DOCSLIB.ORG

Open Thesismaster.Pdf

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Open Thesismaster.Pdf The Pennsylvania State University The Graduate School Department of Biochemistry and Molecular Biology MEMBRANE ASYMMETRY IN APOPTOTIC CELLS, SIGNALS ASSOCIATED WITH PHAGOCYTOSIS AND THEIR IMPACT ON HIV TRANSCRIPTION A Thesis in Biochemistry, Microbiology and Molecular Biology by Bethsebah Nyankomba Gekonge Ó 2006 Bethsebah Nyankomba Gekonge Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2006 The thesis of Bethsebah Nyankomba Gekonge was reviewed and approved* by the following: Andrew J. Henderson Associate Professor of Veterinary Science Thesis Co-advisor Co-chair of Committee Robert A. Schlegel Professor of Biochemistry and Molecular Biology Head of the Department of Biochemistry and Molecular Biology Thesis Co-advisor Co-chair of Committee Andrea M. Mastro Professor of Biochemistry and Molecular Biology Pamela H. Correll Associate Professor of Veterinary Science Biao He Assistant Professor of Veterinary Science *Signatures are on file in the Graduate School iii ABSTRACT Transbilayer membrane phospholipid asymmetry is the defining feature between healthy viable cells and their apoptotic counterparts. Healthy cells maintain all their phosphatidylserine (PS) and a large proportion of their phosphatidylethanolamine (PE) in the inner leaflet of the plasma membrane. Loss of asymmetry and exposure of PS on the cell surface targets apoptotic cells for destruction by macrophages. An aminophospholipid translocase is responsible for maintaining this asymmetry. Since all cells maintain this asymmetric distribution, all cells must have a plasma membrane aminophospholipid translocase; however, the protein responsible for this activity has never been identified. Subfamily IV P-type ATPases comprise amphipathic transporters whose members have yet undetermined function. If a type IV P-type ATPase were the plasma membrane aminophospholipid translocase, it should be expressed in all cells. Using RT-PCR to measure gene expression, I demonstrate that multiple subfamily IV P-type ATPase members are simultaneously expressed in a single mammalian cell type. Of the ten genes analyzed, 1c, 1d, 2a, 2b, 5c, 6f, 6g and 6h were expressed in J774 cells, NIH 3T3 cells, and PC12 cells, suggesting a more general or housekeeping role for these genes; however, these genes also stand as candidates for the plasma membrane aminophospholipid translocase. 1a was expressed in J774 cells and PC12 cells and not in the NIH 3T3 cells, suggesting that 1a may have a specialized function in these cells. PS expression on the surface of apoptotic cells is involved in their removal from tissue in order to maintain overall homeostasis. The engagement of phagocytic receptors by ligands such as PS found on the apoptotic cell surface results in the activation of iv signaling cascades that facilitate engulfment. Macrophages and CD4+ T cells are the primary targets of HIV infection; however, unlike HIV infected macrophages which are invulnerable to the cytopathic effects of HIV, infected CD4+ T cells as well as uninfected bystander T cells undergo apoptosis. Consequently, these infected apoptotic cells as well as the HIV virions that are released from them have external PS. I examined how PS associated with virions and apoptotic cells influences HIV replication. Using annexin V and PS vesicles to mask, or compete with virus-associated PS, respectively, I show that PS inhibits HIV infection at a step prior to integration but following the generation of early reverse transcription products such as strong stop DNA. These observations suggest that PS initiated signals participate in HIV infection. Moreover, apoptotic cells inhibited HIV transcription in macrophages that have an established infection. This ability of apoptotic cells to suppress transcription was independent of PS, signifying that signals triggered following the interaction between an undetermined recognition ligand on the apoptotic cell surface and phagocytic receptor on the macrophage surface might affect HIV infection of monocytic cells. Furthermore, Elmo, a key signaling molecule that is associated with phagocytosis inhibited HIV transcription in infected macrophages, demonstrating that signaling events associated with phagocytosis of apoptotic cells participate in the establishment of HIV infection and provirus transcription. v TABLE OF CONTENTS LIST OF FIGURES.........................................................................................................viii LIST OF TABLES .......................................................................................................