Age-Related Decline in Expression of Calnexin

Total Page:16

File Type:pdf, Size:1020Kb

Age-Related Decline in Expression of Calnexin EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 36, No. 5, 499-503, October 2004 Age-related decline in expression of calnexin Bok-Hee Choi1 and Jong-Suk Kim1,2 age-related diseases such as cancer, cardiovascular disorders, sclerosis and nephropathies (Salvioli et al., 1Department of Biochemistry 1994). In deed, cardiovascular diseases represent one Chonbuk National University Medical School of the fatal disease among an ageing population. Jeonju 561-756, Korea Although blockade of the coronary vasculature is 2Correspondence author: Tel, 82-63-270-3085; known as a common cause of damage to myocar- Fax, 82-63-274-9833; E-mail, [email protected] dium, other changes in cellular level that occur during the aging process result in decreasing function of myocardium and rendering it more susceptible to Accepted 21 October 2004 damages (Verbeke et al., 2001; Taylor and Starnes, Abbreviations: ER, endoplasmic reticulum; ESI Q-TOF MS, electro- 2003). Several reports have already described a de- spray ionization quadupole-time of flight mass spectrometry; HDF, creased capacity of the aging myocardium to different human diploid fibroblast; HSP, heat shock protein; MALDI-TOF types of stresses (Gray et al., 2000; Verbeke et al., MS, matrix-assisted laser desorption/ionization-time of flight mass 2001; Boraldi et al., 2003; Taylor and Starnes, 2003). Furthermore, age-related alterations in the expression spectrometry and activation of proteins are known to be important for the cardioprotective process such as heat shock protein (HSP) 70 (Taylor and Starnes, 2003). Abstract Heat shock response is one of primary intracellular defense mechanisms against stressful conditions. Aging is accompanied by the changes in the Number of heat shock proteins and molecular cha- cells that decrease their capacity to respond to perones are known to be induced by variety of stimuli various forms of stress. Cells are known to re- such as elevated temperature, ischemia, hypoxia, spond to stresses through expression of stress- pressure overload and oxidative stress (Walsh et al., response proteins, heat-shock proteins com- 1997; Feder and Hofmann, 1999; Fehrenbach and posed of molecular chaperones. Recent studies Niess, 1999; Gray et al., 2000; Jolly and Morimoto, suggest that chaperone level and stress-induced 2000; Verbeke et al., 2001). Interestingly, cells iso- chaperone expression could decrease with ag- lated from aged tissues and organisms, and cells ing. The aim of the present study is to identify undergoing replicative senescence in vitro, have a chaperones that show a significant change in reduced heat shock response (Liu et al., 1996; protein expression with aging. We used an in Verbeke et al., 2001; Taylor and Starnes, 2003). vitro aging model system of human diploid Proteomics analysis has provided a tool to examine fibroblasts (HDF). Proteome analysis of HDF minor changes in cellular protein expression as the showed that endoplasmic reticulum (ER) chape- technique develops in enhancing sensitivity and al- lowed to utilize the molecular mechanisms of cellular rone, calnexin, significantly decreased with ag- aging (Toda, 2000; Schoneich, 2003). ing. Oxidative stress-induced expression of cal- In this study, proteomics tool was used for the nexin also attenuated in old HDF compared to analysis of HSP in aging cells. The aim of the present young cells. These findings suggest calnexin study is to identify age-related HSP that shows a decreases with aging and might contribute to a significant decline in protein expression with aging by cytoprotection in a variety of human age-related employing an in vitro aging model system of human diseases. diploid fibroblasts (HDF). Proteins of the cells were separated by two-dimensional electrophoresis and Keywords: Calnexin; Chaperone; Heat shock protein; protein identification was performed by matrix-assisted Proteomics; Senescence laser desorption/ionization-time of flight mass spectro- metry (MALDI-TOF MS). Introduction Materials and Methods Aging is related to a decrease in the stress response in vitro as well as in vivo and could be seen via Cell culture several cellular alterations, that can be responsible for HDF was kept in 10-cm plates in Dulbecco's modified 500 Exp. Mol. Med. Vol. 36(5), 499-503, 2004 Eagle's medium supplemented with 10% fetal bovine II (50 mM Tris-HCl (pH 8.8), 6 M Urea, 30% Glycerol, serum and antibiotics at a humid atmosphere (5% 2% SDS, 0.25% IAA). The second dimension was run CO2, 95% air) and subculture at a ratio of 1:4. We according to Ettan DALT II system operating and defined young cells as those resulting from less than maintenance manual. A 12.5% SDS-polyacrylamide 20 population doublings (PD), and