Publications by authors named "Susana Silberstein"

21 Publications

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Stress-Related Brain Neuroinflammation Impact in Depression: Role of the Corticotropin-Releasing Hormone System and P2X7 Receptor.

Neuroimmunomodulation 2021 Apr 12:1-9. Epub 2021 Apr 12.

Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET-Partner Institute of the Max Planck Society, Buenos Aires, Argentina.

Depression and other psychiatric stress-related disorders are leading causes of disability worldwide. Up to date, treatments of mood disorders have limited success, most likely due to the multifactorial etiology of these conditions. Alterations in inflammatory processes have been identified as possible pathophysiological mechanisms in psychiatric conditions. Here, we review the main features of 2 systems involved in the control of these inflammatory pathways: the CRH system as a key regulator of the stress response and the ATP-gated ion-channel P2X7 receptor (P2X7R) involved in the control of immune functions. The pathophysiology of depression as a stress-related psychiatric disorder is depicted in terms of the impact of CRH and P2X7R function on inflammatory pathways in the brain. Understanding pathogenesis of affective disorders will lead to the development of therapies for treatment of depression and other stress-related diseases.
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http://dx.doi.org/10.1159/000515130DOI Listing
April 2021

Assessing real-time signaling and agonist-induced CRHR1 internalization by optical methods.

Methods Cell Biol 2019 17;149:239-257. Epub 2018 Sep 17.

Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET-Partner Institute of the Max Planck Society, Buenos Aires, Argentina; Departamento Fisiología, Biología Molecular y Celular, Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Buenos Aires, Argentina. Electronic address:

The development of live-cell sensors for real-time measurement of signaling responses, with improved spatial and temporal resolution with respect to classical biochemical methods, has changed our understanding of cellular signaling. Examination of cAMP generation downstream activated GPCRs has shown that signaling responses can be short-lived (generated from the cell surface) or prolonged after receptor internalization. Class B secretin-like Corticotropin-releasing hormone receptor 1 (CRHR1) is a key player in stress pathophysiology. By monitoring real-time signaling in living cells, we uncovered cell context-dependent temporal characteristics of CRHR1-elicited cAMP responses and disclosed a specific link between cAMP generation and receptor signaling from internal compartments. We describe technical aspects and elaborate the protocols for cell line expression of Förster resonance energy transfer (FRET)-based biosensors to study the dynamics of cAMP and calcium signaling responses downstream activated CRHR1, live-cell imaging and analysis, and fluorescence flow cytometry to determine receptor levels at the cell surface.
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http://dx.doi.org/10.1016/bs.mcb.2018.08.009DOI Listing
April 2019

A fluorescence nanoscopy marker for corticotropin-releasing hormone type 1 receptor: computer design, synthesis, signaling effects, super-resolved fluorescence imaging, and in situ affinity constant in cells.

Phys Chem Chem Phys 2018 Nov;20(46):29212-29220

Centro de Investigaciones en Bionanociencias-"Elizabeth Jares-Erijman" (CIBION), CONICET, Godoy Cruz 2390, 1425 Ciudad de Buenos Aires, Argentina.

Class B G protein-coupled receptors (GPCRs) are involved in a variety of human pathophysiological states. These groups of membrane receptors are less studied than class A GPCRs due to the lack of structural information, delayed small molecule drug discovery, and scarce fluorescence detection tools available. The class B corticotropin-releasing hormone type 1 receptor (CRHR1) is a key player in the stress response whose dysregulation is critically involved in stress-related disorders: psychiatric conditions (i.e. depression, anxiety, and addictions), neuroendocrinological alterations, and neurodegenerative diseases. Here, we present a strategy to label GPCRs with a small fluorescent antagonist that permits the observation of the receptor in live cells through stochastic optical reconstruction microscopy (STORM) with 23 nm resolution. The marker, an aza-BODIPY derivative, was designed based on computational docking studies, then synthesized, and finally tested in biological cells. Experiments on hippocampal neurons demonstrate antagonist effects in similar concentrations as the well-established antagonist CP-376395. A quantitative analysis of two color STORM images enabled the determination of the binding affinity of the new marker in the cellular environment.
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http://dx.doi.org/10.1039/c8cp06196cDOI Listing
November 2018

Endocrinology and the brain: corticotropin-releasing hormone signaling.