…x LIST OF ABBREVIATIONS…………………………………………..……………....xi ACKNOWLEDGEMENTS ............................................................................................xv Chapter 1: Literature Review ……………………………………………………….…..1 1.1 Plasma membrane transbilayer phospholipid distribution…………………..1 1.2 The Aminophospholipid Translocase……………………………………….4 1.3 Apoptosis…………………………………………………………………….8 1.4 Macrophages……………………………………………………………..…12 1.5 Macrophage recognition of apoptotic cells…………………………………14 1.6 Human Immunodeficiency Virus (HIV)………………………………..…..20 1.7 HIV Lifecycle………………………………………………………….…...24 1.8 HIV and apoptosis………………………………………………………….30 1.9 Role of lipids during HIV infection………………………………………..31 Chapter 2: Identifying plasma membrane aminophospholipid translocase candidate genes…………………………………………………………….33 2.1 Introduction………………………………………………………….……..33 2.2 Materials and Methods………………………………………………….….35 2.2.1 Cell Lines………………………………………………………...35 2.2.2 Library Preparation……………………………………………....35 2.2.3 PCR Amplification and Sequencing…………………………..…37 vi 2.3 Results……………………………………………………………………..37 2.3.1 PCR analysis of the expression of subfamily IV P-type ATPase members in mouse liver and mouse brain…………………....37 2.3.2 Expression of subfamily IV P-type ATPase genes in J774 cells, NIH 3T3 cells and PC12 cells………………………….…39 2.4 Discussion…………………………………………………………………47 Chapter 3: Signal transduction events induced by apoptotic cells inhibit HIV transcription in monocytes/macrophages………………………………….50 3.1 Introduction………………………………………………………………..50 3.2 Materials and Methods…………………………………………………….52 3.2.1 Cell Lines………………………………………………………..53 3.2.2 Preparation of HIV and infections………………………………54 3.2.3 Expression plasmids and transfections………………………….54 3.2.4 Virus-free fusion assay …………………………………………55 3.2.5 PCR detection of reverse transcriptase (RT) intermediates and integrated provirus……………………………………………...55 3.2.6 Cell death induction and analysis……………………………….56 3.3 Results……………………………………………………………………..57 3.3.1 HIV proviral integration in macrophages requires PS…………..57 3.3.2 Apoptotic cells inhibit HIV transcription ………………………59 3.3.3 CrkII, Dock180 and Elmo inhibit HIV transcription……………63 3.4 Discussion…………………………………………………………………67 vii Chapter 4: Conclusions………………………………………………………………..75 Bibliography…………………………………………………………………………..82 viii LIST OF FIGURES Figure 1.1 Plasma membrane phospholipids distribution…………….……...2 Figure 1.2 Amphipath Transporters……………………………………….....7 Figure 1.3 Apoptosis vs. Necrosis…………………………………….……...9 Figure 1.4 Macrophage Receptors…………………………………………..16 Figure 1.5 CrkII-Dock180-Elmo Signaling Pathway……………………….18 Figure 1.6 HIV genes………………………………………………………..23 Figure 1.7 HIV Lifecycle…………………………………………………....27 Figure 2.1 Expression pattern of subfamily IV P-type ATPase genes in mouse liver…………………………………………………….…41 Figure 2.2 Expression pattern of subfamily IV P-type ATPase genes in J774 cells, NIH 3T3 cells and PC12 cells……………………..…45 Figure 3.1 PS is not required for fusion……………………………………..58 Figure 3.2 PS is required for the establishment of HIV provirus in monocytic cells…………………………………………………..60 Figure 3.3 Inhibition of HIV transcription in macrophages by apoptotic cells…………………………………………………....61 Figure 3.4 Apoptotic cells inhibit induction of latent HIV……………….…65 Figure 3.5 PS is not required for inhibition of HIV transcription………..….66 Figure 3.6 CrkII, Dock180 and Elmo inhibit HIV transcription…………….68 Figure 3.7 ELMO inhibits HIV transcription in monocytes/macrophages…………………………………………69 ix Figure 3.8 Rescue of HIV transcription upon diminishing ELMO Expression…………………………………………………….71 Figure 3.9 LPC and TGFb do not influence HIV transcription………….72 Figure 4.1 Model for PS-dependent effects on integration………………77 Figure 4.2 Model for phagocytic pathways that are altered by HIV……..80 x LIST OF TABLES Table 2.1 Gene specific primer pairs used for PCR amplification…………36 Table 2.2 Expression pattern of subfamily IV P-type ATPase genes in mouse liver……………………………………………………….42 Table 2.3 Expression pattern of subfamily IV P-type ATPase genes in mouse brain………………………………………………………43 Table 2.4 Expression pattern of subfamily IV P-type ATPase genes In J774 cells, NIH 3T3 cells and PC12 cells…………….