DOCSLIB.ORG

Two Alternative Processing Pathways for a Preprohormone

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Two Alternative Processing Pathways for a Preprohormone Proc. Natl. Acad. Sci. USA Vol. 92, pp. 11985-11989, December 1995 Biochemistry Two alternative processing pathways for a preprohormone: A bioactive form of secretin VALENTINA BONETTO, HANS JORNVALL, VIKTOR MUTT, AND RANNAR SILLARD* Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77, Stockholm, Sweden Contributed by Viktor Mutt, September 5, 1995 ABSTRACT An N-terminally 9-residue elongated form of a cloned cDNA species (10, 13). This discrepancy was ex- secretin, secretin-(-9 to 27) amide, was isolated from porcine plained in terms of alternative splicing and later the shorter intestinal tissue and characterized. Current knowledge about form of cDNA was also found (14). A proform of secretin peptide processing sites does not allow unambiguous predic- elongated at its C terminus by Gly-Lys-Arg has been described tion of the signal peptide cleavage site in preprosecretin but (15), and it might represent the next product in the C-terminal suggests cleavage in the region ofresidues -10 to -14 counted processing, presumably produced by cleavage with an enzyme upstream from the N terminus of the hormone. However, the specific for a dibasic site. The next, shortened form of the structure of the isolated peptide suggests that the cleavage precursor, secretin extended at its C terminus by glycine, has between the signal peptide and the N-terminal propeptide also been isolated (16). This form is presumably obtained by occurs at the C-terminal side of residue -10. Moreover, the carboxypeptidase action on the Gly-Lys-Arg-extended form. isolated peptide demonstrates that secretin can be fully pro- The mature hormone-C-terminally amidated secretin-is cessed C-terminally prior to the final N-terminal cleavage. produced from the glycine-extended peptide by the combined The results from this report, and those from earlier studies, action of a peptidylglycine a-monooxygenase and a peptidyl- where C-terminally elongated variants were isolated, show amidoglycolate lyase (17). In aHl of these proforms, the N that the processing of the secretin precursor may proceed by terminus starts with secretin itself, and thus cleavage of the one oftwo alternative pathways, in which either ofthe two ends N-terminal flanking peptide has been considered to be the is processed first. The bioactivity of the N-terminally extended initial processing event after removal of the signal peptide (13). peptide on exocrine pancreatic secretion was lower than that In our laboratory, a variant form of secretin in which the of secretin, indicating the importance of the finally processed N-terminal tryptic peptide differs from that of secretin was free N terminus of the hormone for interaction with secretin observed earlier but was not further characterized (18). receptors. The present study demonstrates that an alternative process- ing pathway is possible, where the N terminus remains ex- Secretin, a gastrointestinal hormone, was discovered by Bayliss tended throughout processing of the C terminus, starting with and Starling in 1902 (1), isolated (2), and characterized as a the precursor and ending with arnidation of the C-terminal 27-residue C-terminally amidated peptide (3). It is known to be valine. produced in endocrine S cells of the small intestine (4, 5), pancreatic 13 cells (6), and possibly also in brain (7, 8). Its best MATERIALS AND METHODS known function is stimulation of the secretion of bicarbonate- rich pancreatic fluid (9). Peptide Purification. A concentrate of thermostable intes- The structures of the rat and mouse secretin precursors as tinal peptides, CTIP, was prepared from porcine intestines as deduced from the corresponding cDNA sequences contain a described (19-21). Briefly, the uppermost meter of the intes- signal sequence, an N-terminal flanking peptide, secretin, and tines was boiled for 10 min, frozen, minced, and extracted with a C-terminal extension peptide in that order (10, 11). The 0.5 M acetic acid (20 h at 4°C). Peptides in the extract were amino acid sequence of pig preprosecretin has also been so adsorbed onto alginic acid (pH 2.7 ± 0.1), eluted with 0.2 M deduced, but it is probably lacking a few residues at the N HCl, and precipitated with salt (pH 3.5 ± 0.1) to produce the terminus of the signal peptide (10). The amino acid sequence CTIP. An aqueous solution of CTIP was fractionated with of the precursor is such that current knowledge about signal ethanol as described (22), and the peptides soluble at -20°C peptide cleavage sites (12) does not allow unambiguous pre- were further processed by size-exclusion chromatography on a diction of this site. On the basis of the amino acid