Endocr Connect 2017 Aug 14;6(6):R99-R120. Epub 2017 Jul 14.

Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET-Partner Institute of the Max Planck SocietyBuenos Aires, Argentina

Corticotropin-releasing hormone (CRH) is a key player of basal and stress-activated responses in the hypothalamic-pituitary-adrenal axis (HPA) and in extrahypothalamic circuits, where it functions as a neuromodulator to orchestrate humoral and behavioral adaptive responses to stress. This review describes molecular components and cellular mechanisms involved in CRH signaling downstream of its G protein-coupled receptors (GPCRs) CRHR1 and CRHR2 and summarizes recent findings that challenge the classical view of GPCR signaling and impact on our understanding of CRHRs function. Special emphasis is placed on recent studies of CRH signaling that revealed new mechanistic aspects of cAMP generation and ERK1/2 activation in physiologically relevant contexts of the neurohormone action. In addition, we present an overview of the pathophysiological role of the CRH system, which highlights the need for a precise definition of CRHRs signaling at molecular level to identify novel targets for pharmacological intervention in neuroendocrine tissues and specific brain areas involved in CRH-related disorders.
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http://dx.doi.org/10.1530/EC-17-0111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551434PMC
August 2017

cAMP-dependent cell differentiation triggered by activated CRHR1 in hippocampal neuronal cells.

Sci Rep 2017 05 16;7(1):1944. Epub 2017 May 16.

Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET-Partner Institute of the Max Planck Society, Buenos Aires, Argentina.

Corticotropin-releasing hormone receptor 1 (CRHR1) activates the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). Both cAMP sources were shown to be required for the phosphorylation of ERK1/2 triggered by activated G protein coupled receptor (GPCR) CRHR1 in neuronal and neuroendocrine contexts. Here, we show that activated CRHR1 promotes growth arrest and neurite elongation in neuronal hippocampal cells (HT22-CRHR1 cells). By characterising CRHR1 signalling mechanisms involved in the neuritogenic effect, we demonstrate that neurite outgrowth in HT22-CRHR1 cells takes place by a sAC-dependent, ERK1/2-independent signalling cascade. Both tmACs and sAC are involved in corticotropin-releasing hormone (CRH)-mediated CREB phosphorylation and c-fos induction, but only sAC-generated cAMP pools are critical for the neuritogenic effect of CRH, further highlighting the engagement of two sources of cAMP downstream of the activation of a GPCR, and reinforcing the notion that restricted cAMP microdomains may regulate independent cellular processes.
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http://dx.doi.org/10.1038/s41598-017-02021-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5434020PMC
May 2017

Characterization of a Protein Interactome by Co-Immunoprecipitation and Shotgun Mass Spectrometry.

Methods Mol Biol 2017 ;1546:223-234

Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas (UNICAMP), Rua Monteiro Lobato 255, 13083-862, Campinas, SP, Brazil.

Identifying the partners of a given protein (the interactome) may provide leads about the protein's function and the molecular mechanisms in which it is involved. One of the alternative strategies used to characterize protein interactomes consists of co-immunoprecipitation (co-IP) followed by shotgun mass spectrometry. This enables the isolation and identification of a protein target in its native state and its interactome from cells or tissue lysates under physiological conditions. In this chapter, we describe a co-IP protocol for interactome studies that uses an antibody against a protein of interest bound to protein A/G plus agarose beads to isolate a protein complex. The interacting proteins may be further fractionated by SDS-PAGE, followed by in-gel tryptic digestion and nano liquid chromatography high-resolution tandem mass spectrometry (nLC ESI-MS/MS) for identification purposes. The computational tools, strategy for protein identification, and use of interactome databases also will be described.
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http://dx.doi.org/10.1007/978-1-4939-6730-8_19DOI Listing
February 2018

Different cAMP sources are critically involved in G protein-coupled receptor CRHR1 signaling.

