Biological and structure characterisation of eukaryotic prefoldin
Date
2018
Authors
Tran, Denise Phuong
Editors
Advisors
Pukala, Tara Louise
Booker, Grant William
Booker, Grant William
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Theses
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Abstract
Prefoldin is a hexameric protein complex ubiquitously expressed and found to influence the
conformation of amyloidogenic peptides. Relatively high degrees of sequence identity and
conservation across evolutionary lineages are observed, however differences in binding abilities
have been noted between the homologs. This thesis describes work examining the structure of
eukaryotic prefoldin and its biological activities with respect to interaction with amyloid β. The
structure and biological activities of prefoldin’s individual subunits are also explored.
Although many studies have investigated the structure of prokaryotic prefoldin, there is limited
information available for eukaryotic prefoldin. Two-dimensional ¹H-¹H and ¹H-¹³C nuclear magnetic
resonance (NMR) spectroscopy was utilised to probe the structure of both α and β human prefoldin
subunits. The data revealed the highly alpha helical secondary structure of the subunits, which was
further verified through far-UV circular dichroism. Further thermal aggregation assays utilising this
technique have demonstrated the stability of the prefoldin subunits.
The biological effect of prefoldin on the amyloid fibril formation of the Alzheimer’s disease related
amyloid β peptide was investigated using a combination of dye-binding assays and cytotoxicity
assays. The presence and absence of fibrils was confirmed by transmission electron microscopy. In
terms of fibril formation, prefoldin and its subunits prevented in vitro conversion of the amyloid β
peptide to amyloid fibrils. In some cases, total inhibition of fibril formation occurred and a 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted on the resultant
products. The product was incubated with healthy PC-12 cells and induced cellular death, therefore
establishing the cytotoxicity of the resultant oligomeric amyloid β form.
Previous investigations into the binding capabilities of prokaryotic prefoldin identified the distal tips
as an important structural aspect, interacting with the amyloidogenic peptide. The binding interface
of prefoldin subunits 5 and 6 with amyloid β was probed using chemical cross-linking (CXL)
experiments. Traditional methods to identify cross-linked peptides are challenging and the results
are often ambiguous. In this study, CXL products were analysed by liquid chromatography-ion
mobility-mass spectrometry (LC-IM-MS) to investigate the utility of IM in enhancing the CXL
analytical workflow. The orthogonal separation of ion mobility enabled the identification of the
cross-linked amino acids. The distal end of prefoldin subunit 5 was found to interact with the Nterminus
of the amyloid peptide, whereas prefoldin subunit 6 was identified to interact with the
peptide in the middle of its sequence. Ion mobility-mass spectrometry (IM-MS) analysis of the eukaryotic prefoldin complex identified the
collisional cross section of the intact hexamer. Solution disruption experiments of the intact complex
revealed the disengaging sub-complexes, and information on the intersubunit contacts and relative
interfacial strengths were obtained. A capillary temperature controller (CTC) was developed to
observe the thermal dissociation of the complex using nano-electrospray IM-MS.
The combination of these results confirmed a structural aspect common to both mammalian
prefoldin and prokaryotic prefoldin, despite the primary sequence differences. The biological assays
revealed the ability of prefoldin to prevent the aggregation and amyloid fibril formation of amyloid
β, and low resolution MS techniques were able to postulate the arrangement of the subunits and the
possible interface interactions of the hexameric complex with the amyloidogenic peptide. This thesis
has therefore provided an in-depth investigation of the structural characteristics of eukaryotic
prefoldin and its chaperoning capability, therefore implicating a potential role for prefoldin in
modulating protein misfolding and aggregation.
School/Discipline
School of Physical Sciences
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 2018
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