sequence, Sephadex G-25 column. A fraction of low molecular weight the cleavage of the signal peptide has been proposed to occur peptides was extracted with methanol, and the peptides, at the C-terminal side of any of the residues between -10 and methanol soluble at neutral pH, were applied to a CM-22 -14 (10), most likely after residue -12, -13, or -14. Hence, column (14 x 20 cm). From this chromatography step, three for determination of the actual cleavage site, isolation of the large fractions were collected: presecretin, a second fraction corresponding N-terminally elongated form of secretin is that contains mainly secretin, and a more basic fraction necessary. In this report, we show that cleavage of the signal denoted postsecretin normally used for isolation of vasoactive peptide probably occurs at the C-terminal side of residue -10, intestinal peptide (23). This material was then used for further thus suggesting the position of cleavage and the length of the purification by ion-exchange chromatography on carboxy- N-terminal flanking peptide. methyl cellulose (CM-22, Whatman; 5 x 18 cm) in 0.0125 M It has been shown earlier that other secretin proforms also sodium phosphate (pH 6.4). Peptides were eluted with a linear exist in intestinal tissue. A proform, consisting of the secretin gradient of 0-0.3 M NaCl in the same buffer until the part together with its C-terminal flanking peptide, has been absorbance at 280 nm decreased below 0.1 (24). The column isolated, although the length of the latter was found to be 31 was then washed with 0.2 M HCl. NaCl was removed from the residues shorter than the amino acid sequence deduced from eluted peptides by passing the eluate through a column of Sephadex G-25 coarse in 0.2 M acetic acid and lyophilizing the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviation: TFA, trifluoroacetic acid. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 11985 Downloaded by guest on September 28, 2021 11986 Biochemistry: Bonetto et al. Proc. Natl. Acad. Sci. USA 92 (1995) peptide-containing fractions. The fractions eluting between 78 using an uncoated capillary of 75 ,um diameter essentially as and 92 min contained the secretin-(-9 to 27) amide (N- described (25). Two different buffer systems were used (50 mM prosecretin) and were taken for further purification as de- phosphate buffers at pH 2.5 and pH 7.0). scribed below. Structural Analysis. The purified peptide was degraded in Porcine secretin was isolated as described (2) and used as a an Applied Biosystems sequencer (model 470A) and the standard in structural and bioactivity measurements. fractions were analyzed on a Hewlett-Packard HPLC system Reverse-Phase HPLC. The peptide fractions from cation- HP1090. Amino acid compositions were determined with an exchange chromatography were applied to LKB Ultropac TSK LKB Alpha Plus 4151 analyzer. The molecular mass of the ODS (7.8 x 300 mm). The column was eluted with a gradient peptide was measured on a Finnigan-MAT (San Jose, CA) of acetonitrile in 0.1% aqueous trifluoroacetic acid (TFA) Lasermat 2000, using a-cyano-4-hydroxycinnamic acid as the using buffers A (0.1% TFA/water) and B (0.1% TFA/water/ matrix. An aliquot (0.5 ,ul) of the sample, dissolved in 0.1% 80% acetonitrile) with 25-55% buffer B in 40 min. For TFA/70% acetonitrile/water, and of the matrix-saturated cation-exchange HPLC, Vydac (Hesperia, CA) 400VHP552 solution (0.5 ,ul in the same solvent) were mixed on a stainless (5.0 x 25 mm) was used. A NaCl gradient was employed using steel target and allowed to dry at room temperature. Mass buffers A (0.1% TFA/water/25% acetonitrile) and B (1 M spectra were obtained from the average of 10 laser shot NaCl in buffer A) with 0-30% buffer B in 30 min. The peptide recordings at 337 nm. Bovine insulin (Sigma) was used as an fraction from the ion-exchange HPLC step was then applied to internal standard. a Vydac C18 (4.6 x 250 mm) HPLC column and eluted with a Sequence searches were carried out with the FASTA program methanol gradient using buffers A (0.1% TFA/water) and B of the Genetics Computer Group software package (release (0.1% TFA/water/80% methanol) with 60-100% buffer B in 8.0) (26) in the Swiss-Prot (release 30.0) data base. 40 min. Synthetic Peptide. N-prosecretin was synthesized by Scha- Enzymatic Digestion. The isolated prosecretin, its synthetic fer-N (Copenhagen). The crude peptide was purified by re- replica, and secretin were each dissolved (1 mg/ml) in 0.1 M verse-phase HPLC on a Vydac C18 (4.6 x 250 mm) column ammonium bicarbonate (pH 7.9) containing 5% acetonitrile. using 0.1% TFA/water as buffer A and 0.1% TFA/water/80% Endoproteinase Glu-C (analytical grade; Boehringer Mann- acetonitrile as buffer B with a gradient of 35-48% buffer B in heim) was added at an enzyme/substrate ratio of 1:10.
