Cell Biology, General Biochemistry, Genetics and Molecular Biology, Molecular Medicine, Cancer Research
13
Scopus Publications
326
Scholar Citations
9
Scholar h-index
9
Scholar i10-index
Scopus Publications
New Insights into the Molecular Actions of Grosheimin, Costunolide, and α- and β-Cyclocostunolide on Primary Cilia Structure and Hedgehog Signaling Marina Murillo-Pineda, Joel Martínez-Miralles, Zahara Medina-Calzada, Rosa María Varela, Francisco Antonio Macías, et al. International Journal of Molecular Sciences, 2025 Sesquiterpene lactones (SLs) are plant-derived metabolites with recognized pharmacological properties. Dysfunction of the primary cilium (PC), a solitary sensory organelle essential for development, is associated with disorders such as ciliopathies and tumors. While previous studies have shown that certain SLs can alter PC structure in human retinal cells, their influence on ciliary signaling pathways remains unclear. In this study, we examined the effect of four SLs—grosheimin, costunolide, α-cyclocostunolide (α-C), and β-cyclocostunolide (β-C)—on ciliary function in human primary fibroblasts. Using immunofluorescence and qPCR to assess cilia structure and Hedgehog (Hh) pathway activation, we found that grosheimin enhanced ciliogenesis without affecting Hh signaling. In contrast, costunolide, α-C, and β-C disrupted ciliary structure and suppressed the Hh pathway transcripts Gli1 and Ptch1. RNA sequencing revealed that grosheimin upregulated genes related to microtubule binding and ciliogenesis, whereas α-C downregulated tubulin subunit transcripts. These findings suggest distinct molecular mechanisms through which SLs affect ciliary structure and function. Collectively, this study highlights the potential of specific SLs as modulators of ciliary signaling, offering promising leads for therapeutic strategies targeting ciliopathies and tumors.
Effects of Sesquiterpene Lactones on Primary Cilia Formation (Ciliogenesis) Marina Murillo-Pineda, Juan M. Coto-Cid, María Romero, Jesús G. Zorrilla, Nuria Chinchilla, et al. Toxins, 2023 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.
The Habc domain of syntaxin 3 is a ubiquitin binding domain Adrian J. Giovannone, Elena Reales, Pallavi Bhattaram, Sirpi Nackeeran, Adam B. Monahan, et al. Scientific Reports, 2020 Syntaxins 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.
Soluble syntaxin 3 functions as a transcriptional regulator Adrian J. Giovannone, Christine Winterstein, Pallavi Bhattaram, Elena Reales, Seng Hui Low, et al. Journal of Biological Chemistry, 2018 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.
Monoubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes Adrian J. Giovannone, Elena Reales, Pallavi Bhattaram, Alberto Fraile-Ramos, Thomas Weimbs Molecular Biology of the Cell, 2017 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.
The MAL protein is crucial for proper membrane condensation at the ciliary base, which is required for primary cilium elongation Elena Reales, Miguel Bernabé-Rubio, Javier Casares-Arias, Carles Rentero, Jaime Fernández-Barrera, et al. Journal of Cell Science, 2015 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.
Syntaxin-4 is implicated in the secretion of antibodies by human plasma cells Laura Gómez-Jaramillo, Luis Delgado-Pérez, Elena Reales, Francisco Mora-López, Rosa M Mateos, et al. Journal of Leukocyte Biology, 2014 PCs 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.
Basolateral sorting of syntaxin 4 is dependent on its N-terminal domain and the AP1B clathrin adaptor, and required for the epithelial cell polarity Elena Reales, Nikunj Sharma, Seng Hui Low, Heike Fölsch, Thomas Weimbs Plos One, 2011 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.
