N'est plus au Laboratoire.
HENRI Pauline
Fonction : ASI Techniques Biologiques
Thème de Recherche: Pharmacologie Cellulaire
pauline.henri
umontpellier.fr
0448792236
Bureau: N2J16, Etg: 2 - Site : Pôle Chimie Balard Recherche
Domaines de Recherche: - Chimie/Chimie analytique
- Sciences du Vivant/Biochimie, Biologie Moléculaire/Biophysique
- Sciences du Vivant/Biotechnologies
- Sciences du Vivant/Immunologie/Immunité innée
- sdv/sdv.sp/sdv.sp.med
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Productions scientifiques :
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Fluorescent peptide biosensor for monitoring CDK4/cyclin D kinase activity in melanoma cell extracts, mouse xenografts and skin biopsies
Auteur(s): Prevel C., Pellerano M., González-Vera Juan, Henri P., Meunier L., Vollaire Julien, Josserand Veronique, Morris C. M.
(Article) Publié:
Biosensors And Bioelectronics / Biosensors & Bioelectronics, vol. 85 p.371 - 380 (2016)
Ref HAL: hal-01905259_v1
PMID 27203461
DOI: 10.1016/j.bios.2016.04.050
Résumé: Melanoma constitutes the most aggressive form of skin cancer, which further metastasizes into a deadly form of cancer. The p16(INK4a)-Cyclin D-CDK4/6-pRb pathway is dysregulated in 90% of melanomas. CDK4/Cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of Cyclin D or loss of p16(INK4a) leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker in melanoma and an emerging pharmacological target, however there are no tools enabling direct detection or quantification of its activity. Here we report on the design and application of a fluorescent peptide biosensor to quantify CDK4 activity in melanoma cell extracts, skin biopsies and melanoma xenografts. This biosensor provides sensitive means of comparing CDK4 activity between different melanoma cell lines and further responds to CDK4 downregulation by siRNA or small-molecule inhibitors. By affording means of monitoring CDK4 hyperactivity consequent to cancer-associated molecular alterations in upstream signaling pathways that converge upon this kinase, this biosensor offers an alternative to immunological identification of melanoma-specific biomarkers, thereby constituting an attractive tool for diagnostic purposes, providing complementary functional information to histological analysis, of particular utility for detection of melanoma onset in precancerous lesions. This is indeed the first fluorescent peptide biosensor which has been successfully implemented to monitor kinase activity in skin samples and melanoma tumour xenografts. Moreover by enabling to monitor response to CDK4 inhibitors, this biosensor constitutes an attractive companion assay to identify compounds of therapeutic relevance for melanoma.
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MC1R expression in HaCaT keratinocytes inhibits UVA-induced ROS production via NADPH oxidase- and cAMP-dependent mechanisms.
Auteur(s): Henri P., Beaumel Sylvain, Guezennec Anne, Poumès Carine, Stoebner P., Stasia Marie-José, Guesnet Joëlle, Martinez J., Meunier L.
(Article) Publié:
Journal Of Cellular Physiology, vol. 227 p.2578-85 (2012)
Ref HAL: hal-00809500_v1
PMID 21898403
DOI: 10.1002/jcp.22996
Résumé: Ultraviolet A (UVA) radiations are responsible for deleterious effects, mainly due to reactive oxygen species (ROS) production. Alpha-melanocyte stimulating hormone (α-MSH) binds to melanocortin-1 receptor (MC1R) in melanocytes to stimulate pigmentation and modulate cutaneous inflammatory responses. MC1R may be induced in keratinocytes after UV exposure. To investigate the effect of MC1R signaling on UVA-induced ROS (UVA-ROS) production, we generated HaCaT cells that stably express human MC1R (HaCaT-MC1R) or the Arg151Cys (R(151)C) non-functional variant (HaCaT-R(151)C). We then assessed ROS production immediately after UVA exposure and found that: (1) UVA-ROS production was strongly reduced in HaCaT-MC1R but not in HaCaT-R(151)C cells compared to parental HaCaT cells; (2) this inhibitory effect was further amplified by incubation of HaCaT-MC1R cells with α-MSH before UVA exposure; (3) protein kinase A (PKA)-dependent NoxA1 phosphorylation was increased in HaCaT-MC1R compared to HaCaT and HaCaT-R(151)C cells. Inhibition of PKA in HaCaT-MC1R cells resulted in a marked increase of ROS production after UVA irradiation; (4) the ability of HaCaT-MC1R cells to produce UVA-ROS was restored by inhibiting epidermal growth factor receptor (EGFR) or extracellular signal-regulated kinases (ERK) activity before UVA exposure. Our findings suggest that constitutive activity of MC1R in keratinocytes may reduce UVA-induced oxidative stress via EGFR and cAMP-dependent mechanisms.