old cells were from slab gel was used for the second dimension gel more than 40 PD. Cellular senescence of all old cells electrophoresis. The IPG strips were placed on the was confirmed by their delayed population doubling surface of the second dimension gel, and then the times and by a senescence-associated β-galacto- IPG strips were sealed with 0.5% agarose in SDS- sidase activity assay, as described by Dimri et al. electrophresis buffer (25 mM Tris base, 192 mM (1995). glycine, 0.1% SDS). The gels were placed into Ettan DALT II system chamber containing 1×SDS electro- phoresis buffer. The gels were run overnight at 110 Western blotting V until the front dye reached the bottom of the gel. Cells were harvested in the lysis buffer (20 mM Hepes pH 7.2, 1% TritonX-100, 150 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, and Silver staining 1 µg/ml aprotinin). After incubation for 30 min at 4oC, Silver staining of gels was performed as modified cellular debris was removed by centrifugation at silver staining protocol using Silver Stain Plus One kit. 100,000 g for 30 min, and supernatants were ana- The use of glutaraldehyde in the sensitization step lyzed by sodium dodesyl sulfate-polyacrylamide gel and formaldehyde in the silver impregnation step was electrophoresis (SDS-PAGE). The protein concentra- omitted. After electrophoresis, the gels were fixed with tion was determined by the Bio-Rad protein assay 40% methanol/10% acetic acid for 30 min. The gels reagent (Bio-Rad Laboratories, LA). Samples (50 µg) were sensitized by incubating in sensitizing solution were prepared with the four volume of 0.5 M Tris-HCl (0.2% sodium thiosulphate, 30% methanol, sodium buffer (pH 6.8) containing 4% SDS, 20% glycerol and acetate 68 g/l), and it was then rinsed with three 0.05% bromophenol blue at 95oC for 5 min. SDS- changes of distilled water for 5 min each. After PAGE was performed in 10% slab gel. Proteins were rinsing, the gels were incubated in 0.25% silver nitrate transferred to nitrocellulose paper. The membrane for 20 min. After incubation, the silver nitrate was was washed in blocking buffer (10 mM Tris-HCl, pH discarded, and the gels were rinsed twice with dis- 8.0, 150 mM NaCl, 5% fat-free milk) for 60 min at tilled water for 1 min and then developed in 0.15% room temperature with shaking and then washed with formaldehyde in 2.5% sodium carbonate with inten- TBST (TBS, 0.01% Tween 20). Blots were incubated sive shaking. After the desired intensity of staining with the primary antibodies against calnexin (Santa was achieved, the development was terminated with Cruz, CA) followed by horseradish peroxidase-con- 1.46% EDTA. jugated secondary antibody. Image analysis Two-dimensional SDS-PAGE We digitized the silver-stained gel image using a After lysis buffer (8 M Urea, 4% CHAPS, 40 mM Tris scanner (UMAX Power Look III). Image Master 2D base, 1% DTT, 0.5% immobilized pH gradient (IPG) (Amersham Pharmacia) was used to analyze the gel buffer, protease inhibitor) was added to HDF, the images. sample suspension was sonicated in short bursts on ice. The lysate was centrifuged in a microcentrifuge o Destaining at 30,000 g for 15 min at 18 C. The supernatant was saved at -80oC until used. The first dimension of 2-D Silver-stained proteins were destained with chemical electrophoresis was performed on an Amersham Pha- reducers to remove the silver as described previously macia Biotech IPG phor Isoelectric Focusing System. with following critical modifications (Scheler et al., Linear pH 3-10 Immobiline Dry Strips (24 cm) were 1998; Song and Lee, 2001). Potassium ferricyanide rehydrated overnight at room temperature in rehy- and sodium thiosulfate were prepared as two stock drating buffer (8 M Urea, 1% DTT, 2% CHAPS, 0.5% solutions of 30 mM and 100 mM, respectively and IPG buffer, BPB). Sample (500 µg) was applied dur- both dissolved in water. A working solution was pre- ing rehydration. The first dimension was run for pared by mixing a 1:1 ratio prior to use. After in- 53,500 V/h using the following conditions at 20oC: 500 teresting protein spots were excised from the gel, V for 1 h, 1,000 V for 1 h; 8,000 V for 6 h 30 min. thirty to fifty microliters of working solution were Next, gels were equilibrated for 30 min in equilibration added to cover the gels and occasionally vortexed. buffer I (50 mM Tris-HCl (pH 8.8), 6 M Urea, 30% The stain intensity was monitored until the brownish Glycerol, 2% SDS, 0.1% DTT) and equilibration buffer color disappeared, then the gel was rinsed several Age-related decline in expression of calnexin 501 Figure 1. Typical electrophoretic patterns of proteins in young HDF cells (PD 21) and old HDF cells (PD 45) on a 2-D gel. Cellular proteins were separated by 2-D PAGE using IPG-Dalt, digested on membranes in the presence of 50% acetonitrile, then analyzed by MALDI-TOF MS.