………46 xi LIST OF ABBREVIATIONS ABC ATP binding cassette ACAMPS Apoptotic cell associated molecular pattern AIDS Acquired immunodeficiency syndrome AS Angelman syndrome ATP Adenosine triphosphate AV Annexin V BRIC Benign recurrent intrahepatic cholestasis Ca2+ Calcium CDK9 Cyclin dependent kinase 9 CDNA Complimentary DNA C/EBP CCAAT/enhancer binding protein Ced Cell death abnormal CNS Central nervous system DC-SIGN Dendritic cell specific ICAM-1-grabbing nonintegrin DMEM Dulbecco’s modified Eagle’s medium DNA Deoxyribonucleic acid ELISA Enzyme linked immunosorbent assay Env Envelope FACS Fluorescent activated cell sorter FBS Fetal bovine serum FIC1 Familial intrahepatic cholestasis 1 xii FITC Fluorescein isothiocyanate
Load more

Recommended publications
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Research Article Identification of ATP8B1 As a Tumor Suppressor Gene for Colorectal Cancer and Its Involvement in Phospholipid Homeostasis
    Hindawi BioMed Research International Volume 2020, Article ID 2015648, 16 pages https://doi.org/10.1155/2020/2015648 Research Article Identification of ATP8B1 as a Tumor Suppressor Gene for Colorectal Cancer and Its Involvement in Phospholipid Homeostasis Li Deng,1,2 Geng-Ming Niu,1 Jun Ren,1 and Chong-Wei Ke 1 1Department of General Surgery, The Fifth People’s Hospital of Shanghai, Fudan University, Shanghai, China 2Department of General Surgery, The Shanghai Public Health Clinical Center, Fudan University, Shanghai, China Correspondence should be addressed to Chong-Wei Ke; [email protected] Received 5 May 2020; Revised 16 July 2020; Accepted 17 August 2020; Published 29 September 2020 Academic Editor: Luis Loura Copyright © 2020 Li Deng et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Homeostasis of membrane phospholipids plays an important role in cell oncogenesis and cancer progression. The flippase ATPase class I type 8b member 1 (ATP8B1), one of the P4-ATPases, translocates specific phospholipids from the exoplasmic to the cytoplasmic leaflet of membranes. ATP8B1 is critical for maintaining the epithelium membrane stability and polarity. However, the prognostic values of ATP8B1 in colorectal cancer (CRC) patients remain unclear. We analyzed transcriptomics, genomics, and clinical data of CRC samples from The Cancer Genome Atlas (TCGA). ATP8B1 was the only potential biomarker of phospholipid transporters in CRC. Its prognostic value was also validated with the data from the Gene Expression Omnibus (GEO). Compared to the normal group, the expression of ATP8B1 was downregulated in the tumor group and the CRC cell lines, which declined with disease progression.
    [Show full text]
  • ATP8B3 Blocking Peptide (N-Term) Synthetic Peptide Catalog # BP20206A
    10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 ATP8B3 Blocking Peptide (N-term) Synthetic peptide Catalog # BP20206A Specification ATP8B3 Blocking Peptide (N-term) - ATP8B3 Blocking Peptide (N-term) - Product Background Information The protein encoded by this gene belongs to Primary Accession O60423 the family of Other Accession NP_620168.1 P-type cation transport ATPases, and to the subfamily of aminophospholipid-transporting ATPases. The ATP8B3 Blocking Peptide (N-term) - Additional Information aminophospholipid translocases transport phosphatidylserine and phosphatidylethanolamine from one side of a Gene ID 148229 bilayer to another. This gene encodes the member 3 of the Other Names phospholipid-transporting Phospholipid-transporting ATPase IK, ATPase 8B. Alternatively spliced transcript ATPase class I type 8B member 3, ATP8B3, variants encoding ATP1K, FOS37502_2 different isoforms have been found for this Target/Specificity gene. [provided by The synthetic peptide sequence is selected RefSeq]. from aa 71-84 of HUMAN ATP8B3 ATP8B3 Blocking Peptide (N-term) - Format References Peptides are lyophilized in a solid powder format. Peptides can be reconstituted in Scott, L.J., et al. Proc. Natl. Acad. Sci. U.S.A. solution using the appropriate buffer as 106(18):7501-7506(2009) needed. Harris, M.J., et al. Biochim. Biophys. Acta 1633(2):127-131(2003) Storage Halleck, M.S., et al. Physiol. Genomics Maintain refrigerated at 2-8°C for up to 6 1(3):139-150(1999) months. For long term storage store at Fries, A.S., et al. Lab. Anim. 12(1):1-4(1978) -20°C. Precautions This product is for research use only.
