Elena Reales Rodriguez
@inibica.es
INIBICA-HU Puerta del Mar-Cádiz
RESEARCH, TEACHING, or OTHER INTERESTS
Cell Biology, General Biochemistry, Genetics and Molecular Biology, Molecular Medicine, Cancer Research
Scopus Publications
Scholar Citations
Scholar h-index
Scholar i10-index
Scopus Publications
Marina Murillo-Pineda, Juan M. Coto-Cid, María Romero, Jesús G. Zorrilla, Nuria Chinchilla, Zahara Medina-Calzada, Rosa M. Varela, Álvaro Juárez-Soto, Francisco A. Macías, and Elena Reales
MDPI AG
Sesquiterpene lactones (SLs), plant-derived metabolites with broad spectra of biological effects, including anti-tumor and anti-inflammatory, hold promise for drug development. Primary cilia, organelles extending from cell surfaces, are crucial for sensing and transducing extracellular signals essential for cell differentiation and proliferation. Their life cycle is linked to the cell cycle, as cilia assemble in non-dividing cells of G0/G1 phases and disassemble before entering mitosis. Abnormalities in both primary cilia (non-motile cilia) and motile cilia structure or function are associated with developmental disorders (ciliopathies), heart disease, and cancer. However, the impact of SLs on primary cilia remains unknown. This study evaluated the effects of selected SLs (grosheimin, costunolide, and three cyclocostunolides) on primary cilia biogenesis and stability in human retinal pigment epithelial (RPE) cells. Confocal fluorescence microscopy was employed to analyze the effects on primary cilia formation (ciliogenesis), primary cilia length, and stability. The effects on cell proliferation were evaluated by flow cytometry. All SLs disrupted primary cilia formation in the early stages of ciliogenesis, irrespective of starvation conditions or cytochalasin-D treatment, with no effect on cilia length or cell cycle progression. Interestingly, grosheimin stabilized and promoted primary cilia formation under cilia homeostasis and elongation treatment conditions. Thus, SLs have potential as novel drugs for ciliopathies and tumor treatment.
Félix E. Rivera-Molina, Zhiqun Xi, Elena Reales, Bryan Wang, and Derek Toomre
Elsevier BV
Marian Ponce, Eugenia Zuasti, Elena Reales, Victoria Anguís, and Catalina Fernández-Díaz
Elsevier BV
Adrian J. Giovannone, Elena Reales, Pallavi Bhattaram, Sirpi Nackeeran, Adam B. Monahan, Rashid Syed, and Thomas Weimbs
Springer Science and Business Media LLC
AbstractSyntaxins are a family of membrane-anchored SNARE proteins that are essential components required for membrane fusion in eukaryotic intracellular membrane trafficking pathways. Syntaxins contain an N-terminal regulatory domain, termed the Habc domain that is not highly conserved at the primary sequence level but folds into a three-helix bundle that is structurally conserved among family members. The syntaxin Habc domain has previously been found to be structurally very similar to the GAT domain present in GGA family members and related proteins that are otherwise completely unrelated to syntaxins. Because the GAT domain has been found to be a ubiquitin binding domain we hypothesized that the Habc domain of syntaxins may also bind to ubiquitin. Here, we report that the Habc domain of syntaxin 3 (Stx3) indeed binds to monomeric ubiquitin with low affinity. This domain binds efficiently to K63-linked poly-ubiquitin chains within a narrow range of chain lengths but not to K48-linked poly-ubiquitin chains. Other syntaxin family members also bind to K63-linked poly-ubiquitin chains but with different chain length specificities. Molecular modeling suggests that residues of the GGA3-GAT domain known to be important for ionic and hydrophobic interactions with ubiquitin may have equivalent, conserved residues within the Habc domain of Stx3. We conclude that the syntaxin Habc domain and the GAT domain are both structurally and functionally related, and likely share a common ancestry despite sequence divergence. Binding of Ubiquitin to the Habc domain may regulate the function of syntaxins in membrane fusion or may suggest additional functions of this protein family.
