Additional binding sites for anionic phospholipids and calcium ions in the crystal structures of complexes of the C2 domain of protein kinase Cα

The C2 domain of protein kinase Cα (PKCα) corresponds to the regulatory sequence motif, found in a large variety of membrane trafficking and signal transduction proteins, that mediates the recruitment of proteins by phospholipid membranes. In the PKCα isoenzyme, the Ca2+-dependent binding to membranes is highly specific to 1,2-sn-phosphatidyl-L-serine. Intrinsic Ca2+ binding tends to be of low affinity and non-cooperative, while phospholipid membranes enhance the overall affinity of Ca2+ and convert it into cooperative binding. The crystal structure of a ternary complex of the PKCα-C2 domain showed the binding of two calcium ions and of one 1,2-dicaproyl-sn-phosphatidyl-L-serine (DCPS) molecule that was coordinated directly to one of the calcium ions. The structures of the C2 domain of PKCα crystallised in the presence of Ca2+ with either 1,2-diacetyl-sn-phosphatidyl-L-serine (DAPS) or 1,2-dicaproyl-sn-phosphatidic acid (DCPA) have now been determined and refined at 1.9 Å and at 2.0 Å, respectively. DAPS, a phospholipid with short hydrocarbon chains, was expected to facilitate the accommodation of the phospholipid ligand inside the Ca2+-binding pocket. DCPA, with a phosphatidic acid (PA) head group, was used to investigate the preference for phospholipids with phosphatidyl-L-serine (PS) head groups. The two structures determined show the presence of an additional binding site for anionic phospholipids in the vicinity of the conserved lysine-rich cluster. Site-directed mutagenesis, on the lysine residues from this cluster that interact directly with the phospholipid, revealed a substantial decrease in C2 domain binding to vesicles when concentrations of either PS or PA were increased in the absence of Ca2+. In the complex of the C2 domain with DAPS a third Ca2+, which binds an extra phosphate group, was identified in the calcium-binding regions (CBRs). The interplay between calcium ions and phosphate groups or phospholipid molecules in the C2 domain of PKCα is supported by the specificity and spatial organisation of the binding sites in the domain and by the variable occupancies of ligands found in the different crystal structures. Implications for PKCα activity of these structural results, in particular at the level of the binding affinity of the C2 domain to membranes, are discussed. © 2002 Elsevier Science Ltd. All rights reserved.This research was supported by grants PB98-0389 to the Universidad de Murcia, and BIO099-0865 to the IBMB and by 1FD97-1558 from DGESIC (Spain) to a collaborative project between the Universidad de Murcia and the IBMB. Data were collected at the EMBL protein crystallography beamlines at ESRF (Grenoble) within a Block Allocation Group (BAG Barcelona), as at ESRF BM14. This work was supported financially by the ESRF and by grant HPRI-CT-1999-00022 of the European Union.Peer Reviewe

Synthesis of steroid-oligonucleotide conjugates for a DNA site-encoded SPR immunosensor

The excellent self-assembling properties of DNA and the excellent specificity of the antibodies to detect analytes of small molecular weight under competitive conditions have been combined in this study. Three oligonucleotide sequences (N1up, N2up, and N3up) have been covalently attached to three steroidal haptens (8, hG, and 13) of three anabolic-androgenic steroids (AAS), stanozolol (ST), tetrahydrogestrinone (THG), and boldenone (B), respectively. The synthesis of steroid-oligonucleotide conjugates has been performed by the reaction of oligonucleotides carrying amino groups with carboxyl acid derivatives of steroidal haptens. Due to the chemical nature of the steroid derivatives, two methods for coupling the haptens and the ssDNA have been studied: a solid-phase coupling strategy and a solution-phase coupling strategy. Specific antibodies against ST, THG, and B have been used in this study to asses the possibility of using the self-assembling properties of the DNA to prepare biofunctional SPR gold chips based on the immobilization of haptens, by hybridization with the complementary oligonucleotide strands possessing SH groups previously immobilized. The capture of the steroid-oligonucleotide conjugates and subsequent binding of the specific antibodies can be monitored on the sensogram due to variations produced on the refractive index on top of the gold chip. The resulting steroid-oligonucleotide conjugates retain the hybridization and specific binding properties of oligonucleotides and haptens as demonstrated by thermal denaturation experiments and surface plasmon resonance (SPR).This work was supported by grants from the Ministry of Science and Innovation, MICINN (MAT2011-29335-C03-01 and CTQ2010-20541-C03-01), CCEE (FUNMOL, FP7-NMP-213382-2), Generalitat de Catalunya (2009/SGR/1343, 2009/SGR/208), and CIBER-BBN. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. Núria Tort has a FI_B fellowship from the AGAUR (Agència de Gestió d’Ajuts Universitaris i de Recerca) of the (Generalitat de Catalunya) Government of Catalonia.Peer reviewe