    [Show full text]
  • Inhibin, Activin, Follistatin and FSH Serum Levels and Testicular
    REPRODUCTIONRESEARCH Expression of Atp8b3 in murine testis and its characterization as a testis specific P-type ATPase Eun-Yeung Gong, Eunsook Park, Hyun Joo Lee and Keesook Lee School of Biological Sciences and Technology, Hormone Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea Correspondence should be addressed to K Lee; Email: [email protected] Abstract Spermatogenesis is a complex process that produces haploid motile sperms from diploid spermatogonia through dramatic morphological and biochemical changes. P-type ATPases, which support a variety of cellular processes, have been shown to play a role in the functioning of sperm. In this study, we isolated one putative androgen-regulated gene, which is the previously reported sperm-specific aminophospholipid transporter (Atp8b3, previously known as Saplt), and explored its expression pattern in murine testis and its biochemical characteristics as a P-type ATPase. Atp8b3 is exclusively expressed in the testis and its expression is developmentally regulated during testicular development. Immunohistochemistry of the testis reveals that Atp8b3 is expressed only in germ cells, especially haploid spermatids, and the protein is localized in developing acrosomes. As expected, from its primary amino acid sequence, ATP8B3 has an ATPase activity and is phosphorylated by an ATP-producing acylphosphate intermediate, which is a signature property of the P-Type ATPases. Together, ATP8B3 may play a role in acrosome development and/or in sperm function during fertilization. Reproduction (2009) 137 345–351 Introduction the removal of testosterone by hypophysectomy or ethane dimethanesulfonate (EDS) treatment in rats leads to the Spermatogenesis is one of most complex processes of cell failure of spermatogenesis (Russell & Clermont 1977, differentiation in animals, in which diploid spermatogo- Bartlett et al.
    [Show full text]
  • 1 Chapter I Introduction to the Role of P4-Atpases And
    CHAPTER I INTRODUCTION TO THE ROLE OF P4-ATPASES AND OXYSTEROL BINDING PROTEIN HOMOLOGUES IN PROTEIN TRANSPORT The plasma membrane and membranes of organelles compartmentalize eukaryotic cells to allow formation of specialized microenvironments in the cytosol and organelle lumens. Specific reactions including glycosylation, proteolytic cleavage and degradation take place in the organellar compartments. The functional identity of each organelle is conferred by their unique set of proteins and lipids which must be transported from common site of synthesis to multiple destinations (Owen et al., 2004). Proteins are transported between organelles of the secretory and endocytic pathways by transport vesicles. Vesicles are often identified by the cytosolic coat proteins help form them, with the best characterized examples being COPI, COPII and clathrin. Clathrin is required to bud vesicles from the trans-Golgi network (TGN), endosomes, and plasma membrane for receptor mediated endocytosis. Several years ago our lab disvoered that a budding yeast protein called Drs2 is required for budding of specific class of exocytic vesicles from the TGN (Chen et al., 1999) and clathrin coated vesicles mediating vesicle transport between the TGN and early endosomes (Liu et al., 2008b). Drs2 is a type IV P-type ATPase (P4-ATPase) and lipid flippase that helps generate membrane asymmetry in biological membranes by flipping specific lipids from one leaflet to the other. Moreover DRS2 is an essential gene at low temperature (Ripmaster et al., 1993). Yeast strains harboring a distruption of DRS2 (drs2∆) grow well from 23C to 37C, but cannot grow at temperatures below 23C. My research is focused on 1 understanding why drs2∆ cells exhibit this unusually strong cold-sensitive growth defect.