Adrian J. Giovannone, Christine Winterstein, Pallavi Bhattaram, Elena Reales, Seng Hui Low, Julie E. Baggs, Mimi Xu, Matthew A. Lalli, John B. Hogenesch, and Thomas Weimbs
Elsevier BV
Syntaxins are a conserved family of SNARE proteins and contain C-terminal transmembrane anchors required for their membrane fusion activity. Here we show that Stx3 (syntaxin 3) unexpectedly also functions as a nuclear regulator of gene expression. We found that alternative splicing creates a soluble isoform that we termed Stx3S, lacking the transmembrane anchor. Soluble Stx3S binds to the nuclear import factor RanBP5 (RAN-binding protein 5), targets to the nucleus, and interacts physically and functionally with several transcription factors, including ETV4 (ETS variant 4) and ATF2 (activating transcription factor 2). Stx3S is differentially expressed in normal human tissues, during epithelial cell polarization, and in breast cancer versus normal breast tissue. Inhibition of endogenous Stx3S expression alters the expression of cancer-associated genes and promotes cell proliferation. Similar nuclear-targeted, soluble forms of other syntaxins were identified, suggesting that nuclear signaling is a conserved, novel function common among these membrane-trafficking proteins.
Adrian J. Giovannone, Elena Reales, Pallavi Bhattaram, Alberto Fraile-Ramos, and Thomas Weimbs
American Society for Cell Biology (ASCB)
Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes/lysosomes, although how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes monoubiquitination in a conserved polybasic domain. Stx3 present at the basolateral—but not the apical—plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs), and excreted in exosomes. A nonubiquitinatable mutant of Stx3 (Stx3-5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV/exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3-5R mutant acts as a dominant-negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3-5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploits this pathway for virion excretion.
Elena Reales, Miguel Bernabé-Rubio, Javier Casares-Arias, Carles Rentero, Jaime Fernández-Barrera, Laura Rangel, Isabel Correas, Carlos Enrich, Germán Andrés, and Miguel A. Alonso
The Company of Biologists
ABSTRACT The base of the primary cilium contains a zone of condensed membranes whose importance is not known. Here, we have studied the involvement of MAL, a tetraspanning protein that exclusively partitions into condensed membrane fractions, in the condensation of membranes at the ciliary base and investigated the importance of these membranes in primary cilium formation. We show that MAL accumulates at the ciliary base of epithelial MDCK cells. Knockdown of MAL expression resulted in a drastic reduction in the condensation of membranes at the ciliary base, the percentage of ciliated cells and the length of the cilia, but did not affect the docking of the centrosome to the plasma membrane or produce missorting of proteins to the pericentriolar zone or to the membrane of the remaining cilia. Rab8 (for which there are two isoforms, Rab8A and Rab8b), IFT88 and IFT20, which are important components of the machinery of ciliary growth, were recruited normally to the ciliary base of MAL-knockdown cells but were unable to elongate the primary cilium correctly. MAL, therefore, is crucial for the proper condensation of membranes at the ciliary base, which is required for efficient primary cilium extension.
Laura Gómez-Jaramillo, Luis Delgado-Pérez, Elena Reales, Francisco Mora-López, Rosa M Mateos, Antonio García-Poley, José A Brieva, and Antonio Campos-Caro
Oxford University Press (OUP)
ABSTRACTPCs are responsible for the production and secretion of antibodies, the effector molecules of the humoral immune response. The molecular mechanisms responsible for vesicle docking and secretion implicated in the antibody-secretion process are not well-known, as they have not been studied, but it is known that SNARE proteins are responsible for many membrane-fusion processes in the cell. We show here that freshly isolated human colon LP-PCs and T-PCs from MM-PC patients and the U266 cell line, as a model for PC secretion, contain a set of these proteins. SNAP23, STX3, and STX4 were localized mainly in the plasma membrane of PCs, and interactions of SNAP23 with STX3 and with STX4 were proven by IP. Interaction between SNAP23 and STX4 was also confirmed in situ. With the use of siRNA, as well as shRNA, the functional role of SNAP23, STX3, and STX4 in antibody secretion was also examined. The findings demonstrate that in addition to SNAP23, STX4 is implicated in the antibody secretion by a myeloma cell line and by normal human colon LP-PCs.
