DOCSLIB.ORG

Identification of Potential Glycoprotein Biomarkers in Estrogen Receptor

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Identification of Potential Glycoprotein Biomarkers in Estrogen Receptor Journal of Cancer 2012, 3 269 Ivyspring International Publisher Journal of Cancer 2012; 3: 269-284. doi: 10.7150/jca.4592 Research Paper Identification of Potential Glycoprotein Biomarkers in Estrogen Receptor Positive (ER+) and Negative (ER-) Human Breast Cancer Tissues by LC-LTQ/FT-ICR Mass Spectrometry Suzan M. Semaan1, Xu Wang1, Alan G. Marshall1,2, Qing-Xiang Amy Sang1 1. Department of Chemistry and Biochemistry and Institute of Molecular Biophysics; 2. Center for Interdisciplinary Magnetic Resonance, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32306-4390, USA. Corresponding author: Tel: +1-850-644-8683. Fax: +1-850-644-8281. Email: [email protected]. Room 3501, Chemical Sciences Labor- atory Building, 102 Varsity Way, Florida State University, Tallahassee, Florida 32306-4390, U.S.A. © Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/ licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. Received: 2012.05.14; Accepted: 2012.06.20; Published: 2012.06.21 Abstract Breast cancer is the second most fatal cancer in American women. To increase the life ex- pectancy of patients with breast cancer new diagnostic and prognostic biomarkers and drug targets must be identified. A change in the glycosylation on a glycoprotein often causes a change in the function of that glycoprotein; such a phenomenon is correlated with cancerous transformation. Thus, glycoproteins in human breast cancer estrogen receptor positive (ER+) tissues and those in the more advanced stage of breast cancer, estrogen receptor negative (ER-) tissues, were compared. Glycoproteins showing differences in glycosylation were examined by 2-dimensional gel electrophoresis with double staining (glyco- and total protein staining) and identified by reversed-phase nano-liquid chromatography coupled with a hybrid linear quadrupole ion trap/ Fourier transform ion cyclotron resonance mass spectrometer. Among the identified glycosylated proteins are alpha 1 acid glycoprotein, alpha-1-antitrypsin, calmodulin, and superoxide dismutase mitochondrial precursor that were further verified by Western blotting for both ER+ and ER- human breast tissues. Results show the presence of a possible glycosylation difference in alpha-1-antitrypsin, a potential tumor-derived biomarker for breast cancer progression, which was expressed highest in the ER- samples. Key words: Two-dimensional gel electrophoresis; Estrogen receptor positive and negative breast cancer; Biomarkers; Alpha-1-antitrypsin; Fourier transform ion cyclotron resonance mass spec- trometry; Proteomics Introduction Breast cancer, if cancers of the skins are exclud- carcinomas will be diagnosed in 2012 [1]. Based on ed, is the most frequently diagnosed cancer in Amer- proteomic and genomic studies, prognostic biomarker ican women and ranks immediately after lung cancer candidates identified from breast cancer include in mortality. According to the American Cancer Soci- growth factor receptors, steroid receptors, p53, Ki-67, ety Breast Cancer Facts and Figures, approximately cyclins, BRCA1 and BRCA2, urokinase plasminogen 39,510 women (i.e., 14.4% of those diagnosed) will die activator, p21, and pro- and anti-apoptotic factors [2]. from breast cancer in the USA. In 2012, estimated new The Food and Drug Administration has approved breast cancer occurrences will reach 226,870, not in- biomarkers CA 15.3, human epidermal growth factor cluding in situ carcinoma. About 63,300 in situ breast receptor 2 (Her-2/neu), and CA27-29. However, the http://www.jcancer.org Journal of Cancer 2012, 3 270 combination of several biomarkers may produce the Furthermore, those cancer-related peptide epitopes best prognosis [3]. have been exploited as immunogens for cancer [32]. Breast cancer development and progression are Therefore, identification of glycoproteins in the ER+ regulated by estrogen and its receptor (ER) that is one and ER- breast cancer tissues might reveal possible of the predictive markers including progesterone re- target(s) and identify putative biomarkers or tumor ceptor (PR), Her-2/neu, and ER for breast cancer. Yet, suppressors for pro- and pre-aggressive breast cancer the anti-estrogen drug tamoxifen is often ineffective stages. Here, we compare glycoproteins in breast even after estrogen