Doctor of Philosophy
School of Chemistry and Molecular Bioscience
The accumulation of misfolded proteins has profoundly toxic consequences for organisms and has been linked to over 40 human disorders. Pregnancy is a unique physiological state involving elevated stresses that are capable of inducing protein misfolding and aggregation. However, essentially nothing is known about how the maternal body adapts during the stresses of pregnancy to maintain protein homeostasis (proteostasis). Uncovering how the maternal body maintains extracellular proteostasis during gestation is important, because the accumulation of misfolded proteins in biological fluids has been reported as a pathological feature of the pregnancy-specific disorder, pre-eclampsia. Pregnancy zone protein (PZP) and plasminogen activator inhibitor type-2 (PAI-2), are two enigmatic proteins attributed with various biological functions that are upregulated in pregnancy and in non-pregnancy associated inflammatory states. Both proteins are known for their protease inhibitory activity in vitro, but there are indications that they potentially contribute to extracellular proteostasis via other mechanisms including holdase-type chaperone activity. Thus, the overarching goal of this project was to provide the first evidence of a pregnancy-associated extracellular chaperone network by characterising the novel holdase-type chaperone activity of PZP and extracellular glycosylated PAI-2 in vitro.
The work described in Chapter 3 includes strategic attempts to optimise the purification of endogenous PZP from human pregnancy plasma. The newly devised procedure operates under non-denaturing conditions, allowing for the purification of native PZP with 40–75% increased yield compared to previously published approaches. Given that pregnancy plasma is technically and ethically challenging to acquire, novel attempts to produce recombinant PZP in human cells are also described in this chapter. The results showed that 6His-tagged recombinant PZP could be produced in HEK293-based cell lines in vitro, which was of a similar size to the endogenous form of PZP found in pregnancy plasma as assessed by native Western blot analysis. However, there were some issues with the quality of recombinant PZP-6His obtained, which was heterogeneous and exhibited formation of undesirable disulfide bonds with other proteins constitutively secreted by the HEK293-based expression systems.
Cater, Jordan Harry, Investigations of the roles of pregnancy zone protein and plasminogen activator inhibitor type-2 in extracellular proteostasis, Doctor of Philosophy thesis, School of Chemistry and Molecular Bioscience, University of Wollongong, 2021. https://ro.uow.edu.au/theses1/1263
FoR codes (2020)
3101 Biochemistry and cell biology
Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.