J Cell Biol 2016 07 11;214(2):181-95. Epub 2016 Jul 11.

Instituto de Investigación en Biomedicina de Buenos Aires-CONICET-Partner Institute of the Max Planck Society, C1425FQD Buenos Aires, Argentina Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EHA Buenos Aires, Argentina

Corticotropin-releasing hormone receptor 1 (CRHR1) activates G protein-dependent and internalization-dependent signaling mechanisms. Here, we report that the cyclic AMP (cAMP) response of CRHR1 in physiologically relevant scenarios engages separate cAMP sources, involving the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). cAMP produced by tmACs and sAC is required for the acute phase of extracellular signal regulated kinase 1/2 activation triggered by CRH-stimulated CRHR1, but only sAC activity is essential for the sustained internalization-dependent phase. Thus, different cAMP sources are involved in different signaling mechanisms. Examination of the cAMP response revealed that CRH-activated CRHR1 generates cAMP after endocytosis. Characterizing CRHR1 signaling uncovered a specific link between CRH-activated CRHR1, sAC, and endosome-based signaling. We provide evidence of sAC being involved in an endocytosis-dependent cAMP response, strengthening the emerging model of GPCR signaling in which the cAMP response does not occur exclusively at the plasma membrane and introducing the notion of sAC as an alternative source of cAMP.
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http://dx.doi.org/10.1083/jcb.201512075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4949449PMC
July 2016

In silico structural and functional characterization of the RSUME splice variants.

PLoS One 2013 28;8(2):e57795. Epub 2013 Feb 28.

Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET, Buenos Aires, Argentina.

RSUME (RWD-containing SUMO Enhancer) is a small protein that increases SUMO conjugation to proteins. To date, four splice variants that codify three RSUME isoforms have been described, which differ in their C-terminal end. Comparing the structure of the RSUME isoforms we found that, in addition to the previously described RWD domain in the N-terminal, all these RSUME variants also contain an intermediate domain. Only the longest RSUME isoform presents a C-terminal domain that is absent in the others. Given these differences, we used the shortest and longest RSUME variants for comparative studies. We found that the C-terminal domain is dispensable for the SUMO-conjugation enhancer properties of RSUME. We also demonstrate that these two RSUME variants are equally induced by hypoxia. The NF-κB signaling pathway is inhibited and the HIF-1 pathway is increased more efficiently by the longest RSUME, by means of a greater physical interaction of RSUME267 with the target proteins. In addition, the mRNA and protein levels of these isoforms differ in human glioma samples; while the shortest RSUME isoform is expressed in all the tumors analyzed, the longest variant is expressed in most but not all of them. The results presented here show a degree of redundancy of the RSUME variants on the SUMO pathway. However, the increased inhibition conferred by RSUME267 over the NF-κB signaling pathway, the increased activation over the HIF-1 pathway and the different expression of the RSUME isoforms suggest specific roles for each RSUME isoform which may be relevant in certain types of brain tumors that express RSUME, like human pituitary adenomas and gliomas.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0057795PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3585135PMC
August 2013

B-Raf and CRHR1 internalization mediate biphasic ERK1/2 activation by CRH in hippocampal HT22 Cells.

Mol Endocrinol 2013 Mar 31;27(3):491-510. Epub 2013 Jan 31.

Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA), CONICET, Partner Institute of the Max Planck Society, Godoy Cruz 2390, C1425FQA Buenos Aires, Argentina.