Recommended publications
  • Profiling of Transcripts and Proteins Modulated by the E7 Oncogene in the Lung Tissue of E7-Tg Mice by the Omics Approach
    MOLECULAR MEDICINE REPORTS 2: 129-137, 2009 129 Profiling of transcripts and proteins modulated by the E7 oncogene in the lung tissue of E7-Tg mice by the omics approach EUNJIN KIM1*, JEONGWOO KANG1,3*, MINCHUL CHO1, SOJUNG LEE1, EUNHEE SEO1, HEESOOK CHOI1, YUMI KIM1, JUNGHEE KIM1, KUM YONG KANG2, KWANG PYO KIM2, JAEYONG HAN3, YHUNYHONG SHEEN4, YOUNG NA YUM5, SUE-NIE PARK5 and DO-YOUNG YOON1 Departments of 1Bioscience and Biotechnology, and 2Molecular Biotechnology, Konkuk University, Hwayang-dong 1, Gwangjin-gu, Seoul 143-701; 3Laboratory of Animal Genetic Engineering, Department of Food and Animal Biotechnology, Seoul National University, Seoul 151-742; 4School of Pharmacy, Ewha Womans University, Seoul 120-750; 5Korea Food and Drug Administration, #194 Tongil-ro, Eunpyung-gu, Seoul 122-704, Korea Received August 18, 2008; Accepted November 10, 2008 DOI: 10.3892/mmr_00000073 Abstract. The E6 and E7 oncoproteins of human papilloma suggest that the E7 oncogene modulates the expression levels virus (HPV) type 16 have been known to cooperatively induce of cell cycle-related (cyclin B1, cyclin E2) and cell adhesion- the immortalization and transformation of primary keratino- and migration-related (actinin ·1, CD166) factors, which may cytes. We established an E7 transgenic mouse model to play important roles in cellular transformation in cancer. In screen HPV-related biomakers using the omics approach. addition, the solubilization of the rigid intermediate filament The methods used to identify HPV-modulated factors were network by specific proteolysis mediated via up-regulating genomics analysis by microarray using the Affymetrix 430 gelsolin and down-regulating cofilin-1, as well as increased 2.0 array to screen E7-modulated genes, and proteomics levels of endoplasmic reticulum protein calnexin with chap- analysis using nano-LC-ESI-MS/MS to screen E7-modulated erone functions, might also be involved in E7-lung epithelial proteins with the lung tissue of E7 transgenic mice.