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RECEPTEURS CUTANES A LA MELANOCORTINE DE TYPE 1 (MC1R) ET REPONSES OXYDATIVES AUX UVA DANS DES KERATINOCYTES HUMAINS HaCaT
Auteur(s): Henri P.
(Thèses)
, 2010
Ref HAL: tel-00560959_v1
Résumé: Les ultraviolets A (UVA) sont carcinogènes et produisent des espèces réactives de l'oxygène (ERO). Le récepteur à la mélanocortine de type 1 (MC1R) est un récepteur couplé aux protéines G (RCPG) qui est impliqué dans la mélanogénèse et dans l'inflammation cutanée. Certains variants du gène sont associés à un risque accru de mélanomes et de carcinomes cutanés. Le MC1R est exprimé surtout dans les mélanocytes mais son expression peut être induite par les UV in vitro dans les kératinocytes et in vivo dans la peau. Le récepteur MC1R est activé par l'α-MSH. L'objectif de ce travail de thèse a été d'étudier les effets du récepteur MC1R sur le stress oxydatif induit par les UVA dans des lignées kératinocytaires humaines HaCaT exprimant le récepteur MC1R ou son variant non fonctionnel Arg151Cys. Nous avons montré que la production d'ERO intracellulaire induite par les UVA est fortement inhibée dans les cellules HaCaT-MC1R et que cette inhibition est renforcée en présence d'α-MSH. L'inhibition du stress oxydatif induit par les UVA dans les cellules transfectées par le MC1R est en partie dépendante de la phosphorylation de la sous-unité activatrice, NoxA1 de la NADPH oxydase. Le traitement des cellules HaCaT-MC1R par un inhibiteur du récepteur au facteur de croissance épidermique (EGFR) restaure l'habilité de ces cellules à induire un stress oxydatif après irradiation UVA. Ces résultats montrent que l'activité constitutive du récepteur MC1R dans des kératinocytes pourrait inhiber le stress oxydatif induit par les UVA via des mécanismes dépendants de l'AMPc et de l'EGFR.
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Differential effects of phosphorylation on DNA binding properties of N Oct-3 are dictated by protein/DNA complex structures.
Auteur(s): Nieto Laurence, Joseph Gérard, Stella Alexandre, Henri P., Burlet-schiltz Odile, Monsarrat Bernard, Clottes Eric, Erard Monique
(Article) Publié:
Journal Of Molecular Biology, vol. 370 p.687-700 (2007)
PMID 17543985
DOI: 10.1016/j.jmb.2007.04.072
Résumé: N Oct-3, a transcription factor member of the POU protein family, is implicated in normal central nervous system development but also in melanoma growth. Its DNA-binding domain (DBD) comprises two subdomains, POUs and POUh, joined by a linker peptide. We have previously shown that N Oct-3 can interact with the already described PORE and MORE DNA motifs, but also with a new structural element we have termed NORE. Having observed that both the PORE and NORE DNA-association modes depend on a strong anchoring of the POUh subdomain rigid arm into the DNA-target minor groove, in contrast to the MORE mode, we have formulated the hypothesis that phosphorylation of the conserved Ser101 residue located in the N Oct-3 POUh arm could lead to differential results in DNA binding according to the type of target. Here we demonstrate that, in vitro, Ser101 is phosphorylated by protein kinase A (PKA), either purified or contained in melanoma (624 mel) nuclear extract, and that this phosphorylation indeed significantly reduced N Oct-3 DBD binding to PORE and NORE motifs, most likely by hampering the POUh rigid arm insertion in the DNA minor groove. Conversely, no effect was observed on the binding of N Oct-3 DBD to MORE sequences. Finally, once bound to its DNA targets, N Oct-3 DBD is less susceptible to PKA activity. We conclude that transcription of genes exhibiting a MORE motif in their promoter should be less affected by N Oct-3 phosphorylation than that of genes switched on by PORE or NORE sequences.
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