CRH is a key regulator of neuroendocrine, autonomic, and behavioral response to stress. CRH-stimulated CRH receptor 1 (CRHR1) activates ERK1/2 depending on intracellular context. In a previous work, we demonstrated that CRH activates ERK1/2 in limbic areas of the mouse brain (hippocampus and basolateral amygdala). ERK1/2 is an essential mediator of hippocampal physiological processes including emotional behavior, synaptic plasticity, learning, and memory. To elucidate the molecular mechanisms by which CRH activates ERK1/2 in hippocampal neurons, we used the mouse hippocampal cell line HT22. We document for the first time that ERK1/2 activation in response to CRH is biphasic, involving a first cAMP- and B-Raf-dependent early phase and a second phase that critically depends on CRHR1 internalization and β-arrestin2. By means of mass-spectrometry-based screening, we identified B-Raf-associated proteins that coimmunoprecipitate with endogenous B-Raf after CRHR1 activation. Using molecular and pharmacological tools, the functional impact of selected B-Raf partners in CRH-dependent ERK1/2 activation was dissected. These results indicate that 14-3-3 proteins, protein kinase A, and Rap1, are essential for early CRH-induced ERK1/2 activation, whereas dynamin and vimentin are required for the CRHR1 internalization-dependent phase. Both phases of ERK1/2 activation depend on calcium influx and are affected by calcium/calmodulin-dependent protein kinase II inactivation. Thus, this report describes the dynamics and biphasic nature of ERK1/2 activation downstream neuronal CRHR1 and identifies several new critical components of the CRHR1 signaling machinery that selectively controls the early and late phases of ERK1/2 activation, thus providing new potential therapeutic targets for stress-related disorders.
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http://dx.doi.org/10.1210/me.2012-1359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5416933PMC
March 2013

The corticotropin-releasing hormone network and the hypothalamic-pituitary-adrenal axis: molecular and cellular mechanisms involved.

Neuroendocrinology 2011 13;94(1):12-20. Epub 2011 May 13.

Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología y Biología Molecular y Celular, Universidad de Buenos Aires, Argentina.

Corticotropin-releasing hormone (CRH) plays a key role in adjusting the basal and stress-activated hypothalamic-pituitary-adrenal axis (HPA). CRH is also widely distributed in extrahypothalamic circuits, where it acts as a neuroregulator to integrate the complex neuroendocrine, autonomic, and behavioral adaptive response to stress. Hyperactive and/or dysregulated CRH circuits are involved in neuroendocrinological disturbances and stress-related mood disorders such as anxiety and depression. This review describes the main physiological features of the CRH network and summarizes recent relevant information concerning the molecular mechanism of CRH action obtained from signal transduction studies using cells and wild-type and transgenic mice lines. Special focus is placed on the MAPK signaling pathways triggered by CRH through the CRH receptor 1 that plays an essential role in CRH action in pituitary corticotrophs and in specific brain structures. Recent findings underpin the concept of specific CRH-signaling pathways restricted to specific anatomical areas. Understanding CRH action at molecular levels will not only provide insight into the precise CRH mechanism of action, but will also be instrumental in identifying novel targets for pharmacological intervention in neuroendocrine tissues and specific brain areas involved in CRH-related disorders.
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http://dx.doi.org/10.1159/000328226DOI Listing
January 2012

Characterization of the B-Raf interactome in mouse hippocampal neuronal cells.

J Proteomics 2011 Feb 3;74(2):186-98. Epub 2010 Nov 3.

Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología y Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales (FCEN), Universidad de Buenos Aires, IFIBYNE-CONICET, Buenos Aires, Argentina.

B-Raf links a variety of extracellular stimuli downstream of cell surface receptors, constituting a determining factor in the ability of neurons to activate ERK. A detailed study of the B-Raf interactome is necessary to clarify the intricacy of B-Raf-dependent signal transduction. We used a mouse hippocampal cell line (HT22) that expresses B-Raf at high levels, to identify B-Raf associated proteins under endogenous expression conditions, avoiding artificial interactions from overexpression studies. We used stringent procedures to co-immunoprecipitate proteins that specifically associate with endogenous B-Raf with the help of gel electrophoresis separation and off-line LC-MALDI-MS/MS proteomic analysis. Our stringent protein identification criteria allowed confident identification of B-Raf interacting proteins under non-stimulating conditions. The presence of previously reported B-Raf interactors among the list of proteins identified confirms the quality of proteomic data. We identified tubulin and actin as B-Raf interactors for the first time, among structural and accessory proteins of cell cytoskeleton, molecular chaperones (Hsc70, GRP78), and cellular components involved in aspects of mRNA metabolism and translation. Interactions were validated in HT22 cells and in the neuronal cell line Neuro-2a providing further evidence that the identified proteins are B-Raf interactors, which constitute a basis for understanding MAPK pathway regulation in neurons.
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http://dx.doi.org/10.1016/j.jprot.2010.10.006DOI Listing
February 2011

Immunology, signal transduction, and behavior in hypothalamic-pituitary-adrenal axis-related genetic mouse models.