Load more

Recommended publications
  • (GLP-1) in Transgenic Plants
    The Open Biotechnology Journal, 2009, 3, 57-66 57 Open Access A Proficient Approach to the Production of Therapeutic Glucagon-Like Peptide-1 (GLP-1) in Transgenic Plants M. Brandsma1, X. Wang1, H. Diao2,3, S.E. Kohalmi1, A.M. Jevnikar2,3 and S. Ma1,2,3,* 1Department of Biology, University of Western Ontario, London, Ontario, N6A 5B7, Canada 2Transplantation Immunology Group, Lawson Health Research Institute, London, Ontario, N6A 4G5, Canada 3Plantigen Inc., 700 Collip Circle, London, Ontario, N6G 4X8, Canada Abstract: Glucagon-like peptide-1 (GLP-1) is a small peptide hormone with potent insulinotropic activity and represents a promising new therapeutic tool for the treatment of diabetes. Like many other therapeutic peptides, GLP-1 is commonly produced using chemical synthesis methods, but is limited by product quantity and cost. The advent of recombinant DNA technology offers the possibility of producing GLP-1 inexpensively and in vast quantities. In this study, transgenic plants were used as a recombinant expression platform for the production of GLP-1 as a large multimeric protein. A synthetic gene encoding ten sequential tandem repeats of GLP-1 sequence (GLP-1x10) was produced and introduced into tobacco plants. Transcriptional expression of the GLP1x10 gene in transgenic plants was confirmed by RT-PCR. Western blot analysis showed that the GLP-1x10 protein efficiently accumulated in transgenic plants, with an accumulation level as high as 0.15% of total soluble protein in leaves. Importantly, insulin secretion assays using a mouse pancreatic cell line (MIN6), showed that plant-derived GLP-1 in its synthetic decamer form, retained its ability to stimulate cellular insulin secretion, although with reduced efficacy.
    [Show full text]
  • Ectopic Expression of Glucagon-Like Peptide 1 for Gene Therapy of Type II Diabetes
    Gene Therapy (2007) 14, 38–48 & 2007 Nature Publishing Group All rights reserved 0969-7128/07 $30.00 www.nature.com/gt ORIGINAL ARTICLE Ectopic expression of glucagon-like peptide 1 for gene therapy of type II diabetes GB Parsons, DW Souza, H Wu, D Yu, SG Wadsworth, RJ Gregory and D Armentano Department of Molecular Biology, Genzyme Corporation, Framingham, MA, USA Glucagon-like peptide 1 (GLP-1) is a promising candidate for lowered both the fasting and random-fed hyperglycemia the treatment of type II diabetes. However, the short in vivo present in these animals. Because the insulinotropic actions half-life of GLP-1 has made peptide-based treatments of GLP-1 are glucose dependent, no evidence of hypogly- challenging. Gene therapy aimed at achieving continuous cemia was observed. Improved glucose homeostasis was GLP-1 expression presents one way to circumvent the rapid demonstrated by improvements in %HbA1c (glycated he- turnover of GLP-1. We have created a GLP-1 minigene that moglobin) and in glucose tolerance tests. GLP-1-treated can direct the secretion of active GLP-1 (amino acids 7–37). animals had higher circulating insulin levels and increased Plasmid and adenoviral expression vectors encoding the 31- insulin immunostaining of pancreatic sections. GLP-1-treated amino-acid peptide linked to leader sequences required for ZDF rats showed diminished food intake and, in the first few secretion of GLP-1 yielded sustained levels of active GLP-1 weeks following vector administration, a diminished weight that were significantly greater than endogenous levels. gain. These results demonstrate the feasibility of gene Systemic administration of expression vectors to animals therapy for type II diabetes using GLP-1 expression vectors.
