IN VITRO BIOSYNTHESIS OF INSULIN AND GLUCAGON

Project: Research project

Project Details

Description

The pancreatic islet hormones somatostatin (SRIF), glucagon and insulin are
synthesized as larger precursors. Our long term goal is to determine the
role of the presursors in mediating intracellular transport,
post-translational processing and secretion of the hormones. PreproSRIF is
a useful model for these studies since it is one of the simplest peptide
hormone precursors, containing only a single bioactive peptide and one
proteolytic processing site for excision of the mature hormone. Using
recombinant DNA techniques a series of mutated preproSRIF molecules will be
constructed to investigate functional domains in the precursor.
Site-directed mutagenesis will be used to alter individual amino acids in
the proteolytic processing site; deletion mutants lacking defined regions
of the propeptide will be constructed and the propeptide will be fused with
non-secretory proteins. These cDNAs will be transfected into heterologous
cells and the parameters of secretion investigated. Common sorting and
processing domains in yeast preproalpha factor and preproSRIF will be
identified by expressing native prepro-SRIF cDNAs and chimeric cDNAs
encoding variable amounts of these respective precursors in yeast. cDNA
encoding preproglucagons will be introduced into different cell types and
their post-translational processing and secretion investigated. In
contrast to preproSRIF, preproglucagons encode several glucagon-related
peptides and thus represent the next order of complexity of precursor
processing. Translation stop codons will be introduced into preproinsulin
cDNA which will be used in a coupled transcription-translation system to
generate truncated preproinsulins. These will be used to investigate the
minimum size of nascent polypeptide chains that can be transfered across
the ER membrane. Prohormone processing enzymes will be identified using a
molecular approach: A cDNA library from cells exhibiting high levels of
processing activity will be inserted into a retrovirus expression vector
and recomtinant virus used to infect L cells in which preproSRIF cDNA has
been stably integrated and which secrete only proSRIF, since they lack
prohormone processing activity. Secretion of mature SRIF will be assayed
using an antibody immunoblotting procedure; DNA will be isolated from
positive cells and characterized. Identifying structural determinants that
mediate processing of islet hormone presursors had direct relevance to
understanding their synthesis and secretion and has important implications
concerning the etiology of diabetes.
StatusFinished
Effective start/end date12/31/896/30/10

Funding

  • National Institute of Diabetes and Digestive and Kidney Diseases: $609,021.00

ASJC

  • Biochemistry
  • Cell Biology
  • Medicine(all)

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