Ann N Y Acad Sci 2009 Feb;1153:120-30

Departamento de Fisiología y Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.

A classical view of the neuroendocrine-immune network assumes bidirectional interactions where pro-inflammatory cytokines influence hypothalamic-pituitary-adrenal (HPA) axis-derived hormones that subsequently affect cytokines in a permanently servo-controlled circle. Nevertheless, this picture has been continuously evolving over the last years as a result of the discovery of redundant expression and extended functions of many of the molecules implicated. Thus, cytokines are not only expressed in cells of the immune system but also in the central nervous system, and many hormones present at hypothalamic-pituitary level are also functionally expressed in the brain as well as in other peripheral organs, including immune cells. Because of this intermingled network of molecules redundantly expressed, the elucidation of the unique roles of HPA axis-related molecules at every level of complexity is one of the major challenges in the field. Genetic engineering in the mouse offers the most convincing method for dissecting in vivo the specific roles of distinct molecules acting in complex networks. Thus, various immunological, behavioral, and signal transduction studies performed with different HPA axis-related mutant mouse lines to delineate the roles of beta-endorphin, the type 1 receptor of corticotropin-releasing hormone (CRHR1), and its ligand CRH will be discussed here.
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http://dx.doi.org/10.1111/j.1749-6632.2008.03967.xDOI Listing
February 2009

Point mutations in the barley HvHAK1 potassium transporter lead to improved K+-nutrition and enhanced resistance to salt stress.

FEBS Lett 2008 Nov 31;582(28):3922-8. Epub 2008 Oct 31.

Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto Tecnológico de Chascomús, Camino Circunvalación km 6, Chascomús, Provincia de Buenos Aires 7130, Argentina.

Members of group I KT-HAK-KUP transporters play an important role in K+ acquisition by plant roots, a process that is strongly affected by salt stress. A PCR-based random mutagenesis approach on HvHAK1 allowed identification of V366I and R591C substitutions, which confer enhanced K+-capture, and improved NaCl, LiCl and NH4Cl tolerance, to yeast cells. Improved K+-capture was linked to an enhanced Vmax. Results reveal an intrinsic protective effect of K+, and assign an important role to the 8th transmembrane domain, as well as the C-terminus, in determining the maximum capacity for the transport of K+ in KT-HAK-KUP transporters.
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http://dx.doi.org/10.1016/j.febslet.2008.10.036DOI Listing
November 2008

RSUME, a small RWD-containing protein, enhances SUMO conjugation and stabilizes HIF-1alpha during hypoxia.

Cell 2007 Oct;131(2):309-23

Departamento de Fisiología y Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.

SUMO conjugation to proteins is involved in the regulation of diverse cellular functions. We have identified a protein, RWD-containing sumoylation enhancer (RSUME), that enhances overall SUMO-1, -2, and -3 conjugation by interacting with the SUMO conjugase Ubc9. RSUME increases noncovalent binding of SUMO-1 to Ubc9 and enhances Ubc9 thioester formation and SUMO polymerization. RSUME enhances the sumoylation of IkB in vitro and in cultured cells, leading to an inhibition of NF-kB transcriptional activity. RSUME is induced by hypoxia and enhances the sumoylation of HIF-1alpha, promoting its stabilization and transcriptional activity during hypoxia. Disruption of the RWD domain structure of RSUME demonstrates that this domain is critical for RSUME action. Together, these findings point to a central role of RSUME in the regulation of sumoylation and, hence, several critical regulatory pathways in mammalian cells.
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http://dx.doi.org/10.1016/j.cell.2007.07.044DOI Listing
October 2007

Molecular transduction mechanisms of cytokine-hormone interactions: role of gp130 cytokines.

