Dennis Shields - US grants

Total poly A containing mRNA isolated from Angler Fish and Cod islets of Langerhans will be translated in the wheat germ cell-free protein synthesizing system to characterize putative preproinsulin and preproglucagon and to utilize this system for testing predictions of the "signal hypothesis". These precursor molecules will be identified from the total translation products by a variety of analytical procedures including specific antibody precipitation SDS-polyacrylamide gel electrophoresis and peptide mapping. The complete amino acid sequence of the N-terminal signal sequences of these molecules will be determined using automatic sequencing techniques. Mammalian "stripped" microsomal membranes will be added to the cell-free system cotranslationally to determine and identify the processed and segregated products of proglucagon. The fate and ultimate sub-cellular localization of the signal peptide(s) following its removal from preproinsulin and preproglucagon, will be investigated by isolating the exogenously added microsomal vesicles from the translation system. The processed and segregated products will be characterized by SDS-gel electrophoresis, gel filtration and automatic Edman degradation. If the signal peptide is not immediately degraded, its subsequent localization will be determined using cell-fractionation and experiments will be performed to determine if a possibly intact signal peptide might be secreted.

Messenger RNA isolated from islets of Langerhans will be translated in a cell-free protein synthesizing system to study the biosynthesis and post-translational modifications of multiple precursors to glucagon and somatostatin. Three proglucagon molecules will be characterized by two-dimensional gel electrophoresis, peptide mapping and automated microsequensing techniques, to determine the structural relationship of each precursor to mature glucagon. The preproglucagons undergo different post-translational modifications in vitro and possible glycosylation, phosphorylation and/or lipid addition will be investigated. The in vitro biosynthesis of somatostatin from islet and brain mRNA's will be compared to determine (a) if multiple preprosomatostatin molecules are also synthesized in the brain (b) the similarity to the islet precursors. In parallel studies, specific cDNA clones to two different species of islet preprosomatostatin mRNA will be used to probe brain mRNA to analyze the expression of distinct somatostatin genes. The complete amino acid sequence of brain preprosomatostatin(s) will be determined using recombinant DNA methodology. These experiments will show if there is tissue-specific expression of the somatostatin genes. Three polypeptides corresponding to conserved proregions of fish, rat and human preprosomatostatin have been synthesized. Each of these polypeptides will be used in the production of specific antibodies to defined regions of prosomatostatin. The antisera will be used to study the fate of the propeptide during somatostatin secretion from a rat islet tumor cell line. Techniques of immunoprecipitation, immunofluorescence and electron microscopy immunocytochemical localization using colloidal gold will be employed to determine if prosomatostatin or fragments of the precursor are degraded intracellularly or are secreted along with the mature hormone. Results obtained with antisera directed against profragments will be correlated with those using antisomatostatin antibodies. Should evidence be found for secretion of propeptides, in addition to the mature hormone, this might suggest such peptides could have physiological functions.

The objective of this proposal is to establish a "state of the art facility" for the analysis and characterization of proteins and peptides using automated microsequencing techniques, determination of amino acid composition, high performance liquid chromatography and DNA oligonucleotide synthesis. Our goal Is to establish a centralized protein structure facility housed in one laboratory. The major instrumentation requested is: (i) an Applied Biosystems Pulsed Liquid Phase Protein/Peptide Sequencer with on-line identification of phenylthlohydantoin derivatives; (ii) an Applied Biosystems Derivatizer Amino Acid Analyzer System for the determination of amino acid composition; iii) a Waters High Performance Liquid Chromatography System for preparative and analytical purification of proteins, peptides, phenylthiohydantoins and oligonucleotides and (iv) an Applied Biosystems DNA synthesizer for the preparation of oligonucleotides. This instrumentation and a recently acquired peptide synthesizer will provide investigators with three types of essential methodologies: protein sequencing, peptide synthesis and DNA synthesis. The facility will ensure that investigators can start at the protein, messenger RNA or gene levels to generate "genes from proteins" or "proteins from genes". The availability of this protein structure instrumentation is essential for continued productivity and optimal progress in the broad spectrum of research programs at Albert Einstein College of Medicine that are addressing fundamental problems in biological regulation. Presently, this Institution possesses one eight year old Beckman 890 C spinning-cup sequencer; although this instrument is still operational it is not sufficiently sensitive or reliable for modern sequencing applications. This application (a) documents the need for an up-dated centralized facility which will be available to our entire scientific community; (b) describes the feasibility of installing, operating and maintaining such a facility and (c) outlines a series of projects that require protein sequencing and which will make immediate use of the facility.