    [Show full text]
  • Signaling Peptides in Plants
    lopmen ve ta e l B D io & l l o l g e y C Cell & Developmental Biology Ghorbani, et al., Cell Dev Biol 2014, 3:2 ISSN: 2168-9296 DOI: 10.4172/2168-9296.1000141 Review Article Open Access Signaling Peptides in Plants Sarieh Ghorbani1,2, Ana Fernandez1,2, Pierre Hilson3,4 and Tom Beeckman1,2* 1Department of Plant Systems Biology, VIB, B-9052 Ghent, Belgium 2Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium 3INRA, UMR1318, Institut Jean-Pierre Bourgin, RD10, F-78000 Versailles, France 4AgroParisTech, Institut Jean-Pierre Bourgin, RD10, F-78000 Versailles, France *Corresponding author: Tom Beeckman, Department of Plant Systems Biology, VIB, B-9052 Ghent, Belgium, Tel: 32-0-93313830; E-mail: [email protected] Rec date: May 03, 2014; Acc date: Jun 16, 2014; Pub date: Jun 18, 2014 Copyright: © 2014 Ghorbani S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract In multicellular organisms, growth and development need to be precisely coordinated and are strongly relying on positional information. Positional control is achieved through exchanges of molecular messages between cells and tissues by means of cell-to-cell communication mechanisms. Especially in plants, accurate and well-controlled cell-to-cell communication networks are essential because of the complete absence of cell mobility and the presence of rigid cell walls. For many years, phytohormones were thought to be the main messengers exchanged between cells.
    [Show full text]
  • Glucagon-Like Peptide-1 and Its Class BG Protein–Coupled Receptors
    1521-0081/68/4/954–1013$25.00 http://dx.doi.org/10.1124/pr.115.011395 PHARMACOLOGICAL REVIEWS Pharmacol Rev 68:954–1013, October 2016 Copyright © 2016 by The Author(s) This is an open access article distributed under the CC BY-NC Attribution 4.0 International license. ASSOCIATE EDITOR: RICHARD DEQUAN YE Glucagon-Like Peptide-1 and Its Class B G Protein–Coupled Receptors: A Long March to Therapeutic Successes Chris de Graaf, Dan Donnelly, Denise Wootten, Jesper Lau, Patrick M. Sexton, Laurence J. Miller, Jung-Mo Ahn, Jiayu Liao, Madeleine M. Fletcher, Dehua Yang, Alastair J. H. Brown, Caihong Zhou, Jiejie Deng, and Ming-Wei Wang Division of Medicinal Chemistry, Faculty of Sciences, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands (C.d.G.); School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom (D.D.); Drug Discovery Biology Theme and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia (D.W., P.M.S., M.M.F.); Protein and Peptide Chemistry, Global Research, Novo Nordisk A/S, Måløv, Denmark (J.La.); Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (L.J.M.); Department of Chemistry and Biochemistry, University of Texas at Dallas, Richardson, Texas (J.-M.A.); Department of Bioengineering, Bourns College of Engineering, University of California at Riverside, Riverside, California (J.Li.); National Center for Drug Screening and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China (D.Y., C.Z., J.D., M.-W.W.); Heptares Therapeutics, BioPark, Welwyn Garden City, United Kingdom (A.J.H.B.); and School of Pharmacy, Fudan University, Zhangjiang High-Tech Park, Shanghai, China (M.-W.W.) Downloaded from Abstract.