New Insights into the Molecular Actions of Grosheimin, Costunolide, and α-and β-Cyclocostunolide on Primary Cilia Structure and Hedgehog Signaling M Murillo-Pineda, J Martínez-Miralles, Z Medina-Calzada, RM Varela, ... International Journal of Molecular Sciences 26 (23), 11754 , 2025 2025
Effects of sesquiterpene lactones on primary cilia formation (ciliogenesis) M Murillo-Pineda, JM Coto-Cid, M Romero, JG Zorrilla, N Chinchilla, ... Toxins 15 (11), 632 , 2023 2023 Citations: 4
Exocyst complex mediates recycling of internal cilia FE Rivera-Molina, Z Xi, E Reales, B Wang, D Toomre Current Biology 31 (24), 5580-5589. e5 , 2021 2021 Citations: 17
Evaluation of an oral DNA nanovaccine against photobacteriosis in Solea senegalensis M Ponce, E Zuasti, E Reales, V Anguís, C Fernández-Díaz Fish & Shellfish Immunology 117, 157-168 , 2021 2021 Citations: 8
The H abc domain of syntaxin 3 is a ubiquitin binding domain AJ Giovannone, E Reales, P Bhattaram, S Nackeeran, AB Monahan, ... Scientific reports 10 (1), 21350 , 2020 2020 Citations: 8
Soluble syntaxin 3 functions as a transcriptional regulator AJ Giovannone, C Winterstein, P Bhattaram, E Reales, SH Low, JE Baggs, ... Journal of Biological Chemistry 293 (15), 5478-5491 , 2018 2018 Citations: 20
Tracking Endocytosis and Intracellular Trafficking of Epitope-tagged Syntaxin 3 by Antibody Feeding in Live, Polarized MDCK Cells AJ Giovannone, E Reales, P Bhattaram, A Fraile-Ramos, T Weimbs Bio-protocol 8 (3), e2453-e2453 , 2018 2018 Citations: 2
The H abc Domain of Syntaxin 3 is a Ubiquitin Binding Domain with Specificity for K63-Linked Poly-Ubiquitin Chains AJ Giovannone, E Reales, P Bhattaram, S Nackeeran, AB Monahan, ... bioRxiv, 219642 , 2017 2017
Monoubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes AJ Giovannone, E Reales, P Bhattaram, A Fraile-Ramos, T Weimbs Molecular biology of the cell 28 (21), 2843-2853 , 2017 2017 Citations: 38
Monoubiquitination of syntaxin 3 leads to targeting into the apical endosomal/exosomal pathway: role in cargo recruitment. AJ Giovannone, E Reales-Rodriguez, P Bhattaram, A Fraile-Ramos, ... MOLECULAR BIOLOGY OF THE CELL 27 , 2016 2016
The MAL protein is crucial for proper membrane condensation at the ciliary base, which is required for primary cilium elongation E Reales, M Bernabé-Rubio, J Casares-Arias, C Rentero, ... Journal of Cell Science 128 (12), 2261-2270 , 2015 2015 Citations: 21
Syntaxin-3 acts as a transcriptional regulator AJ Giovannone, C Winterstein, P Bhattaram, E Reales, S Low, M Lalli, ... MOLECULAR BIOLOGY OF THE CELL 26 , 2015 2015
Syntaxin-4 is implicated in the secretion of antibodies by human plasma cells L Gómez-Jaramillo, L Delgado-Pérez, E Reales, F Mora-López, ... Journal of leukocyte biology 95 (2), 305-312 , 2014 2014 Citations: 25
Implication of proteins syntaxin-3 and syntaxin-4 in constitutive secretion of ig from human plasma cells ML Gomez Jaramillo, L Delgado-Perez, G Jimenez Gomez, ... IMMUNOLOGY 137, 334-334 , 2012 2012
Monoubiquitination of syntaxin 3 regulates its basolateral endocytosis and sorting to intraluminal vesicles in MDCK cells. AJ Giovannone, E Reales-Rodriguez, B Pallavi, T Weimbs MOLECULAR BIOLOGY OF THE CELL 23 , 2012 2012
Soluble SNAREs can function as transcription regulators C Wintersteini, P Bhattaram, E Reales, S Low, J Baggs, J Hogenesch, ... MOLECULAR BIOLOGY OF THE CELL 23 , 2012 2012
Basolateral sorting of syntaxin 4 is dependent on its N-terminal domain and the AP1B clathrin adaptor, and required for the epithelial cell polarity E Reales, N Sharma, SH Low, H Fölsch, T Weimbs PLoS One 6 (6), e21181 , 2011 2011 Citations: 44
Human BSAP and BLIMP1 conform an autoregulatory feedback loop F Mora-López, E Reales, JA Brieva, A Campos-Caro Blood, The Journal of the American Society of Hematology 110 (9), 3150-3157 , 2007 2007 Citations: 54
Identification of Soluble N -Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Exocytotic Machinery in Human Plasma Cells: SNAP-23 Is Essential for … E Reales, F Mora-López, V Rivas, A García-Poley, JA Brieva, ... The Journal of Immunology 175 (10), 6686-6693 , 2005 2005 Citations: 49
Persistent polyclonal B lymphocytosis: an expansion of cells showing IgVH gene mutations and phenotypic features of normal lymphocytes from the CD27+ marginal zone B‐cell … I Salcedo, A Campos‐Caro, A Sampalo, E Reales, JA Brieva British journal of haematology 116 (3), 662-666 , 2002 2002 Citations: 36
