Role of Phosphorylation in the Control of Clathrin-Mediated Internalization of GPCR

The process by which G protein-coupled receptors (GPCRs) are internalized through the clathrin-coated vesicles involves interactions of multifunctional adaptor proteins. These interactions are tightly controlled by phosphorylation and dephosphorylation mechanisms resulting in the regulation of receptor endocytosis. However, the identities of the kinases involved in this process remained largely unknown until recently. This paper discusses advances in our knowledge of the important role played by protein phosphorylation in the regulation of the endocytic machinery and how phosphorylation controls the coated vesicle cycle

Sulconazole inhibits PD-1 expression in immune cells and cancer cells malignant phenotype through NF-κB and calcium activity repression

The overexpression of the immunoinhibitory receptor programmed death-1 (PD1) on T-cells is involved in immune evasion in cancer. The use of anti-PD-1/PDL-1 strategy has deeply changed the therapies of cancers and patient survival. However, their efficacy diverges greatly along with tumor type and patient populations. Thereby, novel treatments are needed to interfere with the anti-tumoral immune responses and propose an adjunct therapy. In the current study, we found that the antifungal drug Sulconazole (SCZ) inhibits PD-1 expression on activated PBMCs and T cells at the RNA and protein levels. SCZ repressed NF-κB and calcium signaling, both, involved in the induction of PD-1. Further analysis revealed cancer cells treatment with SCZ inhibited their proliferation, and migration and ability to mediate tumor growth in zebrafish embryos. SCZ found also to inhibit calcium mobilization in cancer cells. These results suggest the SCZ therapeutic potential used alone or as adjunct strategy to prevent T-cell exhaustion and promotes cancer cell malignant phenotype repression in order to improve tumor eradication.The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by La Region Nouvelle Aquitaine, Planet Vegetal, Ligue Contre le cancer and INSERM.Peer reviewe

Les organoïdes normaux et leurs applications dans la recherche sur le cancer

National audienceThree-dimensional (3D) culture of organoids from primary cells (wild type) or tumoroids from tumor cells, is used to study the physiological mechanisms in vivo, in order to model normal or tumor tissues more accurately than conventional two-dimensional (2D) culture. The features of this 3D culture, such as the three-dimensional structure, the self-renewal capacity and differentiation are preserved and appropriate to cancer study since their cellular characteristics are very similar to in vivo models. Here, we summarize the recent advances in the rapidly evolving field of organoids and their applications to cancer biology, clinical research and personalized medicine.La culture tridimensionnelle (3D) des organoïdes à partir de cellules primaires (de type sauvage) et des tumoroïdes (d’origine tumorale) est utilisée pour l’étude et la compréhension des mécanismes physiologiques in vivo, afin de modéliser les tissus d’origine avec plus de précision que la culture bi-dimensionnelle classique (2D). Les caractéristiques de ce modèle, comme la structure tridimensionnelle, la capacité d’auto-renouvellement et de différenciation, rendent les organoïdes et les tumoroïdes éminemment mieux adaptés à l’étude du cancer, apportant des caractéristiques cellulaires très proches des modèles in vivo. Nous résumons ici les avancées récentes dans le domaine des organoïdes, en constante évolution, et les applications de ces modèles à la biologie du cancer, à la recherche clinique et à la médecine de précision

Integrative analysis of genomic and transcriptomic alterations of AGR2 and AGR3 in cancer