    [Show full text]
  • Islet Amyloid Polypeptide (Amylin): No Evidence of an Abnormal Precursor Sequence in 25 Type 2 (Non-Insulin-Dependent) Diabetic Patients
    Diabetologia (1990) 33:628-630 Diabetologia Springer-Verlag1990 Islet amyloid polypeptide (amylin): no evidence of an abnormal precursor sequence in 25 Type 2 (non-insulin-dependent) diabetic patients M. Nishi 1, G. I. Bell 1'2'3 and D. E Steiner ~'2'3 Departments of Biochemistry and Molecular Biology and 2 Medicine, and 3 Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois, USA Summary. Islet amyloid polypeptide is the major protein quenced. The nucleotide sequences of the amplified regions component of the islet amyloid of patients with Type 2 (non- of both alleles of the islet amyloid polypeptide gene of these insulin-dependent) diabetes mellitus. Since the synthesis of a 25 patients were identical to one another and to the sequence structurally abnormal or mutant protein may contribute to of an islet amyloid polypeptide allele isolated from a human the formation of amyloid deposits, we have examined the fetal liver genomic library. These findings suggest that a pri- possibility that a mutant form of islet amytoid polypeptide or mary structural abnormality of islet amyloid polypeptide or its precursor contributes to the formation of islet amyloid in its precursor is unlikely to play a significant role in the forma- Type 2 diabetic patients. We have sequenced the islet amy- tion of islet amyloid in Type 2 diabetic patients. loid polypeptide precursor coding regions of the gene of 25 patients with Type 2 diabetes. Genomic DNA fragments Key words: Islet amyloid polypeptide, Type 2 (non-insulin- corresponding to exon 2 and 3 of the islet amyloid polypep- dependent) diabetes mellitus, polymerase chain reaction, tide gene were amplified from patients' peripheral blood direct sequencing, maturity onset diabetes mellitus of the leucocyte DNAs using the polymerase chain reaction and young (MODY), Pima Indian.
    [Show full text]
  • Cloning and Characterization of Cdnas Encoding Human Gastrin-Releasing Peptide (Bombesin/Mrna/Neuropeptide) ELIOT R
    Proc. Nati. Acad. Sci. USA Vol. 81, pp. 5699-5703, September 1984 Biochemistry Cloning and characterization of cDNAs encoding human gastrin-releasing peptide (bombesin/mRNA/neuropeptide) ELIOT R. SPINDEL*, WILLIAM W. CHIN*, JANET PRICE+, LESLEY H. REESt, GORDON M. BESSERt, AND JOEL F. HABENER* *Laboratory of Molecular Endocrinology, Massachusetts General Hospital and Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02114, and Departments of tChemical Endocrinology and tEndocrinology, St. Bartholomew's Hospital, London, England Communicated by Herman M. Kalckar, June 6, 1984 ABSTRACT We have prepared and cloned cDNAs de- As is characteristic of many neuropeptides, mammalian rived from poly(A)+ RNA from a human pulmonary carcinoid bombesin-like peptides exist in multiple forms. Bombesin- tumpor rich in immunoreactivity to gastrin-releasing peptide, a related peptides of 10 and 27 amino acids have been isolated peptide closely related in structure to amphibian bombesin. from porcine (16) and canine (4) tissues. Analyses by high- Mixtures of synthetic oligodeoxyribonucleotides correspond- pressure liquid chromatography of fetal lung (17) and lung ing to amphibian bombesin were used as hybridization probes tumors (15, 18, 19) yield multiple peaks of immunoreactivity; to screen a cDNA library prepared from the tumor RNA. Se- one peak elutes near bombesin and one elutes near GRP. quencing of the recombinant plasmids shows that human gas- Multiple immunoreactive forms are also detected in gastroin- trin-releasing peptide (hGRP) mRNA encodes a precursor of testinal tissue (9, 20). In brain, immunohistochemistry re- 148 amino acids containing a typical signal sequence, hGRP veals some distinct regions that react to bombesin antisera consisting of 27 or 28 amino acids, and a carboxyl-terminal and other regions that react only with antisera to ranatensin extension peptide.
    [Show full text]
  • Structure and Dynamics of the Insulin Receptor: Implications for Receptor Activation and Drug Discovery
    REVIEWS Drug Discovery Today Volume 22, Number 7 July 2017 Reviews Structure and dynamics of the insulin POST SCREEN receptor: implications for receptor activation and drug discovery 1 1 2 2 Libin Ye , Suvrajit Maji , Narinder Sanghera , Piraveen Gopalasingam , 3 3 4 Evgeniy Gorbunov , Sergey Tarasov , Oleg Epstein and Judith Klein- Seetharaman1,2 1 Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA 2 Division of Metabolic and Vascular Health & Systems, Medical School, University of Warwick, Coventry CV4 7AL, UK 3 OOO ‘NPF ‘MATERIA MEDICA HOLDING’, 47-1, Trifonovskaya St, Moscow 129272, Russian Federation 4 The Institute of General Pathology and Pathophysiology, 8, Baltiyskaya St, 125315 Moscow, Russian Federation Recently, major progress has been made in uncovering the mechanisms of how insulin engages its receptor and modulates downstream signal transduction. Here, we present in detail the current structural knowledge surrounding the individual components of the complex, binding sites, and dynamics during the activation process. A novel kinase triggering mechanism, the ‘bow-arrow model’, is proposed based on current knowledge and computational simulations of this system, in which insulin, after its initial interaction with binding site 1, engages with site 2 between the fibronectin type III (FnIII)- 1 and -2 domains, which changes the conformation of FnIII-3 and eventually translates into structural changes across the membrane. This model provides a new perspective on the process of insulin binding to its receptor and, thus, could lead to future novel drug discovery efforts. Introduction upon insulin binding [8]. The first crystal structure of the basal The insulin receptor (IR) has a pivotal role in the regulation of (inactive) TK was reported in 1994 [9].
