragmentation in mass spectrometry were identical to those of the authentic derivative hydroxylated at the butyl group ofthe C53side chain. Kinetic measurements and chemical and Immunological inhibitions dem onstrated that CYP3A was implicated in the hydroxylation of docetaxel: Km (2 ELM)and Vm values of docetaxel for human liver microsomes were comparable to those calculated for the formation of metabolitep-hydroxy phenyl C3' paditaxel (M4). Docetaxel hydroxylation correlated only with the CYP3A content of microsomes and with CYP3A-dependent 6(1-hy. droxylation of testosterone and 16-hydroxylation of dehydroepiandros terone. The formation of hydroxydocetaxel was strongly reduced by CYP3A inhibitors such as ketoconazole, midaZOlam, erythromycin, tes tosterone, orphenadrine, and troleandomycin, whereas quinidine (CYP2D6), hexobarbital, tolbutamide, and mephenytoin (CYP2C) had no orlittle effect. The hydroxylation of docetaxel exhibited a highly positive correlation with the formation of metabolite M4 of paditaxel (r = 0.929, P < 0.0001, n 12), but not with its 6-hydroxylation (r 0.48, P > 0.15). Docetaxel abolished the hydroxylation of paclitaxel metabolite M4, but was totally inactive on its 6a-hydroxylation. Conversely, pacitaxel reduced signifi cantly the hydroxylation of docetaxel. We examined in vitro the possible interaction among docetaxel, paclitaxel, and drugs which could be ama dated during chemotherapy. Cisplatin, verapamil, doxorubicin, vinblas. tine, and vincristine at concentrations usually recommended did not markedly modify taxoid metabolism. Ranitidine and diphenylhydramine bad no effect, but 100 @LM cimetidine partially inhibited the formation of 6a.hydroxypaclitaxel. Pretreatment of patients with barbiturates strikingly stimulated do cetaxel hydroxylation, whereas no acceleration of docetaxel hydroxylation was noticed in a patient receiving steroids.

Bulletin Du Cancer, Feb 4, 1997

The metabolism of paclitaxel and docetaxel by human liver microsomes was investigated in vitro. T... more The metabolism of paclitaxel and docetaxel by human liver microsomes was investigated in vitro. The main metabolite of paclitaxel formed in vitro was the 6 alpha-hydroxypaclitaxel: its formation largely exceeded the formation of other metabolites hydroxylated on the lateral chain by rat liver microsomes and initially characterized in rat bile. In contrast, in vitro studied showed that the initial metabolite of docetaxel resulted from the hydroxylation of the tert-butyl of the lateral chain at C13 and that the same metabolites were formed in human and animal models. Comparison of individual CYP protein content of human microsomes and catalytic activities with taxoid biotransformation, showed that 2 distinct isoforms were assigned to the 6 alpha-hydroxylation (CYP2C) and to the hydroxylation of the lateral chain (CYP3A4). Chemical and immunological inhibitions confirmed these assumptions. The effect of antineoplastic drugs potentially associated with taxoids during chemotherapy has been tested in vitro on paclitaxel and docetaxel biotransformations. In the therapeutic range, vincristine, vinblastine, doxorubicine and cisplatin elicited a moderate or no inhibition of paclitaxel and docetaxel metabolism, as well as cimetidine, ranitidine and diphenylhydramine used to prevent major side effects associated with taxoid therapy. In patients given barbiturates, the hydroxylation on the lateral chain of paclitaxel and docetaxel was markedly stimulated and resulted from the induction of CYP3A isoforms. These results clearly demonstrated that the biotransformation of paclitaxel and docetaxel by human liver microsomes was supported by 2 distinct CYP proteins and that drug interactions could modify the therapeutic efficiency of taxoids during chemotherapy.

Oncogene, 2005

Rad51 protein plays an essential role in recombination repair of DNA double-strand breaks and DNA... more Rad51 protein plays an essential role in recombination repair of DNA double-strand breaks and DNA crosslinking adducts. It is part of complexes which can vary with the stage of the cell cycle and the nature of the DNA lesions. During a search for Rad51-associated proteins in CHO nuclear extracts of S-phase cells by mass spectrometry of proteins immunoprecipitated with Rad51