Elena Reales, Nikunj Sharma, Seng Hui Low, Heike Fölsch, and Thomas Weimbs
Public Library of Science (PLoS)
Generation of epithelial cell polarity requires mechanisms to sort plasma membrane proteins to the apical and basolateral domains. Sorting involves incorporation into specific vesicular carriers and subsequent fusion to the correct target membranes mediated by specific SNARE proteins. In polarized epithelial cells, the SNARE protein syntaxin 4 localizes exclusively to the basolateral plasma membrane and plays an important role in basolateral trafficking pathways. However, the mechanism of basolateral targeting of syntaxin 4 itself has remained poorly understood. Here we show that newly synthesized syntaxin 4 is directly targeted to the basolateral plasma membrane in polarized Madin-Darby canine kidney (MDCK) cells. Basolateral targeting depends on a signal that is centered around residues 24–29 in the N-terminal domain of syntaxin 4. Furthermore, basolateral targeting of syntaxin 4 is dependent on the epithelial cell-specific clathrin adaptor AP1B. Disruption of the basolateral targeting signal of syntaxin 4 leads to non-polarized delivery to both the apical and basolateral surface, as well as partial intercellular retention in the trans-Golgi network. Importantly, disruption of the basolateral targeting signal of syntaxin 4 leads to the inability of MDCK cells to establish a polarized morphology which suggests that restriction of syntaxin 4 to the basolateral domain is required for epithelial cell polarity.
Francisco Mora-López, Elena Reales, José A. Brieva, and Antonio Campos-Caro
American Society of Hematology
AbstractB-lymphocyte–induced maturation protein-1 (BLIMP1), encoded by the PRDM1 gene, is a transcriptional repressor considered a master regulator that is required and sufficient for plasma cell (PC) differentiation. BLIMP1 represses the PAX5 gene, coding for the B-cell lineage–specific activator protein (BSAP), which is required for B-cell identity and survival. Mutations in PAX5 gene as well as in PRDM1 gene have been recently implicated in lymphomas. In the present study, sequence analysis of PRDM1 gene revealed a binding site for BSAP transcription factor. By analyzing different human cell lines, we have found that a specific nuclear factor for B-cell lines binds to a site on the PRDM1 promoter. Electrophoretic mobility shift assays identified this factor as BSAP, and chromatin immunoprecipitation assays confirmed its binding in vivo to the human PRDM1 promoter. Moreover, by ectopically expressing BSAP, and using a PRDM1 promoter with the BSAP-binding site mutated, we demonstrated that this factor represses the expression of BLIMP1. Therefore, repression of PRDM1 by BSAP reveals an autoregulatory negative-feedback loop that could play a relevant role in controlling human PC differentiation.
Elena Reales, Francisco Mora-López, Verónica Rivas, Antonio García-Poley, José A. Brieva, and Antonio Campos-Caro
The American Association of Immunologists
Abstract Plasma cells (PC) are B-lymphocytes terminally differentiated in a postmitotic state, with the unique purpose of manufacturing and exporting Igs. Despite the importance of this process in the survival of vertebrates, no studies have been made to understand the molecular events that regulate Ig exocytosis by PC. The present study explores the possible presence of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) system in human PC, and examines its functional role in Ig secretion. Syntaxin-2, Syntaxin-3, Syntaxin-4, vesicle-associated membrane protein (VAMP)-2, VAMP-3, and synaptosome-associated protein (SNAP)-23 could be readily detected in normal human PC obtained from intestinal lamina propria and blood, as well as in human PC lines. Because SNAP-23 plays a central role in SNAREs complex formation, it was chosen to examine possible functional implications of the SNARE system in PC Ig secretion. When recombinant SNAP-23 fusion protein was introduced into the cells, a complete abolishment of Ig production was observed in the culture supernatants of PC lines, as well as in those of normal PC. These results provide insights, for the first time, into the molecular machinery of constitutive vesicular trafficking in human PC Ig secretion and present evidence indicating that at least SNAP-23 is essential for Ab production.
Irene Salcedo, Antonio Campos-Caro, Almudena Sampalo, Elena Reales, and José A. Brieva
Wiley
Summary. Persistent polyclonal B‐cell lymphocytosis (PPBL) is an unusual and benign lymphoproliferation characterized by a polyclonal expansion of B lymphocytes, whose nature remains undetermined. The phenotypic analysis of three cases revealed that these cells were CD27+ IgMhigh CD21high CD5low and CD23low, a phenotype associated with the normal marginal zone (MZ) B‐cell compartment. As MZ B cells have initiated immunoglobulin (Ig)V gene somatic mutations, PPBL IgVH genes were sequenced. An average of 73% of these sequences were mutated. The mean number of mutation per sequence was 6·9, a number similar to those observed in the MZ B‐cell compartment.