    [Show full text]
  • Calreticulin—Multifunctional Chaperone in Immunogenic Cell Death: Potential Significance As a Prognostic Biomarker in Ovarian
    cells Review Calreticulin—Multifunctional Chaperone in Immunogenic Cell Death: Potential Significance as a Prognostic Biomarker in Ovarian Cancer Patients Michal Kielbik *, Izabela Szulc-Kielbik and Magdalena Klink Institute of Medical Biology, Polish Academy of Sciences, 106 Lodowa Str., 93-232 Lodz, Poland; [email protected] (I.S.-K.); [email protected] (M.K.) * Correspondence: [email protected]; Tel.: +48-42-27-23-636 Abstract: Immunogenic cell death (ICD) is a type of death, which has the hallmarks of necroptosis and apoptosis, and is best characterized in malignant diseases. Chemotherapeutics, radiotherapy and photodynamic therapy induce intracellular stress response pathways in tumor cells, leading to a secretion of various factors belonging to a family of damage-associated molecular patterns molecules, capable of inducing the adaptive immune response. One of them is calreticulin (CRT), an endoplasmic reticulum-associated chaperone. Its presence on the surface of dying tumor cells serves as an “eat me” signal for antigen presenting cells (APC). Engulfment of tumor cells by APCs results in the presentation of tumor’s antigens to cytotoxic T-cells and production of cytokines/chemokines, which activate immune cells responsible for tumor cells killing. Thus, the development of ICD and the expression of CRT can help standard therapy to eradicate tumor cells. Here, we review the physiological functions of CRT and its involvement in the ICD appearance in malignant dis- ease. Moreover, we also focus on the ability of various anti-cancer drugs to induce expression of surface CRT on ovarian cancer cells. The second aim of this work is to discuss and summarize the prognostic/predictive value of CRT in ovarian cancer patients.
    [Show full text]
  • A Selective ER-Phagy Exerts Procollagen Quality Control Via a Calnexin-FAM134B Complex
    Article A selective ER-phagy exerts procollagen quality control via a Calnexin-FAM134B complex Alison Forrester1,†, Chiara De Leonibus1,†, Paolo Grumati2,†, Elisa Fasana3,†, Marilina Piemontese1, Leopoldo Staiano1, Ilaria Fregno3,4, Andrea Raimondi5, Alessandro Marazza3,6, Gemma Bruno1, Maria Iavazzo1, Daniela Intartaglia1, Marta Seczynska2, Eelco van Anken7, Ivan Conte1, Maria Antonietta De Matteis1,8, Ivan Dikic2,9,* , Maurizio Molinari3,10,** & Carmine Settembre1,11,*** Abstract The EMBO Journal (2019) 38:e99847 Autophagy is a cytosolic quality control process that recognizes substrates through receptor-mediated mechanisms. Procollagens, Introduction the most abundant gene products in Metazoa, are synthesized in the endoplasmic reticulum (ER), and a fraction that fails to attain Macroautophagy (hereafter referred to as autophagy) is a homeostatic the native structure is cleared by autophagy. However, how auto- catabolic process devoted to the sequestration of cytoplasmic material phagy selectively recognizes misfolded procollagens in the ER in double-membrane vesicles (autophagic vesicles, AVs) that eventu- lumen is still unknown. We performed siRNA interference, CRISPR- ally fuse with lysosomes where cargo is degraded (Mizushima, 2011). Cas9 or knockout-mediated gene deletion of candidate autophagy Autophagy is essential to maintain tissue homeostasis and counter- and ER proteins in collagen producing cells. We found that the ER- acts both the onset and progression of many disease conditions, such resident lectin chaperone Calnexin (CANX) and the ER-phagy as ageing, neurodegeneration and cancer (Levine et al, 2015). receptor FAM134B are required for autophagy-mediated quality Substrates can be selectively delivered to AVs through receptor- control of endogenous procollagens. Mechanistically, CANX acts as mediated processes. Autophagy receptors harbour a LC3 or GABARAP co-receptor that recognizes ER luminal misfolded procollagens and interaction motif (LIR or GIM, respectively) that facilitate binding of interacts with the ER-phagy receptor FAM134B.