receptor positivity [2]. Previous cancer ER+ tissue and those in ER- tissue, the more research has revealed that the tumorigenic epithelial aggressive breast cancer, after sample cells in the breast duct are ER positive (ER+) cells N-deglycosylation to compare glycosylation differ- overlying an intact myoepithelial membrane, but ences with controls and double staining of the some adjacent cells overlying a focally disrupted two-dimensional electrophoresis (2-DE) gels. Glyco- myoepithelial cell layer have lost their response to proteins were identified by reversed-phase anti-estrogen drugs because they no longer express nano-liquid chromatography coupled with a hybrid the ER. Moreover, the loss of the ER of those once linear quadrupole ion trap/ Fourier transform ion tumorigenic cells at the invasion site contributes to cyclotron resonance mass spectrometer their higher potential for aggressiveness and invasion (LC-LTQ/FT-ICR MS) and the glycosylation type for [4-6]. Therefore, it is important to search for new selected breast cancer-related proteins was verified by prognostic and predictive markers for breast cancer Western blotting. progression from ER+ to ER-. Many fundamental cellular processes such as Materials and Methods cellular differentiation, control of cell division, Chemicals and materials cell–cell recognition, adhesion, and malignant trans- Ready immobilized pH gradient (IPG) Strips, pH formation are controlled by oligosaccharides [7-10]. In 4-7, 11 cm long, were purchased from Bio-Rad. Dithi- most cases, oligosaccharides are conjugated with othreitol was purchased from Promega Corporation other biomolecules known as glycol-conjugates. This (Madison, WI). Bromophenol blue, Tris, SDS, Tricine, process is observed within the endoplasmic reticu- Ponseau S solution, bovine serum albumin (BSA), and lum, Golgi apparatus, nucleus, and the cytoplasm of a cholamidopropyl dimethylammonio propanesul- cell. More than half of all proteins are glycosylated, fonate hydrate (CHAPS) were purchased from Sig- either N- and/or O-linked [11]. Within the endo- ma-Aldrich. Glacial acetic acid was purchased from plasmic reticulum and Golgi apparatus, O-linked EMD chemicals Inc. Urea was purchased from Fisher glycans are usually attached to the peptide chain by a Scientific. Primary polyclonal anti-human rabbit an- serine or threonine hydroxyl group. Mostly within the tibodies against ER-α MC-20 (MW 66 kDa), calmodu- endoplasmic reticulum, N-linked glycans, the pre- lin FL-149 (MW 17 kDa), and superoxide dismutase dominant carbohydrate attachment in mammalian FL-222 (MW 25 kDa) were purchased from Santa Cruz cells [12], are linked to the side chain by means of an Biotechnology, Inc. (Santa Cruz, CA). Polyclonal rab- amide bond to asparagine in a 60-65% conserved bit anti-human alpha-1-antitrypsin A0012 (MW ~57 Asn-Xxx-Ser/Thr motif (in which Xxx can be any kDa) was purchased from DakoCytomation, Den- amino acid except proline) [11, 13-14]. N-linked car- mark. Rabbit polyclonal anti-human alpha-1-acid bohydrates are frequently found together with glycoprotein ab440-2 (MW ~50 kDa) was bought from O-linked oligosaccharides in proteins such as fetuin Abcam. Anti-rabbit IgG (whole molecule) peroxidase [15], insulin receptor [16], IgG immunoglobulins [17], antibody A0545 produced in goat and monoclonal and O-linked glycoproteins include collagens [18], mouse anti-β-actin antibody A5316 (MW 42 kDa) mucins [19], and proteoglycans [20]. were purchased from Sigma-Aldrich. Protease and In breast cancer cells, changes in O-linked [21-26] phosphatase inhibitor cocktails were purchased from and N-linked [27-28] oligosaccharide structures are Thermo-Pierce (Rockford, IL). frequently correlated with cancerous transformation. For example, breast carcinomas may display altered Sample preparation expression of secreted and membrane-bound mucins, Human ductal breast carcinoma tissue samples such as MUC1 and MUC2 [29]. When epithelial were obtained from the Cooperative Human Tissue cell-surface mucin MUC1 is abnormally glycosylated, Network-Midwestern Division. Tissues were cut into its expression shows up-regulation in breast cancer 1 mm cubes on ice [33]. Tissues were suspended in [30]. As a result, new epitopes are exposed leading to cold tissue lysis buffer in 10 mM Tris pH 7.5, 130 mM humoral and cell-mediated immune responses [31]. NaCl, 1% octylglucopyranoside (OG), 1% CHAPS, 10 http://www.jcancer.org Journal of Cancer 2012, 3 271 mM sodium phosphate, 10 mM sodium fluoride, and teins, and antibodies were diluted in 1% BSA in TBST. 1x phosphatase and protease inhibitor (EDTA-free). For multiple probing on the same nitrocellulose, the Samples were centrifuged for 15 min at 14,000 rpm at membrane was treated with 30% H2O2 for 15 min at 37 4 ˚C to remove insoluble material. The supernatant ºC while covered, then rinsed with TBST, followed by layer was transferred to a new tube, and the pellet and blocking and normal probing procedure [35]. The the top lipid layer were discarded. Samples were then H2O2 inactivates the
