Walker, Kelly L. (2013) Molecular characterisation of PDIp : the pancreas-specific isoform of PDI. PhD thesis, University of Warwick.

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Abstract

PDIp is a close homolog of the well known protein folding catalyst PDI. Unlike PDI
however, PDIp exhibits restricted protein expression and is found predominantly in
the exocrine pancreas. Currently, the physiological function of PDIp is unknown but
previous work has shown a clear specificity for substrates containing a hydroxyaryl
group. Again this is in contrast to PDI which exhibits more general specificity. This
work to investigate the in vitro activities of PDIp, was stimulated by the hypothesis
that PDIp has an essential role in folding a specific subset of secretory proteins. The
identity of these proteins is currently unknown.
In this work, the redox-mediated conformational changes of PDIp have been studied
and compared using far UV CD, dynamic light scattering and limited proteolysis.
Compared to PDI, these changes are conservative in PDIp. Also, unlike PDI for
which b’xa’c is the minimal redox-active cassette, the PDIp a and b domains seem to
be involved in modulating these conformational changes. This may indicate that
PDIp has a unique substrate binding mechanism that may work synergistically with
its restricted substrate specificity.
Using the insulin reduction assay, PDIp was shown to have ~50% of the oxidoreductase
activity of PDI and this was not due to the aberrant threonine residue in the
a’ domain active site motif (CTHC). Further investigation by stopped flow kinetic
studies showed that the low activity could be due to the abnormally high pKa for the
PDIp a domain N-terminal catalytic cysteine. This result was unexpected because
PDI and PDIp a share the same catalytic active site motif (CGHC) indicating that
nearby residues may act as mediators of activity. Future work to clarify this will be
essential.
This is the first study of the structure of PDIp and its molecular basis for activity.
Through investigation of these two areas, it is hoped that the general understanding
of the role of PDIp and its contribution to oxidative folding in secretory tissues will
be improved.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QH Natural history > QH301 Biology Q Science > QP Physiology
Library of Congress Subject Headings (LCSH):	Protein disulfide isomerase -- Structure -- Research, Protein folding, Catalysts, Molecular biology
Official Date:	July 2013
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Freedman, R. B.
Sponsors:	Medical Research Council (MRC)
Extent:	xvi, 218 leaves : illustrations, charts.
Language:	eng

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