    [Show full text]
  • Neurotensin/Neuromedin N Precursor (Peptide Hormones/Enteric Mucosa/Polyprotein Precursor) PAUL R
    Proc. Nati. Acad. Sci. USA Vol. 84, pp. 3516-3520, May 1987 Neurobiology Cloning and sequence analysis of cDNA for the canine neurotensin/neuromedin N precursor (peptide hormones/enteric mucosa/polyprotein precursor) PAUL R. DOBNER*t, DIANE L. BARBERt, LYDIA VILLA-KOMAROFF§, AND COLLEEN MCKIERNAN* Departments of *Molecular Genetics and Microbiology, and *Physiology, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA 01605; and §Department of Neuroscience, The Children's Hospital, 300 Longwood Avenue, Boston, MA 02115 Communicated by Sanford L. Palay, February 5, 1987 ABSTRACT Cloned cDNAs encoding neurotensin were related peptides possess common carboxyl-terminal se- isolated from a cDNA library derived from primary cultures of quences but differ at the amino terminus. canine enteric mucosa cells. Nucleotide sequence analysis has A method for obtaining primary cultures enriched in revealed the primary structure of a 170-amino acid precursor neurotensin-containing cells from the canine enteric mucosa protein that encodes both neurotensin and the neurotensin-like has been described (13). We have used these enriched cells peptide neuromedin N. The peptide-coding domains are locat- to isolate cDNA clones encoding neurotensin. Nucleotide ed in tandem near the carboxyl terminus of the precursor and sequence analysis of the cloned cDNA has revealed the are bounded and separated by the paired, basic amino acid primary structure of a precursor protein that encodes both residues Lys-Arg. An additional coding domain, resembling neurotensin and neuromedin N. neuromedin N, occurs immediately after an Arg-Arg basic amino acid pair located in the central region of the precursor. MATERIALS AND METHODS Additional amino acid homologies suggest that tandem dupli- cations have contributed to the structure of the gene.
    [Show full text]
  • Impaired Cleavage of Preproinsulin Signal Peptide Linked To
    CORE Metadata, citation and similar papers at core.ac.uk Provided by Caltech Authors - Main ORIGINAL ARTICLE Impaired Cleavage of Preproinsulin Signal Peptide Linked to Autosomal-Dominant Diabetes Ming Liu,1,2 Roberto Lara-Lemus,1 Shu-ou Shan,3 Jordan Wright,1 Leena Haataja,1 Fabrizio Barbetti,4 Huan Guo,1 Dennis Larkin,1 and Peter Arvan1 Recently, missense mutations upstream of preproinsulin’s signal the ER exit of wild-type (WT) proinsulin and decreasing peptide (SP) cleavage site were reported to cause mutant INS insulin production (21). Moreover, recent studies indicate gene-induced diabetes of youth (MIDY). Our objective was to that insulin deficiency precedes a net loss of b-cell mass understand the molecular pathogenesis using metabolic labeling (22,23), suggesting that this dominant-negative blockade and assays of proinsulin export and insulin and C-peptide pro- caused by mutants is sufficient to account for initial onset duction to examine the earliest events of insulin biosynthesis, of diabetes in MIDY (24,25). highlighting molecular mechanisms underlying b-cell failure plus b fi In -cells, insulin synthesis begins with the precursor a novel strategy that might ameliorate the MIDY syndrome. We nd preproinsulin, which must undergo cotranslational trans- that whereas preproinsulin-A(SP23)S is efficiently cleaved, pro- ducing authentic proinsulin and insulin, preproinsulin-A(SP24)D location into the ER, signal peptide (SP) cleavage and is inefficiently cleaved at an improper site, producing two subpo- downstream proinsulin folding. These earliest events are pulations of molecules. Both show impaired oxidative folding and critical to insulin biosynthesis, but they are relatively are retained in the endoplasmic reticulum (ER).