    [Show full text]
  • Detection of Pro Angiogenic and Inflammatory Biomarkers in Patients With
    www.nature.com/scientificreports OPEN Detection of pro angiogenic and infammatory biomarkers in patients with CKD Diana Jalal1,2,3*, Bridget Sanford4, Brandon Renner5, Patrick Ten Eyck6, Jennifer Laskowski5, James Cooper5, Mingyao Sun1, Yousef Zakharia7, Douglas Spitz7,9, Ayotunde Dokun8, Massimo Attanasio1, Kenneth Jones10 & Joshua M. Thurman5 Cardiovascular disease (CVD) is the most common cause of death in patients with native and post-transplant chronic kidney disease (CKD). To identify new biomarkers of vascular injury and infammation, we analyzed the proteome of plasma and circulating extracellular vesicles (EVs) in native and post-transplant CKD patients utilizing an aptamer-based assay. Proteins of angiogenesis were signifcantly higher in native and post-transplant CKD patients versus healthy controls. Ingenuity pathway analysis (IPA) indicated Ephrin receptor signaling, serine biosynthesis, and transforming growth factor-β as the top pathways activated in both CKD groups. Pro-infammatory proteins were signifcantly higher only in the EVs of native CKD patients. IPA indicated acute phase response signaling, insulin-like growth factor-1, tumor necrosis factor-α, and interleukin-6 pathway activation. These data indicate that pathways of angiogenesis and infammation are activated in CKD patients’ plasma and EVs, respectively. The pathways common in both native and post-transplant CKD may signal similar mechanisms of CVD. Approximately one in 10 individuals has chronic kidney disease (CKD) rendering CKD one of the most common diseases worldwide1. CKD is associated with a high burden of morbidity in the form of end stage kidney disease (ESKD) requiring dialysis or transplantation 2. Furthermore, patients with CKD are at signifcantly increased risk of death from cardiovascular disease (CVD)3,4.
    [Show full text]
  • Genetic, Cytogenetic and Physical Refinement of the Autosomal Recessive CMT Linked to 5Q31ð Q33: Exclusion of Candidate Genes I
    European Journal of Human Genetics (1999) 7, 849–859 © 1999 Stockton Press All rights reserved 1018–4813/99 $15.00 t http://www.stockton-press.co.uk/ejhg ARTICLE Genetic, cytogenetic and physical refinement of the autosomal recessive CMT linked to 5q31–q33: exclusion of candidate genes including EGR1 Ang`ele Guilbot1, Nicole Ravis´e1, Ahmed Bouhouche6, Philippe Coullin4, Nazha Birouk6, Thierry Maisonobe3, Thierry Kuntzer7, Christophe Vial8, Djamel Grid5, Alexis Brice1,2 and Eric LeGuern1,2 1INSERM U289, 2F´ed´eration de Neurologie and 3Laboratoire de Neuropathologie R Escourolle, Hˆopital de la Salpˆetri`ere, Paris 4Laboratoire de cytog´en´etique, Villejuif 5G´en´ethon, Evry, France 6Service de Neurologie, Hˆopital des Sp´ecialit´es, Rabat, Morocco 7Service de Neurologie, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland 8Service D’EMG et de pathologie neuromusculaire, Hˆopital neurologique Pierre Wertheimer, Lyon, France Charcot-Marie-Tooth disease is an heterogeneous group of inherited peripheral motor and sensory neuropathies with several modes of inheritance: autosomal dominant, X-linked and autosomal recessive. By homozygosity mapping, we have identified, in the 5q23–q33 region, a third locus responsible for an autosomal recessive form of demyelinating CMT. Haplotype reconstruction and determination of the minimal region of homozygosity restricted the candidate region to a 4 cM interval. A physical map of the candidate region was established by screening YACs for microsatellites used for genetic analysis. Combined genetic, cytogenetic and physical mapping restricted the locus to a less than 2 Mb interval on chromosome 5q32. Seventeen consanguineous families with demyelinating ARCMT of various origins were screened for linkage to 5q31–q33.