Exp Physiol 2007 Sep;92(5):801-6

Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología y Biología Molecular y Celular, FCEN, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina.

Highly sophisticated mechanisms confer on the immune system the capacity to respond with a certain degree of autonomy. However, the final outcome of an immune response depends on the interaction of the immune system with other systems. The immune and neuroendocrine systems have an intimate cross-communication that makes possible a satisfactory response to environmental changes. Part of this interaction occurs through cytokines and steroid hormones. The last step of this cross-talk is the molecular level. As a model of interaction, this review focuses on the gp130 cytokine family. These cytokines, as well as their receptors, are expressed in pituitary cells. They regulate hormone production as well as growth of pituitary cells. During acute or chronic inflammation or infection, systemic, hypothalamic and hypophyseal gp130 cytokines act on anterior pituitary cells, integrating the neuroendocrine-immune response. Disruptions of these pathways may lead not only to abnormal growth of pituitary cells but also to immune disorders, for which, based on recent findings, targeting these cytokines might be a novel therapeutic approach.
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http://dx.doi.org/10.1113/expphysiol.2007.036236DOI Listing
September 2007

Pituitary action of cytokines: focus on BMP-4 and gp130 family.

Neuroendocrinology 2007 28;85(2):94-100. Epub 2007 Feb 28.

Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología y Biología Molecular y Celular, FCEN, Universidad de Buenos Aires, Buenos Aires, Argentina.

The anterior pituitary can develop benign tumors of different sizes, classified as micro- and macroadenomas, frequently associated with high levels of hormone production, leading to different associated syndromes like Cushing's disease, acromegaly or prolactinomas. Much work has been done in order to understand the signaling pathways and the factors and hormones involved in the pituitary tumorigenic process. In recent years, much evidence has been collected and it is now well documented that cytokines of the gp130 family, such as interleukin-6, that use gp130 as a common signaling protein stimulate not only the proliferation but also the hormone secretion of pituitary cells. Experiments in vivo have shown that the overexpression of the gp130 receptor resulted in pituitary abnormal growth. Moreover, it has been recently described that bone morphogenetic protein-4 (BMP-4), a member of the TGF-beta family, has a stimulatory role on lactosomatotropic cells promoting the development of prolactinomas but it has an inhibitory action on the corticotropic lineage. This inhibitory action prevents Cushing's disease progression. Furthermore, BMP-4 mediates the antiproliferative action of retinoic acid in these cells. The present review highlights the most recent work about gp130 and TGF-beta cytokine families and their role in pituitary tumorigenesis.
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http://dx.doi.org/10.1159/000100428DOI Listing
June 2007

Molecular understanding of cytokine-steroid hormone dialogue: implications for human diseases.

Ann N Y Acad Sci 2006 Nov;1088:297-306

Laboratorio de Fisiología y Biología Molecular, Departmento de Fisiologiía y Biooogía Molecular y Celular, Universidad de Buenos Aires, Ciudad Universitaria, Argentina.

Highly sophisticated mechanisms confer upon the immune system the capacity to respond with a certain degree of autonomy. However, the final outcome of an adaptative immune response depends on the interaction with other systems of the organism. The immune-neuroendocrine systems have an intimate cross-communication, making possible a satisfactory response to environmental changes. Part of this interaction occurs through cytokines and steroid hormones. The last step of this crosstalk is at the molecular level. In this article we will focus on the physical and functional interrelationship between cytokine signaling pathway-activated transcription factors (TFs) and steroid receptors in different cell models, where the signals triggered by cytokines and steroid hormones have major roles: (1) the ligand-dependent-activated glucocorticoid receptor (GR) influence the genetic program that specifies lineage commitment in T helper (Th) cell differentiation. How posttranslational modifications of several TFs as well as nuclear hormone receptors could be implicated in the molecular crosstalk between the immune-neuroendocrine messengers is discussed. (2) glucocorticoid (GC) antagonism on the TCR-induced T cell apoptosis. (3) estrogen receptor/TGF-beta family proteins molecular interaction implicated on pituitary prolactinomas pathogenesis. The functional crosstalk at the molecular level between immune and steroids signals is essential to determine an integrative response to both mediators (which in the last instance results in a new gene activation/repression profile) and constitutes the ultimate integrative level of interaction between the immune and neuroendocrine systems.
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http://dx.doi.org/10.1196/annals.1366.007DOI Listing
November 2006

