BIOSYNTHESIS AND PROCESSING OF PEPTIDE HORMONE

Project: Research project

Description

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.
StatusFinished
Effective start/end date7/1/786/30/10

Funding

  • National Institutes of Health: $609,021.00

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Phosphatidic Acids
Peptide Hormones
Golgi Apparatus
Secretory Vesicles
trans-Golgi Network
Phospholipase D
Endocrine Cells
Hormones
Phosphatidylinositols
Phosphoric Monoester Hydrolases
Drug Design
Product Packaging
Phosphatidylcholines
Signal Transduction
Phospholipids
Protein Isoforms
Hydrolysis
Phosphotransferases
Maintenance
Cell Membrane

ASJC

  • Medicine(all)