    [Show full text]
  • Neurotensin and Its Receptors Q Philippe Sarret, Florine Cavelier
    Neurotensin and Its Receptors q Philippe Sarret, Florine Cavelier To cite this version: Philippe Sarret, Florine Cavelier. Neurotensin and Its Receptors q. Neuroscience and Biobehavioral Psychology, 2018. hal-03150277 HAL Id: hal-03150277 https://hal.archives-ouvertes.fr/hal-03150277 Submitted on 10 Mar 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Neurotensin and receptors Philippe Sarret* and Florine Cavelier$ *Département de Pharmacologie & Physiologie, Institut de Pharmacologie de Sherbrooke, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4 $Institut des Biomolécules Max Mousseron, UMR-5247, CNRS, Université Montpellier, 34095 Montpellier Cedex 5, France Philippe Sarret, PhD Professeur Titulaire, Département de Pharmacologie & Physiologie Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001, 12Ème Avenue Nord, Sherbrooke, Québec, Canada, J1H 5N4 Tel: (819) 821 8000 ext : 72554 Email: [email protected] Florine Cavelier, PhD Directeur de
    [Show full text]
  • Cell-Free Synthesis of Fish Preproinsulin, and Processing By
    Proc. Natl. Acad. Sci. USA Vol. 74, No. 5, pp. 2059-2063, May 1977 Cell Biology Cell-free synthesis of fish preproinsulin, and processing by heterologous mammalian microsomal membranes (mRNA from islets of Langerhans/wheat germ system/canine pancreatic microsomal membranes/amino-terminal sequences/ sequence homologies) DENNIS SHIELDS AND GUNTER BLOBEL Department of Cell Biology, Rockefeller University, New York, New York 10021 Communicated by Gerald M. Edelman, March 10, 1977 ABSTRACT Poly(A-containing mRNA isolated from the peptidase, resulting in the synthesis of proinsulin rather than islets of Langerhans obtained from two species of fish, angler preproinsulin. fish (Lophius americanus) and sea raven (Hemitripterus amer- icanus), stimulated protein synthesis 16-fold in a wheat germ cell-free system. Characterization of the translation products METHODS by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed a major polypeptide weighing 11,500 daltons that was Extraction of mRNA from Islet Tissue. Approximately 1 specifically precipitated by an antibody against angler fish in- g of islet tissue was homogenized in a Waring Blendor for 1 min sulin. Partial sequence analysis of the amino terminal revealed at room temperature in 30-40 volumes of 150 mM NaCl/50 that this polypeptide is preproinsulin, in which the amino ter- mM Tris-HCI, pH 7.4/5 mM EDTA/1% sodium dodecyl sul- minus of proinsulin is preceded by either 23 (angler fish) or 25 fate. Proteinase K was added to a concentration of 100 ,g/ml (sea raven) amino acid residues. Translation of fish islet mRNA and the solution was incubated at room temperature for 10 min.
    [Show full text]
  • The Role of Residues 23–60 of the Calcitonin Receptor-Like Receptor
    Peptides 31 (2010) 170–176 Contents lists available at ScienceDirect Peptides journal homepage: www.elsevier.com/locate/peptides Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23–60 of the calcitonin receptor-like receptor James Barwell a, Philip S. Miller b, Dan Donnelly b, David R. Poyner a,* a School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK b Institute of Membrane & Systems Biology, LIGHT Laboratories, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK ARTICLE INFO ABSTRACT Article history: The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide Received 18 September 2009 (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor Received in revised form 29 October 2009 activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in Accepted 29 October 2009 this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) Available online 11 November 2009 was examined by an alanine scan and the results were interpreted with the help of a molecular model. The potency of CGRP at stimulating cAMP production was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Keywords: Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also CLR impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was CGRP RAMP1 abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell Family B GPCRs surface expression of Gln45Ala, Cys48Ala and Tyr49Ala.
    [Show full text]