The Saccharomyces cerevisiae YFR041C/ERJ5 gene encoding a type I membrane protein with a J domain is required to preserve the folding capacity of the endoplasmic reticulum.

Biochim Biophys Acta 2007 Feb 26;1773(2):232-42. Epub 2006 Oct 26.

IBYF-CONICET, Cátedra de Microbiología, Facultad de Agronomía, Universidad de Buenos Aires, Avda. San Martín 4453, C1417DSE Buenos Aires, Argentina.

YFR041C/ERJ5 was identified in Saccharomyces cerevisiae as a gene regulated by the unfolded protein response pathway (UPR). The open reading frame of the gene has a J domain characteristic of the DnaJ chaperone family of proteins that regulate the activity of Hsp70 chaperones. We determined the expression and topology of Erj5p, a type I membrane protein with a J domain in the lumen of the endoplasmic reticulum (ER) that colocalizes with Kar2p, the major Hsp70 in the yeast ER. We identified synthetic interactions of Deltaerj5 with mutations in genes involved in protein folding in the ER (kar2-159, Deltascj1Deltajem1) and in the induction of the unfolded protein response (Deltaire1). Loss of Erj5p in yeast cells with impaired ER protein folding capacity increased sensitivity to agents that cause ER stress. We identified the ERJ5 mRNA and confirmed that agents that promote accumulation of misfolded proteins in the ER regulate its abundance. We found that loss of the non-essential ERJ5 gene leads to a constitutively induced UPR, indicating that ERJ5 is required for maintenance of an optimal folding environment in the yeast ER.
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http://dx.doi.org/10.1016/j.bbamcr.2006.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1847348PMC
February 2007

Expression of TPK1 and TPK2 genes encoding PKA catalytic subunits during growth and morphogenesis in Candida albicans.

Yeast 2006 Jun;23(8):591-603

Cátedra de Microbiología, Facultad de Agronomía, Universidad de Buenos Aires, IBYF-CONICET, Avda. San Martín 4453, C1417DSE, Buenos Aires, Argentina.

The transcript levels of Candida albicans TPK1 and TPK2 genes, encoding PKA catalytic subunits, as well as phosphotransferase activity, were measured in the parental strain CAI4 and in homozygous tpk1Delta and tpk2Delta mutants during vegetative growth and during yeast-to-mycelial transition in N-acetylglucosamine liquid inducing medium at 37 degrees C. We observed two TPK2 transcripts, a major one of 1.8 kb and a minor one of 1.4 kb, and established by 3'-RACE that they originate from the recognition of the three polyadenylation signals present in the 3' untranslated region of the gene. During vegetative growth of CAI4 strain, the expression profiles of TPK1 and TPK2 varied similarly, reaching maximal expression at the late logarithmic phase. TPK1 mRNA levels were lower than those of TPK2 at all stages measured. In the corresponding homozygous tpk mutants, mRNA levels and the expression patterns of TPK1 and TPK2 were similar to those of CAI4, suggesting that the loss of one catalytic isoform is not compensated by overexpression of the other. Changes in PKA specific activity roughly correlated with fluctuations of mRNA expression levels. During yeast-to-mycelial transition, a sharp increase in TPK1 mRNA levels and in PKA-specific activity correlated with the onset of germ-tube formation in strain tpk2Delta. We also showed that tpk1Delta strain exhibited a delayed morphogenetic shift in comparison with CAI4 and tpk2Delta strains in several liquid inducing media, reinforcing the idea that Tpk1p is important for faster germ-tube appearance.
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http://dx.doi.org/10.1002/yea.1377DOI Listing
June 2006

Expression levels and subcellular localization of Bcy1p in Candida albicans mutant strains devoid of one BCY1 allele results in a defective morphogenetic behavior.