    [Show full text]
  • Adipose Gene Expression Profiles Reveal Novel Insights Into the Adaptation of Northern Eurasian Semi-Domestic Reindeer
    bioRxiv preprint doi: https://doi.org/10.1101/2021.04.17.440269; this version posted April 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Adipose gene expression profiles reveal novel insights into the 2 adaptation of northern Eurasian semi-domestic reindeer 3 (Rangifer tarandus) 4 Short title: Reindeer adipose transcriptome 5 6 Melak Weldenegodguad1, 2, Kisun Pokharel1¶, Laura Niiranen3¶, Päivi Soppela4, 7 Innokentyi Ammosov5, Mervi Honkatukia6, Heli Lindeberg7, Jaana Peippo1, 6, Tiina 8 Reilas1, Nuccio Mazzullo4, Kari A. Mäkelä3, Tommi Nyman8, Arja Tervahauta2, 9 Karl-Heinz Herzig9, 10,11, Florian Stammler4, Juha Kantanen1* 10 11 1 Natural Resources Institute Finland (Luke), Jokioinen, Finland 12 2 Department of Environmental and Biological Sciences, University of Eastern 13 Finland, Kuopio, Finland 14 3 Research Unit of Biomedicine, Faculty of Medicine, University of Oulu, Oulu, 15 Finland 16 4 Arctic Centre, University of Lapland, Rovaniemi, Finland 17 5 Board of Agricultural Office of Eveno-Bytantaj Region, Batagay-Alyta, The Sakha 18 Republic (Yakutia), Russia 19 6 NordGen—Nordic Genetic Resource Center, Ås, Norway 20 7 Natural Resources Institute Finland (Luke), Maaninka, Finland 21 8 Department of Ecosystems in the Barents Region, Norwegian Institute of 22 Bioeconomy Research, Svanvik, Norway 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.17.440269; this version posted April 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
    [Show full text]
  • Original Article Unclassified Renal Cell Carcinoma: a Clinicopathological, Comparative Genomic Hybridization, and Whole-Genome Exon Sequencing Study
    Int J Clin Exp Pathol 2014;7(7):3865-3875 www.ijcep.com /ISSN:1936-2625/IJCEP0000596 Original Article Unclassified renal cell carcinoma: a clinicopathological, comparative genomic hybridization, and whole-genome exon sequencing study Zhen-Yan Hu1*, Li-Juan Pang1*, Yan Qi1,2, Xue-Ling Kang1, Jian-Ming Hu1,2, Lianghai Wang1, Kun-Peng Liu1, Yuan Ren1, Mei Cui1, Li-Li Song1, Hong-An Li1, Hong Zou1,2, Feng Li1 1Department of Pathology, School of Medicine, Shihezi University, Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Ministry of Education of China, Xinjiang 832002, China; 2Tongji Hospital Cancer Center, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China. *Equal contributors. Received April 22, 2014; Accepted June 2, 2014; Epub June 15, 2014; Published July 1, 2014 Abstract: Unclassified renal cell carcinoma (URCC) is a rare variant of RCC, accounting for only 3-5% of all cases. Studies on the molecular genetics of URCC are limited, and hence, we report on 2 cases of URCC analyzed us- ing comparative genome hybridization (CGH) and the genome-wide human exon GeneChip technique to identify the genomic alterations of URCC. Both URCC patients (mean age, 72 years) presented at an advanced stage and died within 30 months post-surgery. Histologically, the URCCs were composed of undifferentiated, multinucleated, giant cells with eosinophilic cytoplasm. Immunostaining revealed that both URCC cases had strong p53 protein expression and partial expression of cluster of differentiation-10 and cytokeratin. The CGH profiles showed chro- mosomal imbalances in both URCC cases: gains were observed in chromosomes 1p11-12, 1q12-13, 2q20-23, 3q22-23, 8p12, and 16q11-15, whereas losses were detected on chromosomes 1q22-23, 3p12-22, 5p30-ter, 6p, 11q, 16q18-22, 17p12-14, and 20p.