The pancreatic islet hormones somatostatin (SRIF), insulin and glucagon are synthesized as larger precursors. Our long term goal is to elucidate the function of polypeptide hormone precursors in mediating intracellular transport, post-translational processing and secretion of the mature hormone. PreproSRIF is a useful model for these studies since it is one of the simplest peptide hormone precursors. I. In vitro Reconstitution of Prohormone Processing and Sorting. Little is known about the molecular mechanisms whereby endocrine cells discriminate between proteins destined for the constitutive and regulated secretory pathways. To investigate prohormone processing and sorting in the Trans Golgi Network (TGN), we will use a cell-free system derived from GH3 cells expressing proSRIF. We propose to: (i) Characterize this in vitro system in detail; (ii) Investigate proSRIF cleavage in the TGN and the concomitant packaging of mature SRIF and endogenous GH into nascent secretory granules; (iii) Prepare in vitro systems from cells deficient in prohormone processing and by mixing experiments identify cell-specific components involved in prohormone processing and sorting. II. Identification of a Monobasic- residue Specific Prohormone Converting Enzyme. In many prohormones the bioactive peptide is flanked by pairs of basic amino acids, however in several precursors the hormone sequence is cleaved at single basic residues. We identified a novel proteolytic activity in yeast cells which cleaves heterologously expressed proSRIF-II correctly at a sequence the gene encoding this enzyme; (ii) Exploit the yeast gene to identify its mammalian equivalent by screening a pancreatic islet cDNA library; (iii) Co-express the protease cDNA and proSRIF-II in GH3 cells, where proSRIF-II is degraded intracellularly, to determine if the prohormone is then cleaved to SRIF-28. Our goal is not only to identify a novel prohormone processing enzyme(s) but also to understand the molecular basis whereby cells discriminate between prohormones which are substrates for proteolytic processing from those targeted for intracellular degradation. III. Structure-Function Studies on Topogenic Domains in Peptide Hormone Precursors. The SRIF propeptide functions in mediating intracellular transport and correct proteolytic processing. To identify structural features that effect precursor sorting and processing, we have over- expressed several proSRIFs in E.coli. We will: (i) Characterize conserved domains in different species of proSRIF by identifying protease-resistant and -sensitive regions of the precursors; (ii) Purify the proSRIFs to homogeneity, prepare crystals of the purified polypeptides and determine their X-ray crystallographic structure. These studies will enable us to identify structural "domains" which function in mediating intracellular transport. There is now evidence that defects in proinsulin processing lead to certain forms of familial hyperinsulinemia. Therefore understanding the biosynthesis and sorting of the islet hormones has important implications concerning the etiology of diabetes.

DESCRIPTION (provided by applicant): The selective packaging of peptide hormone precursors into secretory vesicles is essential for hormone biosynthesis. In the trans Golgi network (TGN), the precursors are sorted into nascent secretory granules where they are processed to generate bioactive polypeptides. Data from our laboratory has demonstrated that the maintenance of Golgi structure and release of hormone containing vesicles from the TGN requires phosphatidic acid (PA), the product of phospholipase D (PLD)-mediated phosphatidylcholine hydrolysis, as well as synthesis of the inositol phospholipids phosphatidylinositol (4) phosphate, PI(4)P, and phosphatidylinositol (4,5) bisphosphate, PI(4,5)P2. The goal of this proposal is to understand the mechanisms whereby these lipids regulate Golgi structure, budding of secretory vesicles from the TGN and hormone processing. Aim 1: The role of PI(4)P and PI(4,5)P2 in maintaining Golgi structure and function in endocrine cells: We will test the hypothesis that in pituitary GH3 cells both PI(4)P and PI(4,5)P2 are required to maintain Golgi architecture whereas Pi(4,5)P2 also mediates release of post-Golgi vesicles and trafficking to the plasma membrane. Aim I1: The Regulation of Golgi PI(4,5)P2 synthesis: Surprisingly, the Golgi apparatus appears to possess little PI(4,5)P2 and its level maybe tightly controlled, in part, by a potent Pl(4,5)P2-5-phosphatase activity that we have identified. We will test the hypothesis that PI(4,5)P2 synthesis is regulated locally by recruitment of specific PI(4)P 5-kinases to the Golgi apparatus and by the activity of the Pl(4,5)P2-5-phosphatase. Aim III. To determine the role of PLD isoforms and PA in maintaining Golgi structure: PA has been implicated in vesicle trafficking, signal transduction and regulation of PI(4,5)P2 synthesis. We demonstrated in endocrine cells that PLD1 and -2, which generate PA, localize to different regions of the Golgi apparatus. We will determine the mechanism of PLD recruitment to the Golgi apparatus and use novel PA reporter proteins to demonstrate PA itself localizes to the Golgi apparatus. Although multiple enzymes involved in phospholipid biosynthesis have been characterized, in endocrine cells relatively little is known whereby the different activities are coordinated and integrated in the Golgi apparatus to control the formation of nascent secretory vesicles. Consequently, understanding the mechanism of secretory vesicle budding will facilitate the rational design of drugs that could be used to enhance or diminish hormone secretion in a variety of pathological conditions including diabetes.