Load more

Recommended publications
  • Calmodulin and Calmodulin-Dependent Protein Kinase II Inhibit Hormone Secretion in Human Parathyroid Adenoma
    31 Calmodulin and calmodulin-dependent protein kinase II inhibit hormone secretion in human parathyroid adenoma Ming Lu1,2,3, Erik Berglund1, Catharina Larsson1,3, Anders Ho¨o¨g4, Lars-Ove Farnebo1 and Robert Bra¨nstro¨m1 1Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital L1:03, SE-171 76 Stockholm, Sweden 2Department of Geriatric Endocrinology, First Affiliated Hospital of Guangxi Medical University, NanNing, People’s Republic of China 3Center for Molecular Medicine (CMM), Karolinska University Hospital, SE-171 76 Stockholm, Sweden 4Department of Oncology–Pathology, Karolinska Institutet, Karolinska University Hospital, SE-171 76 Stockholm, Sweden (Correspondence should be addressed to M Lu at Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital; Email: [email protected]) Abstract 2C 2C Intracellular calcium ([Ca ]i) is the most relevant modulator adenoma cells in spite of increased [Ca ]i. The inhibitory C of parathyroid hormone (PTH) secretion. Uniquely, an effect of Ca2 calmodulin on PTH secretion may be due to 2C increase in [Ca ]i results in an inhibition of PTH secretion, the absence of synaptotagmin 1 protein in parathyroid and it probably exerts its function via calcium-binding protein adenomas, as demonstrated by western blot analysis. An pathways. The ubiquitous calcium-binding proteins, calmo- increased extracellular calcium level acutely lowered the dulin and calmodulin-dependent protein kinase II (CaMKII), amount of active phosphorylated CaMKII (pCaMKII) in have well-established roles in regulated exocytosis in neurons adenoma cells in vitro, indicating the physiological importance and neuroendocrine cells. However, their roles in parathyroid of this pathway. Moreover, a negative correlation between the cells and PTH secretion are still unclear.
    [Show full text]
  • Bioinformatic Analyses in Early Host Response To
    Schroyen et al. BMC Genomics (2016) 17:196 DOI 10.1186/s12864-016-2547-z RESEARCH ARTICLE Open Access Bioinformatic analyses in early host response to Porcine Reproductive and Respiratory Syndrome virus (PRRSV) reveals pathway differences between pigs with alternate genotypes for a major host response QTL Martine Schroyen1†, Christopher Eisley2†, James E. Koltes3, Eric Fritz-Waters1, Igseo Choi4, Graham S. Plastow5, Leluo Guan5, Paul Stothard5, Hua Bao5, Arun Kommadath5, James M. Reecy1, Joan K. Lunney4, Robert R. R. Rowland6, Jack C. M. Dekkers1 and Christopher K. Tuggle1* Abstract Background: AregiononSus scrofa chromosome 4 (SSC4) surrounding single nucleotide polymorphism (SNP) marker WUR10000125 (WUR) has been reported to be strongly associated with both weight gain and serum viremia in pigs after infection with PRRS virus (PRRSV). A proposed causal mutation in the guanylate binding protein 5 gene (GBP5)is predicted to truncate the encoded protein. To investigate transcriptional differences between WUR genotypes in early host response to PRRSV infection, an RNA-seq experiment was performed on globin depleted whole blood RNA collectedon0,4,7,10and14dayspost-infection(dpi)from eight littermate pairs with one AB (favorable) and one AA (unfavorable) WUR genotype animal per litter. Results: Gene Ontology (GO) enrichment analysis of transcripts that were differentially expressed (DE) between dpi across both genotypes revealed an inflammatory response for all dpi when compared to day 0. However, at the early time points of 4 and 7dpi, several GO terms had higher enrichment scores compared to later dpi, including inflammatory response (p <10-7), specifically regulation of NFkappaB (p < 0.01), cytokine, and chemokine activity (p <0.01).At10and 14dpi, GO term enrichment indicated a switch to DNA damage response, cell cycle checkpoints, and DNA replication.