    [Show full text]
  • P4-Atpases As Phospholipid Flippases—Structure, Function, and Enigmas
    REVIEW published: 08 July 2016 doi: 10.3389/fphys.2016.00275 P4-ATPases as Phospholipid Flippases—Structure, Function, and Enigmas Jens P. Andersen 1, Anna L. Vestergaard 1, Stine A. Mikkelsen 1, Louise S. Mogensen 1, Madhavan Chalat 2 and Robert S. Molday 2* 1 Department of Biomedicine, Aarhus University, Aarhus, Denmark, 2 Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada P4-ATPases comprise a family of P-type ATPases that actively transport or flip phospholipids across cell membranes. This generates and maintains membrane lipid asymmetry, a property essential for a wide variety of cellular processes such as vesicle budding and trafficking, cell signaling, blood coagulation, apoptosis, bile and cholesterol homeostasis, and neuronal cell survival. Some P4-ATPases transport phosphatidylserine and phosphatidylethanolamine across the plasma membrane or intracellular membranes whereas other P4-ATPases are specific for phosphatidylcholine. The importance of P4-ATPases is highlighted by the finding that genetic defects in two P4-ATPases ATP8A2 Edited by: and ATP8B1 are associated with severe human disorders. Recent studies have provided Olga Vagin, insight into how P4-ATPases translocate phospholipids across membranes. P4-ATPases University of California, Los Angeles, USA form a phosphorylated intermediate at the aspartate of the P-type ATPase signature Reviewed by: sequence, and dephosphorylation is activated by the lipid substrate being flipped from Mikhail Y. Golovko, the exoplasmic to the cytoplasmic leaflet similar to the activation of dephosphorylation University of North Dakota, USA + + + Emilia Lecuona, of Na /K -ATPase by exoplasmic K . How the phospholipid is translocated can be Northwestern University, USA understood in terms of a peripheral hydrophobic gate pathway between transmembrane *Correspondence: helices M1, M3, M4, and M6.
    [Show full text]
  • Adipose Gene Expression Profiles Reveal Novel Insights Into the Adaptation of Northern Eurasian Semi-Domestic Reindeer (Rangifer
    bioRxiv preprint doi: https://doi.org/10.1101/2021.04.17.440269; this version posted April 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Adipose gene expression profiles reveal novel insights into the 2 adaptation of northern Eurasian semi-domestic reindeer 3 (Rangifer tarandus) 4 Short title: Reindeer adipose transcriptome 5 6 Melak Weldenegodguad1, 2, Kisun Pokharel1¶, Laura Niiranen3¶, Päivi Soppela4, 7 Innokentyi Ammosov5, Mervi Honkatukia6, Heli Lindeberg1, Jaana Peippo1, 6, Tiina 8 Reilas1, Nuccio Mazzullo4, Kari A. Mäkelä3, Tommi Nyman7, Arja Tervahauta2, 9 Karl-Heinz Herzig8, 9, 10, Florian Stammler4, Juha Kantanen1* 10 11 1 Natural Resources Institute Finland (Luke), Jokioinen, Finland 12 2 Department of Environmental and Biological Sciences, University of Eastern 13 Finland, Kuopio, Finland 14 3 Research Unit of Biomedicine, Faculty of Medicine, University of Oulu, Oulu, 15 Finland 16 4 Arctic Centre, University of Lapland, Rovaniemi, Finland 17 5 Board of Agricultural Office of Eveno-Bytantaj Region, Batagay-Alyta, The Sakha 18 Republic (Yakutia), Russia 19 6 NordGen—Nordic Genetic Resource Center, Ås, Norway 20 7 Department of Ecosystems in the Barents Region, Norwegian Institute of 21 Bioeconomy Research, Svanvik, Norway 22 8 Research Unit of Biomedicine, Medical Research Center, Faculty of Medicine, 23 University of Oulu bioRxiv preprint doi: https://doi.org/10.1101/2021.04.17.440269; this version posted April 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
    [Show full text]
  • STAT3 Targets Suggest Mechanisms of Aggressive Tumorigenesis in Diffuse Large B Cell Lymphoma
    STAT3 Targets Suggest Mechanisms of Aggressive Tumorigenesis in Diffuse Large B Cell Lymphoma Jennifer Hardee*,§, Zhengqing Ouyang*,1,2,3, Yuping Zhang*,4 , Anshul Kundaje*,†, Philippe Lacroute*, Michael Snyder*,5 *Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305; §Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520; and †Department of Computer Science, Stanford University School of Engineering, Stanford, CA 94305 1The Jackson Laboratory for Genomic Medicine, Farmington, CT 06030 2Department of Biomedical Engineering, University of Connecticut, Storrs, CT 06269 3Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT 06030 4Department of Biostatistics, Yale School of Public Health, Yale University, New Haven, CT 06520 5Corresponding author: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305. Email: [email protected] DOI: 10.1534/g3.113.007674 Figure S1 STAT3 immunoblotting and immunoprecipitation with sc-482. Western blot and IPs show a band consistent with expected size (88 kDa) of STAT3. (A) Western blot using antibody sc-482 versus nuclear lysates. Lanes contain (from left to right) lysate from K562 cells, GM12878 cells, HeLa S3 cells, and HepG2 cells. (B) IP of STAT3 using sc-482 in HeLa S3 cells. Lane 1: input nuclear lysate; lane 2: unbound material from IP with sc-482; lane 3: material IP’d with sc-482; lane 4: material IP’d using control rabbit IgG. Arrow indicates the band of interest. (C) IP of STAT3 using sc-482 in K562 cells. Lane 1: input nuclear lysate; lane 2: material IP’d using control rabbit IgG; lane 3: material IP’d with sc-482.