Biochim Biophys Acta 2006 Jan 27;1763(1):64-72. Epub 2005 Dec 27.

Cátedra de Microbiología, Facultad de Agronomía, Universidad de Buenos Aires, IBYF-CONICET, Avda. San Martín 4453, C1417DSE Buenos Aires, Argentina.

We investigated expression, functionality and subcellular localization of C. albicans Bcy1p, the PKA regulatory subunit, in mutant strains having one BCY1 allele fused to a green fluorescent protein (GFP). DE-52 column chromatography of soluble extracts of yeast cells from strains bearing one BCY1 allele (fused or not to GFP) showed co-elution of Bcy1p and Bcy1p-GFP with phosphotransferase activity, suggesting that interaction between regulatory and catalytic subunits was not impaired by the GFP tag. Subcellular localization of Bcy1p-GFP supports our previous hypothesis on the nuclear localization of the regulatory subunit, which can thus tether the PKA catalytic subunit to the nucleus. Protein modeling of CaBcy1p, showed that the fusion of the GFP tag to Bcy1p C-terminus did not significantly disturb its proper folding. Bcy1p levels in mutant strains having one or both BCY1 alleles, led us to establish a direct correlation between the amount of protein and the number of alleles, indicating that deletion of one BCY1 allele is not fully compensated by overexpression of the other. The morphogenetic behavior of several C. albicans mutant strains bearing one or both BCY1 alleles, in a wild-type and in a TPK2 null genetic background, was assessed in N-acetylglucosamine (GlcNAc) liquid medium at 37 degrees C. Strains with one BCY1 allele tagged or not, behaved similarly, displaying pseudohyphae and true hyphae. In contrast, hyphal morphology was almost exclusive in strains having both BCY1 alleles, irrespective of the GFP insertion. It can be inferred that a tight regulation of PKA activity is needed for hyphal growth.
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http://dx.doi.org/10.1016/j.bbamcr.2005.11.016DOI Listing
January 2006

Candida albicans lacking the gene encoding the regulatory subunit of protein kinase A displays a defect in hyphal formation and an altered localization of the catalytic subunit.

Eukaryot Cell 2004 Feb;3(1):190-9

Cátedra de Microbiología, Facultad de Agronomía, Universidad de Buenos Aires, IBYF-CONICET, C1417DSE Buenos Aires, Argentina.

The fungal pathogen Candida albicans switches from a yeast-like to a filamentous mode of growth in response to a variety of environmental conditions. We examined the morphogenetic behavior of C. albicans yeast cells lacking the BCY1 gene, which encodes the regulatory subunit of protein kinase A. We cloned the BCY1 gene and generated a bcy1 tpk2 double mutant strain because a homozygous bcy1 mutant in a wild-type genetic background could not be obtained. In the bcy1 tpk2 mutant, protein kinase A activity (due to the presence of the TPK1 gene) was cyclic AMP independent, indicating that the cells harbored an unregulated phosphotransferase activity. This mutant has constitutive protein kinase A activity and displayed a defective germinative phenotype in N-acetylglucosamine and in serum-containing medium. The subcellular localization of a Tpk1-green fluorescent protein (GFP) fusion protein was examined in wild-type, tpk2 null, and bcy1 tpk2 double mutant strains. The fusion protein was observed to be predominantly nuclear in wild-type and tpk2 strains. This was not the case in the bcy1 tpk2 double mutant, where it appeared dispersed throughout the cell. Coimmunoprecipitation of Bcy1p with the Tpk1-GFP fusion protein demonstrated the interaction of these proteins inside the cell. These results suggest that one of the roles of Bcy1p is to tether the protein kinase A catalytic subunit to the nucleus.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC329502PMC
http://dx.doi.org/10.1128/ec.3.1.190-199.2004DOI Listing
February 2004