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • Serial Analysis of Gene Expression in Normal P53 Null Mammary Epithelium
    Oncogene (2002) 21, 6366 – 6376 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc Serial analysis of gene expression in normal p53 null mammary epithelium C Marcelo Aldaz*,1, Yuhui Hu1, Rachael Daniel1, Sally Gaddis1, Frances Kittrell2 and Daniel Medina2 1The University of Texas M.D. Anderson Cancer Center, Department of Carcinogenesis, Smithville, Texas, TX 78957, USA; 2Baylor College of Medicine Department of Molecular and Cellular Biology, Houston, Texas, TX 77030, USA Much evidence has accumulated implicating the p53 gene function although activating mutations were also as of importance in breast carcinogenesis. However, observed. Usually p53 abnormalities associate with much still remains to be uncovered on the specific poorer clinical outcome. This, likely, is the consequence downstream pathways influenced by this important of the known critical roles of p53 in regulating the cell activator/repressor of transcription. This study investi- cycle, apoptosis, DNA repair and maintaining genome gated the effects of a p53 null genotype on the stability (Levine, 1997). The loss of wild type p53 transcriptome of ‘normal’ mouse mammary epithelium function is clearly an important event in breast using a unique in vivo model of preneoplastic transforma- tumorigenesis as documented both in human and murine tion. We used SAGE for the comparative analysis of p53 systems (Donehower et al., 1995; Elledge and Allred, wild type (wt) and null mammary epithelium unexposed 1994). However, the exact mechanisms by which such and exposed to hormonal stimulation. Analysis of the lack of normal gene function leads to cancer formation hormone exposed samples provided a comprehensive view and progression are only beginning to be understood.
    [Show full text]
  • Versão Do Arquivo Anexado / Version of Attached File: Versão Do Editor / Published Version
    UNIVERSIDADE ESTADUAL DE CAMPINAS SISTEMA DE BIBLIOTECAS DA UNICAMP REPOSITÓRIO DA PRODUÇÃO CIENTIFICA E INTELECTUAL DA UNICAMP Versão do arquivo anexado / Version of attached file: Versão do Editor / Published Version Mais informações no site da editora / Further information on publisher's website: https://physoc.onlinelibrary.wiley.com/doi/full/10.14814/phy2.12409 DOI: 10.14814/phy2.12409 Direitos autorais / Publisher's copyright statement: ©2015 by John Wiley & Sons. All rights reserved. DIRETORIA DE TRATAMENTO DA INFORMAÇÃO Cidade Universitária Zeferino Vaz Barão Geraldo CEP 13083-970 – Campinas SP Fone: (19) 3521-6493 http://www.repositorio.unicamp.br Physiological Reports ISSN 2051-817X ORIGINAL RESEARCH Expression of calcium-buffering proteins in rat intrinsic laryngeal muscles Renato Ferretti1, Maria Julia Marques2, Tejvir S. Khurana3 & Humberto Santo Neto2 1 Departamento de Anatomia, Instituto de Biociencias de Botucatu, Universidade Estadual Paulista, Botucatu, Sao~ Paulo, Brazil 2 Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Sao~ Paulo, Brazil 3 Department of Physiology, Perelman School of Medicine and Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, Pennsylvania Keywords Abstract Calcium, laryngeal muscles, Ncx, Pmca1, Serca, store-operated calcium entry. Intrinsic laryngeal muscles (ILM) are highly specialized muscles involved in phonation and airway protection, with unique properties that allow them to Correspondence perform extremely rapid contractions and to escape from damage in muscle Humberto Santo Neto, Departamento de dystrophy. Due to that, they may differ from limb muscles in several physio- Biologia Estrutural e Funcional, Instituto de logical aspects. Because a better ability to handle intracellular calcium has been Biologia, Universidade Estadual de Campinas suggested to explain ILM unique properties, we hypothesized that the profile (UNICAMP).