    [Show full text]
  • P4 Atpases: Flippases in Health and Disease
    Int. J. Mol. Sci. 2013, 14, 7897-7922; doi:10.3390/ijms14047897 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Review P4 ATPases: Flippases in Health and Disease Vincent A. van der Mark *, Ronald P.J. Oude Elferink and Coen C. Paulusma Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Meibergdreef 69-71, 1105 BK Amsterdam, The Netherlands; E-Mails: [email protected] (R.P.J.O.E.); [email protected] (C.C.P.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +31-205-668-156; Fax: +31-205-669-190. Received: 6 March 2013; in revised form: 28 March 2013 / Accepted: 7 April 2013 / Published: 11 April 2013 Abstract: P4 ATPases catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes, a process termed “lipid flipping”. Accumulating evidence obtained in lower eukaryotes points to an important role for P4 ATPases in vesicular protein trafficking. The human genome encodes fourteen P4 ATPases (fifteen in mouse) of which the cellular and physiological functions are slowly emerging. Thus far, deficiencies of at least two P4 ATPases, ATP8B1 and ATP8A2, are the cause of severe human disease. However, various mouse models and in vitro studies are contributing to our understanding of the cellular and physiological functions of P4-ATPases. This review summarizes current knowledge on the basic function of these phospholipid translocating proteins, their proposed action in intracellular vesicle transport and their physiological role.
    [Show full text]
  • Inner Workings and Biological Impact of Phospholipid Flippases Radhakrishnan Panatala1,2, Hanka Hennrich1 and Joost C
    © 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 2021-2032 doi:10.1242/jcs.102715 COMMENTARY Inner workings and biological impact of phospholipid flippases Radhakrishnan Panatala1,2, Hanka Hennrich1 and Joost C. M. Holthuis1,2,* ABSTRACT phospholipid transport catalyzed by P4-ATPases helps establish the The plasma membrane, trans-Golgi network and endosomal system membrane curvature needed to bud vesicles from the trans-Golgi of eukaryotic cells are populated with flippases that hydrolyze ATP to network (TGN), endosomes and plasma membrane. We also help establish asymmetric phospholipid distributions across the bilayer. describe how P4-ATPase-catalyzed flippase activity is subject to Upholding phospholipid asymmetry is vital to a host of cellular complex regulatory mechanisms that interconnect the establishment processes, including membrane homeostasis, vesicle biogenesis, cell of lipid asymmetry with phosphoinositide metabolism and signaling, morphogenesis and migration. Consequently, defining the sphingolipid homeostasis, allowing cells to cross-regulate multiple identity of flippases and their biological impact has been the subject of key determinants of membrane function. At the organismal level, intense investigations. Recent work has revealed a remarkable degree disruption of P4-ATPase function has been linked to diabetes, of kinship between flippases and cation pumps. In this Commentary, we obesity, immune deficiency, neurological disorders and a potentially review emerging insights into how flippases work, how their activity is fatal liver disease. Recent insights into the molecular basis of these controlled according to cellular demands, and how disrupting flippase diseases are also discussed. activity causes system failure of membrane function, culminating in membrane trafficking defects, aberrant signaling and disease.
    [Show full text]