    [Show full text]
  • Anoctamin 1 (Tmem16a) Ca -Activated Chloride Channel Stoichiometrically Interacts with an Ezrin–Radixin–Moesin Network
    Anoctamin 1 (Tmem16A) Ca2+-activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network Patricia Perez-Cornejoa,1, Avanti Gokhaleb,1, Charity Duranb,1, Yuanyuan Cuib, Qinghuan Xiaob, H. Criss Hartzellb,2, and Victor Faundezb,2 aPhysiology Department, School of Medicine, Universidad Autónoma de San Luis Potosí, San Luis Potosí, SLP 78210, Mexico; and bDepartment of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322 Edited by David E. Clapham, Howard Hughes Medical Institute, Children’s Hospital Boston, Boston, MA, and approved May 9, 2012 (received for review January 4, 2012) The newly discovered Ca2+-activated Cl− channel (CaCC), Anocta- approach to identify Ano1-interacting proteins. We find that min 1 (Ano1 or TMEM16A), has been implicated in vital physiolog- Ano1 forms a complex with two high stochiometry interactomes. ical functions including epithelial fluid secretion, gut motility, and One protein network is centered on the signaling/scaffolding smooth muscle tone. Overexpression of Ano1 in HEK cells or Xen- actin-binding regulatory proteins ezrin, radixin, moesin, and opus oocytes is sufficient to generate Ca2+-activated Cl− currents, RhoA. The ezrin–radixin–moesin (ERM) proteins organize the but the details of channel composition and the regulatory factors cortical cytoskeleton by linking actin to the plasma membrane that control channel biology are incompletely understood. We and coordinate cell signaling events by scaffolding signaling used a highly sensitive quantitative SILAC proteomics approach molecules (19). The other major interactome is centered on the to obtain insights into stoichiometric protein networks associated SNARE and SM proteins VAMP3, syntaxins 2 and -4, and the with the Ano1 channel.
    [Show full text]
  • Profiling of Transcripts and Proteins Modulated by the E7 Oncogene in the Lung Tissue of E7-Tg Mice by the Omics Approach
    MOLECULAR MEDICINE REPORTS 2: 129-137, 2009 129 Profiling of transcripts and proteins modulated by the E7 oncogene in the lung tissue of E7-Tg mice by the omics approach EUNJIN KIM1*, JEONGWOO KANG1,3*, MINCHUL CHO1, SOJUNG LEE1, EUNHEE SEO1, HEESOOK CHOI1, YUMI KIM1, JUNGHEE KIM1, KUM YONG KANG2, KWANG PYO KIM2, JAEYONG HAN3, YHUNYHONG SHEEN4, YOUNG NA YUM5, SUE-NIE PARK5 and DO-YOUNG YOON1 Departments of 1Bioscience and Biotechnology, and 2Molecular Biotechnology, Konkuk University, Hwayang-dong 1, Gwangjin-gu, Seoul 143-701; 3Laboratory of Animal Genetic Engineering, Department of Food and Animal Biotechnology, Seoul National University, Seoul 151-742; 4School of Pharmacy, Ewha Womans University, Seoul 120-750; 5Korea Food and Drug Administration, #194 Tongil-ro, Eunpyung-gu, Seoul 122-704, Korea Received August 18, 2008; Accepted November 10, 2008 DOI: 10.3892/mmr_00000073 Abstract. The E6 and E7 oncoproteins of human papilloma suggest that the E7 oncogene modulates the expression levels virus (HPV) type 16 have been known to cooperatively induce of cell cycle-related (cyclin B1, cyclin E2) and cell adhesion- the immortalization and transformation of primary keratino- and migration-related (actinin ·1, CD166) factors, which may cytes. We established an E7 transgenic mouse model to play important roles in cellular transformation in cancer. In screen HPV-related biomakers using the omics approach. addition, the solubilization of the rigid intermediate filament The methods used to identify HPV-modulated factors were network by specific proteolysis mediated via up-regulating genomics analysis by microarray using the Affymetrix 430 gelsolin and down-regulating cofilin-1, as well as increased 2.0 array to screen E7-modulated genes, and proteomics levels of endoplasmic reticulum protein calnexin with chap- analysis using nano-LC-ESI-MS/MS to screen E7-modulated erone functions, might also be involved in E7-lung epithelial proteins with the lung tissue of E7 transgenic mice.