MOST CITED SCHOLAR PUBLICATIONS
Human BSAP and BLIMP1 conform an autoregulatory feedback loop F Mora-López, E Reales, JA Brieva, A Campos-Caro Blood, The Journal of the American Society of Hematology 110 (9), 3150-3157 , 2007 2007 Citations: 54
Identification of Soluble N -Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Exocytotic Machinery in Human Plasma Cells: SNAP-23 Is Essential for … E Reales, F Mora-López, V Rivas, A García-Poley, JA Brieva, ... The Journal of Immunology 175 (10), 6686-6693 , 2005 2005 Citations: 49
Basolateral sorting of syntaxin 4 is dependent on its N-terminal domain and the AP1B clathrin adaptor, and required for the epithelial cell polarity E Reales, N Sharma, SH Low, H Fölsch, T Weimbs PLoS One 6 (6), e21181 , 2011 2011 Citations: 44
Monoubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes AJ Giovannone, E Reales, P Bhattaram, A Fraile-Ramos, T Weimbs Molecular biology of the cell 28 (21), 2843-2853 , 2017 2017 Citations: 38
Persistent polyclonal B lymphocytosis: an expansion of cells showing IgVH gene mutations and phenotypic features of normal lymphocytes from the CD27+ marginal zone B‐cell … I Salcedo, A Campos‐Caro, A Sampalo, E Reales, JA Brieva British journal of haematology 116 (3), 662-666 , 2002 2002 Citations: 36
Syntaxin-4 is implicated in the secretion of antibodies by human plasma cells L Gómez-Jaramillo, L Delgado-Pérez, E Reales, F Mora-López, ... Journal of leukocyte biology 95 (2), 305-312 , 2014 2014 Citations: 25
The MAL protein is crucial for proper membrane condensation at the ciliary base, which is required for primary cilium elongation E Reales, M Bernabé-Rubio, J Casares-Arias, C Rentero, ... Journal of Cell Science 128 (12), 2261-2270 , 2015 2015 Citations: 21
Soluble syntaxin 3 functions as a transcriptional regulator AJ Giovannone, C Winterstein, P Bhattaram, E Reales, SH Low, JE Baggs, ... Journal of Biological Chemistry 293 (15), 5478-5491 , 2018 2018 Citations: 20
Exocyst complex mediates recycling of internal cilia FE Rivera-Molina, Z Xi, E Reales, B Wang, D Toomre Current Biology 31 (24), 5580-5589. e5 , 2021 2021 Citations: 17
Evaluation of an oral DNA nanovaccine against photobacteriosis in Solea senegalensis M Ponce, E Zuasti, E Reales, V Anguís, C Fernández-Díaz Fish & Shellfish Immunology 117, 157-168 , 2021 2021 Citations: 8
The H abc domain of syntaxin 3 is a ubiquitin binding domain AJ Giovannone, E Reales, P Bhattaram, S Nackeeran, AB Monahan, ... Scientific reports 10 (1), 21350 , 2020 2020 Citations: 8
Effects of sesquiterpene lactones on primary cilia formation (ciliogenesis) M Murillo-Pineda, JM Coto-Cid, M Romero, JG Zorrilla, N Chinchilla, ... Toxins 15 (11), 632 , 2023 2023 Citations: 4
Tracking Endocytosis and Intracellular Trafficking of Epitope-tagged Syntaxin 3 by Antibody Feeding in Live, Polarized MDCK Cells AJ Giovannone, E Reales, P Bhattaram, A Fraile-Ramos, T Weimbs Bio-protocol 8 (3), e2453-e2453 , 2018 2018 Citations: 2
New Insights into the Molecular Actions of Grosheimin, Costunolide, and α-and β-Cyclocostunolide on Primary Cilia Structure and Hedgehog Signaling M Murillo-Pineda, J Martínez-Miralles, Z Medina-Calzada, RM Varela, ... International Journal of Molecular Sciences 26 (23), 11754 , 2025 2025
The H abc Domain of Syntaxin 3 is a Ubiquitin Binding Domain with Specificity for K63-Linked Poly-Ubiquitin Chains AJ Giovannone, E Reales, P Bhattaram, S Nackeeran, AB Monahan, ... bioRxiv, 219642 , 2017 2017
Monoubiquitination of syntaxin 3 leads to targeting into the apical endosomal/exosomal pathway: role in cargo recruitment. AJ Giovannone, E Reales-Rodriguez, P Bhattaram, A Fraile-Ramos, ... MOLECULAR BIOLOGY OF THE CELL 27 , 2016 2016
Syntaxin-3 acts as a transcriptional regulator AJ Giovannone, C Winterstein, P Bhattaram, E Reales, S Low, M Lalli, ... MOLECULAR BIOLOGY OF THE CELL 26 , 2015 2015
Implication of proteins syntaxin-3 and syntaxin-4 in constitutive secretion of ig from human plasma cells ML Gomez Jaramillo, L Delgado-Perez, G Jimenez Gomez, ... IMMUNOLOGY 137, 334-334 , 2012 2012
Monoubiquitination of syntaxin 3 regulates its basolateral endocytosis and sorting to intraluminal vesicles in MDCK cells. AJ Giovannone, E Reales-Rodriguez, B Pallavi, T Weimbs MOLECULAR BIOLOGY OF THE CELL 23 , 2012 2012
Soluble SNAREs can function as transcription regulators C Wintersteini, P Bhattaram, E Reales, S Low, J Baggs, J Hogenesch, ... MOLECULAR BIOLOGY OF THE CELL 23 , 2012 2012