International audienceThe AGR2 and AGR3 genes have been shown by numerous groups to be functionally associated with adenocarcinoma progression and metastasis. In this paper, we explore the data available in databases concerning genomic and transcriptomic features of these two genes: the NCBI dbSNP database was used to explore the presence and roles of constitutional SNPs, and the NCI, Cancer Cell Line Encyclopedia (CCLE) and TCGA databases were used to explore somatic mutations and copy number variations (CNVs), as well as mRNA expression of these genes in human cancer cell lines and tumours. Relationships of AGR2/3 expression with whole-genome mRNA expression and cancer features (i.e. mutations and CNVs of oncogenes and tumour suppressor genes (TSG)) were established using the CCLE and TCGA databases. In addition, the CCLE data concerning CRISPR gene extinction screens (Achilles project) of these two genes and a panel of oncogenes and TSG were explored. We observed that no functional polymorphism or recurrent mutation could be detected in AGR2 or AGR3. The expression of these genes was positively correlated with the expression of epithelial genes and inversely correlated with that of mesenchymal genes. It was also significantly associated with several cancer features, such as TP53 or SMAD4 mutations, depending on the gene and the cancer type. In addition, the CRISPR screens revealed the absence of cell fitness modification upon gene extinction, in contrast with oncogenes (cell fitness decrease) and TSG (cell fitness increase). Overall, these explorations revealed that AGR2 and AGR3 proteins appear as common non-genetic evolutionary factors in the process of human tumorigenesis

Separation, identification and profiling of membrane proteins by GFC/ IEC/SDS-PAGE and MALDI TOF MS

International audienceMembrane protein identification by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) requires that proteins be separated prior to MS analysis. After membrane solubilization with the nondenaturing detergent n-dodecyl-beta-D-maltoside, proteins can be separated by ion-exchange chromatography (IEC) and further resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An additional separation step by gel filtration (GF) before IEC/SDS-PAGE can be required depending on the complexity of the membrane protein mixture. Staining of final SDS-PAGE gels allows one to establish simply the protein expression pattern of a membrane fraction and to profile responses. Moreover, in-gel digestion of hydrophobic integral proteins is valuable. Finally, the resolution capacity of this separation procedure allows identification of proteins by MALDI-TOF MS. The method is illustrated by application to plant and yeast plasma membrane and to plant vacuolar membrane

The Anterior GRadient (AGR) family proteins in epithelial ovarian cancer

International audienceEpithelial ovarian cancer (EOC) is the most common gynecologic disorder. Even with the recent progresses made towards the use of new therapeutics, it still represents the most lethal gynecologic malignancy in women from developed countries.The discovery of the anterior gradient proteins AGR2 and AGR3, which are highly related members belonging to the protein disulfide isomerase (PDI) family, attracted researchers’ attention due to their putative involvement in adenocarcinoma development. This review compiles the current knowledge on the role of the AGR family and the expression of its members in EOC and discusses the potential clinical relevance of AGR2 and AGR3 for EOC diagnosis, prognosis, and therapeutics.A better understanding of the role of the AGR family may thus provide new handling avenues for EOC patients

Modulation of PDI Functions by Localization: The Example of the Anterior Gradient Family

International audienceSignificance: Oxidative folding within the endoplasmic reticulum (ER) introduces disulfide bonds into nascent polypeptides, ensuring proteins' stability and proper functioning. Consequently, this process is critical for maintaining proteome integrity and overall health. The productive folding of thousands of secretory proteins requires stringent quality control measures, such as the Unfolded Protein Response (UPR) and ER-Associated Degradation (ERAD), which contribute significantly to maintaining ER homeostasis. ER-localised protein disulfide isomerases (PDI) play an essential role in each of these processes, thereby contributing to various aspects of ER homeostasis, including maintaining redox balance, proper protein folding, and signaling from the ER to the nucleus.Recent advances: Over the years, there have been increasing reports of the (re)localization of PDI family members and other ER-localized proteins to various compartments. A prime example is the Anterior gradient (AGR) family of PDI proteins, which have been reported to relocate to the cytosol or the extracellular environment, acquiring gain of functions that intersect with various cellular signaling pathways.Critical issues: Here, we summarize the functions of PDIs and their gain or loss of functions in non-ER locations. We will focus on the activity, localization, and function of the AGR proteins: AGR1, AGR2, and AGR3.Future direction: Targeting PDIs in general and AGRs in particular is a promising strategy in different human diseases. Thus, there is a need for innovative strategies and tools aimed at targeting PDIs; those strategies should integrate the specific localization and newly acquired functions of these PDIs rather than solely focusing on their canonical roles

Role of Pro-oncogenic Protein Disulfide Isomerase (PDI) Family Member Anterior Gradient 2 (AGR2) in the Control of Endoplasmic Reticulum Homeostasis

International audienc