The biotransformation of taxol by human liver was investigated in vitro with microsomes isolated ... more The biotransformation of taxol by human liver was investigated in vitro with microsomes isolated from adult and developing human tissues. In vitro, no metabolism was detected with kidney microsomes, whereas two metabolites were generated by liver microsomes. The most prominent metabolite, termed M5, corresponded to an hydroxylation at the C6 position on the taxane ring, while the other metabolite, termed M4, corresponded to an hydroxylation at the para-posifion on the phenyi ring at the C3'-position of the C13 side chain. These two taxol derivatives have been shown to be the major metabolites recovered in bile from a patient infused with taxol. Several approaches have been used to identify the cytochrome P450 (CYP) isozymes involved in these reactions. No positive correlation was observed between the in vitro synthesis of these two metabolites, suggesting that two cytochrome P450 isozymes could be involved, although they could not be distinguished by their apparent affinities (Kin 15 pM). The formation of metabolite M4 was substantially reduced both by antibody directed against CYP3A and by the addition of CYP3A substrates such as orphenadrine, erythromycin, troleandomycin, and testosterone. Conversely, the formation of metabolite M5 remained unaffected by antibodies against CYP3A and by CYP3A substrates but was sensitive to diazepam inhibition, a preferential substrate of CYP2C. Correlation between CYP2C content or diazepam demethylation and the synthesis of metabolite M5 was highly positive. The formation of metabolite M4 developed during the early postnatal period. In contrast, the synthesis of metabolite M5 rose only after 3 months of age. These data clearly implicate CYP3A in the formation of metabolite M4 and CYP2C in the synthesis of metabolite M5. Microsomes from patients treated with barbiturates and benzodiazepines increased the formation of metabolite M4 to the level of metabolite M5, demonstrating that drug interactions could modify the human metabolism of taxol.

Nature Communications, 2016

Obesity favours the occurrence of locally disseminated prostate cancer in the periprostatic adipo... more Obesity favours the occurrence of locally disseminated prostate cancer in the periprostatic adipose tissue (PPAT) surrounding the prostate gland. Here we show that adipocytes from PPAT support the directed migration of prostate cancer cells and that this event is strongly promoted by obesity. This process is dependent on the secretion of the chemokine CCL7 by adipocytes, which diffuses from PPAT to the peripheral zone of the prostate, stimulating the migration of CCR3 expressing tumour cells. In obesity, higher secretion of CCL7 by adipocytes facilitates extraprostatic extension. The observed increase in migration associated with obesity is totally abrogated when the CCR3/CCL7 axis is inhibited. In human prostate cancer tumours, expression of the CCR3 receptor is associated with the occurrence of aggressive disease with extended local dissemination and a higher risk of biochemical recurrence, highlighting the potential benefit of CCR3 antagonists in the treatment of prostate cancer.

The Journal of Immunology, 2015

full#ref-list-1 , 20 of which you can access for free at: cites 46 articles This article average ... more full#ref-list-1 , 20 of which you can access for free at: cites 46 articles This article average * 4 weeks from acceptance to publication Fast Publication! • Every submission reviewed by practicing scientists No Triage! • from submission to initial decision Rapid Reviews! 30 days* • Submit online.

Molecular and Cellular Pharmacology, 2009

The metabolism of taxanes by human liver microsomes is regioselective: the major metabolite of pa... more The metabolism of taxanes by human liver microsomes is regioselective: the major metabolite of paclitaxel formed by CYP2C8 results from the hydroxylation on the taxane ring at C-6. Hydroxylations on the lateral chain at C-13 are catalyzed by CYP3A4 on either the tert-butyl of docetaxel or the C-3' phenyl of paclitaxel. Furthermore, the presence of the acetyl group in position 10, has been shown to play an important role in determining the regioselective oxidation by CYP. The biotransformation of a series of docetaxel analogues by human liver microsomes and recombinant CYP expressed in HEK293 cells was examined by high-performance liquid chromatography/mass spectrometry. The formation of derivatives was lost when the tertbutyl of docetaxel was replaced by an ethyl group. Addition of an aliphatic chain at either position 7 or 10 led to the formation of oxidized metabolites, whereas addition at both 7 and 10 totally impaired the production of derivatives. Similarly, the insertion of a phenylbenzoyl group in position 10 prevented the biotransformation of the molecule. The site of hydroxylation could be clearly located on the aliphatic chain inserted in position 10, but remained on the tert-butyl at C-13 when the aliphatic chain was added in position 7. These reactions were catalyzed only by CYP3A4: recombinant CYP3A4 generated the same metabolites as liver microsomes did, whereas neither CYP3A5 nor 2C8 or 2C9 could form derivatives. In conclusion, the presence of substituents in position 10 of the docetaxel molecule plays a pivotal role in determining the site of oxidation by CYP3A4.

Pharmacochemistry Library, 1995

... LOADING... The Chemistry and Pharmacology of Taxol and its Derivatives V. Farina, editor .9 1... more ... LOADING... The Chemistry and Pharmacology of Taxol and its Derivatives V. Farina, editor .9 1995 Elsevier Science BV All rights reserved 131 4 METABOLISM AND PHARMACOLOGY OF TAXOIDS M. Wright, B. Monsarrat, I. Royer Laboratoire de Pharmacologie et Toxicologie ...