    [Show full text]
  • Transcriptomic Landscape of Breast Cancers Through Mrna Sequencing Jeyanthy Eswaran George Washington University
    Himmelfarb Health Sciences Library, The George Washington University Health Sciences Research Commons Biochemistry and Molecular Medicine Faculty Biochemistry and Molecular Medicine Publications 2-14-2012 Transcriptomic landscape of breast cancers through mRNA sequencing Jeyanthy Eswaran George Washington University Dinesh Cyanam George Washington University Prakriti Mudvari George Washington University Sirigiri Divijendra Natha Reddy George Washington University Suresh Pakala George Washington University See next page for additional authors Follow this and additional works at: http://hsrc.himmelfarb.gwu.edu/smhs_biochem_facpubs Part of the Biochemistry, Biophysics, and Structural Biology Commons Recommended Citation Eswaran, J., Cyanam, D., Mudvari, P., Reddy, S., Pakala, S., Nair, S., Florea, L., & Fuqua, S. (2012). Transcriptomic landscape of breast cancers through mrna sequencing. Scientific Reports, 2, 264. This Journal Article is brought to you for free and open access by the Biochemistry and Molecular Medicine at Health Sciences Research Commons. It has been accepted for inclusion in Biochemistry and Molecular Medicine Faculty Publications by an authorized administrator of Health Sciences Research Commons. For more information, please contact [email protected]. Authors Jeyanthy Eswaran, Dinesh Cyanam, Prakriti Mudvari, Sirigiri Divijendra Natha Reddy, Suresh Pakala, Sujit S. Nair, Liliana Florea, Suzanne A.W. Fuqua, Sucheta Godbole, and Rakesh Kumar This journal article is available at Health Sciences Research Commons: http://hsrc.himmelfarb.gwu.edu/smhs_biochem_facpubs/3
    [Show full text]
  • Proteomics of Serum Extracellular Vesicles Identifies a Novel COPD Biomarker, Fibulin-3 from Elastic Fibres
    ORIGINAL ARTICLE COPD Proteomics of serum extracellular vesicles identifies a novel COPD biomarker, fibulin-3 from elastic fibres Taro Koba 1, Yoshito Takeda1, Ryohei Narumi2, Takashi Shiromizu2, Yosui Nojima 3, Mari Ito3, Muneyoshi Kuroyama1, Yu Futami1, Takayuki Takimoto4, Takanori Matsuki1, Ryuya Edahiro1, Satoshi Nojima5, Yoshitomo Hayama1, Kiyoharu Fukushima1, Haruhiko Hirata1, Shohei Koyama1, Kota Iwahori1, Izumi Nagatomo1, Mayumi Suzuki1, Yuya Shirai1, Teruaki Murakami1, Kaori Nakanishi 1, Takeshi Nakatani1, Yasuhiko Suga1, Kotaro Miyake1, Takayuki Shiroyama1, Hiroshi Kida 1, Takako Sasaki6, Koji Ueda7, Kenji Mizuguchi3, Jun Adachi2, Takeshi Tomonaga2 and Atsushi Kumanogoh1 ABSTRACT There is an unmet need for novel biomarkers in the diagnosis of multifactorial COPD. We applied next-generation proteomics to serum extracellular vesicles (EVs) to discover novel COPD biomarkers. EVs from 10 patients with COPD and six healthy controls were analysed by tandem mass tag-based non-targeted proteomics, and those from elastase-treated mouse models of emphysema were also analysed by non-targeted proteomics. For validation, EVs from 23 patients with COPD and 20 healthy controls were validated by targeted proteomics. Using non-targeted proteomics, we identified 406 proteins, 34 of which were significantly upregulated in patients with COPD. Of note, the EV protein signature from patients with COPD reflected inflammation and remodelling. We also identified 63 upregulated candidates from 1956 proteins by analysing EVs isolated from mouse models. Combining human and mouse biomarker candidates, we validated 45 proteins by targeted proteomics, selected reaction monitoring. Notably, levels of fibulin-3, tripeptidyl- peptidase 2, fibulin-1, and soluble scavenger receptor cysteine-rich domain-containing protein were significantly higher in patients with COPD.