    [Show full text]
  • Gene Expression Signatures and Biomarkers of Noninvasive And
    Oncogene (2006) 25, 2328–2338 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Gene expression signatures and biomarkers of noninvasive and invasive breast cancer cells: comprehensive profiles by representational difference analysis, microarrays and proteomics GM Nagaraja1, M Othman2, BP Fox1, R Alsaber1, CM Pellegrino3, Y Zeng2, R Khanna2, P Tamburini3, A Swaroop2 and RP Kandpal1 1Department of Biological Sciences, Fordham University, Bronx, NY, USA; 2Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI, USA and 3Bayer Corporation, West Haven, CT, USA We have characterized comprehensive transcript and Keywords: representational difference analysis; micro- proteomic profiles of cell lines corresponding to normal arrays; proteomics; breast carcinoma; biomarkers; breast (MCF10A), noninvasive breast cancer (MCF7) and copper homeostasis invasive breast cancer (MDA-MB-231). The transcript profiles were first analysed by a modified protocol for representational difference analysis (RDA) of cDNAs between MCF7 and MDA-MB-231 cells. The majority of genes identified by RDA showed nearly complete con- Introduction cordance withmicroarray results, and also led to the identification of some differentially expressed genes such The transformation of a normal cell into a cancer cell as lysyl oxidase, copper transporter ATP7A, EphB6, has been correlated to altered expression of a variety of RUNX2 and a variant of RUNX2. The altered transcripts genes (Perou et al., 2000; Becker et al., 2005). The identified by microarray analysis were involved in cell–cell expression of some of these genes is a direct result of or cell–matrix interaction, Rho signaling, calcium home- sequence mutation, whereas other changes occur due to ostasis and copper-binding/sensitive activities.
    [Show full text]
  • High-Throughput Screen Detects Calcium Signaling Dysfunction in Hutchinson-Gilford Progeria Syndrome
    International Journal of Molecular Sciences Article High-Throughput Screen Detects Calcium Signaling Dysfunction in Hutchinson-Gilford Progeria Syndrome Juan A. Fafián-Labora , Miriam Morente-López, Fco. Javier de Toro and María C. Arufe * Grupo de Terapia Celular y Medicina Regenerativa, Departamento de Fisioterapia, Ciencias Biomédicas y Medicina, Universdidade da Coruña, Agrupación Estratégica INIBIC-CICA, 15006 A Coruña, Spain; [email protected] (J.A.F.-L.); [email protected] (M.M.-L.); [email protected] (F.J.d.T.) * Correspondence: [email protected] Abstract: Hutchinson–Gilford progeria syndrome (HGPS) is a deadly childhood disorder, which is considered a very rare disease. It is caused by an autosomal dominant mutation on the LMNA gene, and it is characterized by accelerated aging. Human cell lines from HGPS patients and healthy parental controls were studied in parallel using next-generation sequencing (NGS) to unravel new non-previously altered molecular pathways. Nine hundred and eleven transcripts were differentially expressed when comparing healthy versus HGPS cell lines from a total of 21,872 transcripts; ITPR1, ITPR3, CACNA2D1, and CAMK2N1 stood out among them due to their links with calcium signaling, and these were validated by Western blot analysis. It was observed that the basal concentration of intracellular Ca2+ was statistically higher in HGPS cell lines compared to healthy ones. The relationship between genes involved in Ca2+ signaling and mitochondria-associated membranes (MAM) was demonstrated through cytosolic calcium handling by means of an automated fluorescent plate reading system (FlexStation 3, Molecular Devices), and apoptosis and mitochondrial ROS Citation: Fafián-Labora, J.A.; production were examined by means of flow cytometry analysis.
