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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK021860-15
Application #
3483406
Study Section
Endocrinology Study Section (END)
Project Start
1978-07-01
Project End
1996-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
15
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
Radulescu, Andreea E; Shields, Dennis (2012) Clathrin is required for postmitotic Golgi reassembly. FASEB J 26:129-36
Radulescu, Andreea E; Mukherjee, Shaeri; Shields, Dennis (2011) The Golgi protein p115 associates with gamma-tubulin and plays a role in Golgi structure and mitosis progression. J Biol Chem 286:21915-26
How, Poh Choo; Shields, Dennis (2011) Tethering function of the caspase cleavage fragment of Golgi protein p115 promotes apoptosis via a p53-dependent pathway. J Biol Chem 286:8565-76
Riebeling, Christian; Morris, Andrew J; Shields, Dennis (2009) Phospholipase D in the Golgi apparatus. Biochim Biophys Acta 1791:876-80
Mukherjee, Shaeri; Shields, Dennis (2009) Nuclear import is required for the pro-apoptotic function of the Golgi protein p115. J Biol Chem 284:1709-17
Mukherjee, Shaeri; Chiu, Raymond; Leung, Som-Ming et al. (2007) Fragmentation of the Golgi apparatus: an early apoptotic event independent of the cytoskeleton. Traffic 8:369-78
Radulescu, Andreea E; Siddhanta, Anirban; Shields, Dennis (2007) A role for clathrin in reassembly of the Golgi apparatus. Mol Biol Cell 18:94-105
Cai, Dongming; Zhong, Minghao; Wang, Runsheng et al. (2006) Phospholipase D1 corrects impaired betaAPP trafficking and neurite outgrowth in familial Alzheimer's disease-linked presenilin-1 mutant neurons. Proc Natl Acad Sci U S A 103:1936-40
Freyberg, Zachary; Siddhanta, Anirban; Shields, Dennis (2003) ""Slip, sliding away"": phospholipase D and the Golgi apparatus. Trends Cell Biol 13:540-6
Siddhanta, Anirban; Radulescu, Andreea; Stankewich, Michael C et al. (2003) Fragmentation of the Golgi apparatus. A role for beta III spectrin and synthesis of phosphatidylinositol 4,5-bisphosphate. J Biol Chem 278:1957-65

Showing the most recent 10 out of 43 publications