    [Show full text]
  • Human Induced Pluripotent Stem Cell–Derived Podocytes Mature Into Vascularized Glomeruli Upon Experimental Transplantation
    BASIC RESEARCH www.jasn.org Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation † Sazia Sharmin,* Atsuhiro Taguchi,* Yusuke Kaku,* Yasuhiro Yoshimura,* Tomoko Ohmori,* ‡ † ‡ Tetsushi Sakuma, Masashi Mukoyama, Takashi Yamamoto, Hidetake Kurihara,§ and | Ryuichi Nishinakamura* *Department of Kidney Development, Institute of Molecular Embryology and Genetics, and †Department of Nephrology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; ‡Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan; §Division of Anatomy, Juntendo University School of Medicine, Tokyo, Japan; and |Japan Science and Technology Agency, CREST, Kumamoto, Japan ABSTRACT Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in
    [Show full text]
  • Exophagy of Annexin A2 Via RAB11, RAB8A and RAB27A in IFN-Γ
    www.nature.com/scientificreports OPEN Exophagy of annexin A2 via RAB11, RAB8A and RAB27A in IFN-γ- stimulated lung epithelial cells Received: 23 September 2016 Ying-Da Chen1, Yi-Ting Fang2, Yi-Lin Cheng1,2, Chiou-Feng Lin3,4, Li-Jin Hsu1,3,5, Shu-Ying Accepted: 7 June 2017 Wang1,2,3, Robert Anderson3,6, Chih-Peng Chang1,2,3 & Yee-Shin Lin1,2,3 Published: xx xx xxxx Annexin A2 (ANXA2), a phospholipid-binding protein, has multiple biological functions depending on its cellular localization. We previously demonstrated that IFN-γ-triggered ANXA2 secretion is associated with exosomal release. Here, we show that IFN-γ-induced autophagy is essential for the extracellular secretion of ANXA2 in lung epithelial cells. We observed colocalization of ANXA2- containing autophagosomes with multivesicular bodies (MVBs) after IFN-γ stimulation, followed by exosomal release. IFN-γ-induced exophagic release of ANXA2 could not be observed in ATG5-silenced or mutant RAB11-expressing cells. Furthermore, knockdown of RAB8A and RAB27A, but not RAB27B, reduced IFN-γ-triggered ANXA2 secretion. Surface translocation of ANXA2 enhanced efferocytosis by epithelial cells, and inhibition of different exophagic steps, including autophagosome formation, fusion of autophagosomes with MVBs, and fusion of amphisomes with plasma membrane, reduced ANXA2- mediated efferocytosis. Our data reveal a novel route of IFN-γ-induced exophagy of ANXA2. Annexin A2 (ANXA2), a Ca2+-dependent membrane-binding protein, can distribute to the nucleus, the cytosolic membrane of organelles, as well as the inner and outer leaflets of the plasma membrane in different cell types, including macrophages, endothelium, epithelium, and tumor cells1–3.
    [Show full text]
  • Prostate Cancer Cell Extracellular Vesicles Increase Mineralisation of Bone Osteoblast Precursor Cells in an in Vitro Model
    biology Article Prostate Cancer Cell Extracellular Vesicles Increase Mineralisation of Bone Osteoblast Precursor Cells in an In Vitro Model Ben Lanning 1,2, Jason Webber 2,3 , Pinar Uysal-Onganer 4 , Wen Guo Jiang 1 , Aled Clayton 2 and Dafydd Alwyn Dart 1,5,* 1 Cardiff China Medical Research Collaborative, Cardiff University School of Medicine, Cardiff CF14 4YS, UK; [email protected] (B.L.); [email protected] (W.G.J.) 2 Tissue Microenvironment Group, Division of Cancer & Genetics, School of Medicine, Cardiff University, Cardiff CF14 4YS, UK; [email protected] (J.W.); [email protected] (A.C.) 3 Institute of Life Science, Swansea University Medical School, Swansea University, Singleton Park, Swansea SA2 8PP, UK 4 School of Life Sciences, College of Liberal Arts and Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK; [email protected] 5 Institute of Medical and Biomedical Education, St George’s University of London, Cranmer Terrace, Tooting, London SW17 0RE, UK * Correspondence: [email protected] Simple Summary: Prostate cancer frequently metastasizes to the bone, where it forms primarily osteoblastic lesions. Currently there is no real therapeutic option for this late stage of disease, and understanding prostate cancer-bone interaction and communication is vital. Using a simple in vitro model of os-teoblast differentiation and mineralization, we studied this interaction and observed that prostate cancer cells secreted large quantities of extracellular vesicles containing microRNAs. When Citation: Lanning, B.; Webber, J.; ex-posed to the extracellular vesicles, increased osteoblast differentiation and mineralization could be Uysal-Onganer, P.; Jiang, W.G.; observed, and upon RNA-seq several of these microRNAs were implicated as upstream regulators of Clayton, A.; Dart, D.A.
    [Show full text]