    [Show full text]
  • New Approach for Untangling the Role of Uncommon Calcium-Binding Proteins in the Central Nervous System
    brain sciences Review New Approach for Untangling the Role of Uncommon Calcium-Binding Proteins in the Central Nervous System Krisztina Kelemen * and Tibor Szilágyi Department of Physiology, Doctoral School, Faculty of Medicine, George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, 540142 Târgu Mures, , Romania; [email protected] * Correspondence: [email protected]; Tel.: +40-746-248064 Abstract: Although Ca2+ ion plays an essential role in cellular physiology, calcium-binding proteins (CaBPs) were long used for mainly as immunohistochemical markers of specific cell types in different regions of the central nervous system. They are a heterogeneous and wide-ranging group of proteins. Their function was studied intensively in the last two decades and a tremendous amount of informa- tion was gathered about them. Girard et al. compiled a comprehensive list of the gene-expression profiles of the entire EF-hand gene superfamily in the murine brain. We selected from this database those CaBPs which are related to information processing and/or neuronal signalling, have a Ca2+- buffer activity, Ca2+-sensor activity, modulator of Ca2+-channel activity, or a yet unknown function. In this way we created a gene function-based selection of the CaBPs. We cross-referenced these findings with publicly available, high-quality RNA-sequencing and in situ hybridization databases (Human Protein Atlas (HPA), Brain RNA-seq database and Allen Brain Atlas integrated into the HPA) and created gene expression heat maps of the regional and cell type-specific expression levels of the selected CaBPs. This represents a useful tool to predict and investigate different expression patterns and functions of the less-known CaBPs of the central nervous system.
    [Show full text]
  • Calmodulin-Dependent Activation of the Epithelial Calcium-Dependent Chloride Channel TMEM16A
    The FASEB Journal • Research Communication Calmodulin-dependent activation of the epithelial calcium-dependent chloride channel TMEM16A Yuemin Tian,* Patthara Kongsuphol,* Martin Hug,‡ Jiraporn Ousingsawat,* Ralph Witzgall,† Rainer Schreiber,*,1 and Karl Kunzelmann*,1,2 *Institut fu¨r Physiologie and †Institut fu¨r Anatomie, Universita¨t Regensburg, Regensburg, Germany; and ‡Apotheke der Universita¨tsklinik Freiburg, Freiburg, Germany 2ϩ ABSTRACT TMEM16A (anoctamin 1, Ano1), a mem- sion, time dependence and amplitude of the current, Ca ber of a family of 10 homologous proteins, has been dependence, and sensitivity toward inhibitors vary among ؉ shown to form an essential component of Ca2 -activated TMEM16 proteins. Preliminary data suggest that these pro- ؊ Cl channels. TMEM16A-null mice exhibit severe defects teins can exist as heterooligomeric complexes (6). in epithelial transport along with tracheomalacia and TMEM16A shows common characteristics of the native death within 1 mo after birth. Despite its outstanding CaCCs, present in a large number of tissues and cell types physiological significance, the mechanisms for activation (1–3, 7, 8). We and others demonstrated the fundamental ϩ Ϫ of TMEM16A remain obscure. TMEM16A is activated on importance of TMEM16A for Ca2 -dependent Cl secretion ؉ increase in intracellular Ca2 , but it is unclear whether in a number of epithelial tissues (9–11). TMEM16A is ؉ ϩ Ϫ Ca2 binds directly to the channel or whether addi- essential for Ca2 -dependent Cl currents in airways, large tional components are required. We demonstrate intestine, salivary gland, pancreatic gland, and hepatocytes that TMEM16A is strictly membrane localized and (9–11). Severe transport defects were detected in epithelial requires cytoskeletal interactions to be fully acti- tissues of TMEM16A-knockout mice, leading to reduced vated.