Characterizing the Invasive Tumor Front of Aggressive Uterine Adenocarcinoma and Leiomyosarcoma

Perfils epigenètics; Matriu extracel·lular; Cèl·lules immunesPerfiles epigenéticos; Matriz extracelular; Células inmunesEpigenetic profiles; Extracellular matrix; Immune cellsThe invasive tumor front (the tumor–host interface) is vitally important in malignant cell progression and metastasis. Tumor cell interactions with resident and infiltrating host cells and with the surrounding extracellular matrix and secreted factors ultimately determine the fate of the tumor. Herein we focus on the invasive tumor front, making an in-depth characterization of reticular fiber scaffolding, infiltrating immune cells, gene expression, and epigenetic profiles of classified aggressive primary uterine adenocarcinomas (24 patients) and leiomyosarcomas (11 patients). Sections of formalin-fixed samples before and after microdissection were scanned and studied. Reticular fiber architecture and immune cell infiltration were analyzed by automatized algorithms in colocalized regions of interest. Despite morphometric resemblance between reticular fibers and high presence of macrophages, we found some variance in other immune cell populations and distinctive gene expression and cell adhesion-related methylation signatures. Although no evident overall differences in immune response were detected at the gene expression and methylation level, impaired antimicrobial humoral response might be involved in uterine leiomyosarcoma spread. Similarities found at the invasive tumor front of uterine adenocarcinomas and leiomyosarcomas could facilitate the use of common biomarkers and therapies. Furthermore, molecular and architectural characterization of the invasive front of uterine malignancies may provide additional prognostic information beyond established prognostic factors.This research was supported by grants from the ISCIII and ERDF (PI17/01558 and PI20/01107), by the CIBERONC (contracts CB16/12/00484, CB16/12/0328, CB16/12/00363, CB16/12/00364, CB16/12/00481, and CB16/12/00231) and Grupos Coordinados Estables from the Asociación Española Contra el Cáncer (AECC). ÁD-L was funded by a contract “Juan Rodés” from the ISCIII (JR17/00016). The funders had no involvement in the research process nor in the preparation and submission of the article

Metabolomic Analysis Points to Bioactive Lipid Species and Acireductone Dioxygenase 1 (ADI1) as Potential Therapeutic Targets in Poor Prognosis Endometrial Cancer

Metabolomic profiling analysis has the potential to highlight new molecules and cellular pathways that may serve as potential therapeutic targets for disease treatment. In this study, we used an LC-MS/MS platform to define, for the first time, the specific metabolomic signature of uterine serous carcinoma (SC), a relatively rare and aggressive variant of endometrial cancer (EC) responsible for 40% of all endometrial cancer-related deaths. A metabolomic analysis of 31 ECs (20 endometrial endometrioid carcinomas (EECs) and 11 SCs) was performed. Following multivariate statistical analysis, we identified 232 statistically different metabolites among the SC and EEC patient samples. Notably, most of the metabolites identified (89.2%) were lipid species and showed lower levels in SCs when compared to EECs. In addition to lipids, we also documented metabolites belonging to amino acids and purine nucleotides (such as 2-Oxo-4-methylthiobutanoic acid, synthesised by acireductone dioxygenase 1 (ADI1) enzyme), which showed higher levels in SCs. To further investigate the role of ADI1 in SC, we analysed the expression protein levels of ADI1 in 96 ECs (67 EECs and 29 SCs), proving that the levels of ADI1 were higher in SCs compared to EECs. We also found that ADI1 mRNA levels were higher in p53 abnormal ECs compared to p53 wild type tumours. Furthermore, elevated ADI1 mRNA levels showed a statistically significant negative correlation with overall survival and progression-free survival among EEC patients. Finally, we tested the ability of ADI1 to induce migration and invasion capabilities in EC cell lines. Altogether, these results suggest that ADI1 could be a potential therapeutic target in poor-prognosis SCs and other Ecs with abnormal p53 expression.This study was funded by the Instituto de Salud Carlos III (ISCIII) through projects PI20/00502, CP19/00025, CB16/12/00231, PI16/00692, PI18/00573, PI21/00672, CP17/00063 and PI18/00795; and by the Spanish Ministry of Science, Innovation and Universities (Ministerio de Ciencia, Innovación y Universidades, RTI2018-099200-BI00), co-funded by the European Regional Development Fund (ERDF) as part of the “A way to make Europe” programme and the European Social Fund (ESF) as part of the “Investing in Your Future” programme. This study was also supported by the “Xarxa de Bancs de Tumors de Catalunya” and sponsored by “Pla Director d’Oncologia de Catalunya (XBTC)”, “IRBLleida Biobank” (B.0000682) and “Plataforma Biobancos” PT20/00021. We also thank the Generalitat of Catalonia: Agency for Management of University and Research Grants (2017SGR1368 and 2017SGR696) and the “Asociación Española Contra el Cáncer” (AECC; Grupos Estables 2018 and LABAE19004LLOB). M.J. is a Serra Húnter Fellow. N.E. (MS19/00025) and D.L-N. (MS17/00063) are recipients of a Miguel Servet research scheme (co-funded by the ESF program “Investing in Your Future”). C. M-L. holds a predoctoral fellowship from the Generalitat de Catalunya (2020FI_B2 00099) and the predoctoral fellowship “Ajuts 2021 de Promoció de la Recerca en Salut-9a edició” from IRBLleida/Diputació de Lleida. IRBLleida is a CERCA Program/Generalitat of Catalonia

An inducible knockout mouse to model the cellautonomous role of PTEN in initiating endometrial, prostate and thyroid neoplasias

PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. The role of PTEN in carcinogenesis has been validated by knockout mouse models. PTEN heterozygous mice develop neoplasms in multiple organs. Unfortunately, the embryonic lethality of biallelic excision of PTEN has inhibited the study of complete PTEN deletion in the development and progression of cancer. By crossing PTEN conditional knockout mice with transgenic mice expressing a tamoxifen-inducible Cre-ERT under the control of a chicken actin promoter, we have generated a tamoxifeninducible mouse model that allows temporal control of PTEN deletion. Interestingly, administration of a single dose of tamoxifen resulted in PTEN deletion mainly in epithelial cells, but not in stromal, mesenchymal or hematopoietic cells. Using the mT/mG double-fluorescent Cre reporter mice, we demonstrate that epithelial-specific PTEN excision was caused by differential Cre activity among tissues and cells types. Tamoxifen-induced deletion of PTEN resulted in extremely rapid and consistent formation of endometrial in situ adenocarcinoma, prostate intraepithelial neoplasia and thyroid hyperplasia. We also analyzed the role of PTEN ablation in other epithelial cells, such as the tubular cells of the kidney, hepatocytes, colonic epithelial cells or bronchiolar epithelium, but those tissues did not exhibit neoplastic growth. Finally, to validate this model as a tool to assay the efficacy of anti-tumor drugs in PTEN deficiency, we administered the mTOR inhibitor everolimus to mice with induced PTEN deletion. Everolimus dramatically reduced the progression of endometrial proliferations and significantly reduced thyroid hyperplasia. This model could be a valuable tool to study the cell-autonomous mechanisms involved in PTEN-loss-induced carcinogenesis and provides a good platform to study the effect of anti-neoplastic drugs on PTEN-negative tumors

Arbovirus surveillance: first dengue virus detection in local Aedes albopictus mosquitoes in Europe, Catalonia, Spain, 2015

Dengue has emerged as the most important viral mosquito-borne disease globally. The current risk of dengue outbreaks in Europe appeared with the introduction of the vector Aedes albopictus mosquito in Mediterranean countries. Considering the increasing frequency of dengue epidemics worldwide and the movement of viraemic hosts, it is expected that new autochthonous cases will occur in the future in Europe. Arbovirus surveillance started in Catalonia in 2015 to monitor imported cases and detect possible local arboviral transmission. During 2015, 131 patients with a recent travel history to endemic countries were tested for dengue virus (DENV) and 65 dengue cases were detected. Twenty-eight patients with a febrile illness were viraemic, as demonstrated by a positive real-time RT-PCR test for DENV in serum samples. Entomological investigations around the viraemic cases led to the detection of DENV in a pool of local Ae. albopictus captured in the residency of one case. The sequence of the DENV envelope gene detected in the mosquito pool was identical to that detected in the patient. Our results show how entomological surveillance conducted around viraemic travellers can be effective for early detection of DENV in mosquitoes and thus might help to prevent possible autochthonous transmission