    [Show full text]
  • Expression of Calciumbuffering Proteins in Rat Intrinsic Laryngeal
    Physiological Reports ISSN 2051-817X ORIGINAL RESEARCH Expression of calcium-buffering proteins in rat intrinsic laryngeal muscles Renato Ferretti1, Maria Julia Marques2, Tejvir S. Khurana3 & Humberto Santo Neto2 1 Departamento de Anatomia, Instituto de Biociencias de Botucatu, Universidade Estadual Paulista, Botucatu, Sao~ Paulo, Brazil 2 Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Sao~ Paulo, Brazil 3 Department of Physiology, Perelman School of Medicine and Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, Pennsylvania Keywords Abstract Calcium, laryngeal muscles, Ncx, Pmca1, Serca, store-operated calcium entry. Intrinsic laryngeal muscles (ILM) are highly specialized muscles involved in phonation and airway protection, with unique properties that allow them to Correspondence perform extremely rapid contractions and to escape from damage in muscle Humberto Santo Neto, Departamento de dystrophy. Due to that, they may differ from limb muscles in several physio- Biologia Estrutural e Funcional, Instituto de logical aspects. Because a better ability to handle intracellular calcium has been Biologia, Universidade Estadual de Campinas suggested to explain ILM unique properties, we hypothesized that the profile (UNICAMP). Campinas, SP - 13083-970, of the proteins that regulate calcium levels in ILM is different from that in a Brazil. Tel: +55-19-3521-6395 limb muscle. Calcium-related proteins were analyzed in the ILM, cricothyroid Fax: 55-19-3521-6185
    [Show full text]
  • Calcineurin Is an Important Factor Involved in Glucose Uptake in Human Adipocytes
    Molecular and Cellular Biochemistry (2018) 445:157–168 https://doi.org/10.1007/s11010-017-3261-0 Calcineurin is an important factor involved in glucose uptake in human adipocytes Ana Catarina R. G. Fonseca1,2 · Eugénia Carvalho2,3,4,5 · Jan W. Eriksson1 · Maria J. Pereira1 Received: 27 October 2017 / Accepted: 23 December 2017 / Published online: 27 January 2018 © The Author(s) 2018. This article is an open access publication Abstract Calcineurin inhibitors are used in immunosuppressive therapy applied after transplantation, but they are associated with major metabolic side effects including the development of new onset diabetes. Previously, we have shown that the calcineu- rin inhibiting drugs tacrolimus and cyclosporin A reduce adipocyte and myocyte glucose uptakes by reducing the amount of glucose transporter type 4 (GLUT4) at the cell surface, due to an increased internalization rate. However, this happens without alteration in total protein and phosphorylation levels of key proteins involved in insulin signalling or in the total amount of GLUT4. The present study evaluates possible pathways involved in the altered internalization of GLUT4 and consequent reduction of glucose uptake provoked by calcineurin inhibitors in human subcutaneous adipose tissue. Short- and long-term treatments with tacrolimus, cyclosporin A or another CNI deltamethrin (herbicide) decreased basal and insulin- dependent glucose uptake in adipocytes, without any additive effects observed when added together. However, no tacrolimus effects were observed on glucose uptake when gene transcription and protein translation were inhibited. Investigation of genes potentially involved in GLUT4 trafficking showed only a small effect on ARHGEF11 gene expression (p < 0.05). In conlusion, the specific inhibition of calcineurin, but not that of protein phosphatases, decreases glucose uptake in human subcutaneous adipocytes, suggesting that calcineurin is an important regulator of glucose transport.
    [Show full text]
  • Supplemental Figure 1. Protein-Protein Interaction Network with Increased Expression in Fteb During the Luteal Phase
    Supplemental Figure 1. Protein-protein interaction network with increased expression in FTEb during the luteal phase. Supplemental Figure 2. Protein-protein interaction network with decreased expression in FTEb during luteal phase. LEGENDS TO SUPPLEMENTAL FIGURES Supplemental Figure 1. Protein-protein interaction network with increased expression in FTEb during the luteal phase. Submission of probe sets differentially expressed in the FTEb specimens that clustered with SerCa as well as those specifically altered in FTEb luteal samples to the online I2D database revealed overlapping networks of proteins with increased expression in the four FTEb samples and/or FTEb luteal samples overall. Proteins are represented by nodes, and known and predicted first-degree interactions are represented by solid lines. Genes encoding proteins shown as large ovals highlighted in blue were exclusively found in the first comparison (Manuscript Figure 2), whereas those highlighted in red were only found in the second comparison (Manuscript Figure 3). Genes encoding proteins shown as large ovals highlighted in black were found in both comparisons. The color of each node indicates the ontology of the corresponding protein as determined by the Online Predicted Human Interaction Database (OPHID) link with the NAViGaTOR software. Supplemental Figure 2. Protein-protein interaction network with decreased expression in FTEb during the luteal phase. Submission of probe sets differentially expressed in the FTEb specimens that clustered with SerCa as well as those specifically altered in FTEb luteal samples to the online I2D database revealed overlapping networks of proteins with decreased expression in the four FTEb samples and/or FTEb luteal samples overall. Proteins are represented by nodes, and known and predicted first-degree interactions are represented by solid lines.
    [Show full text]