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Extracellular Chaperones and Their Cell Surface Receptors University of Wollongong Research Online University of Wollongong Thesis Collection 2017+ University of Wollongong Thesis Collections 2020 Extracellular Chaperones and their Cell Surface Receptors Jennifer Renee West Follow this and additional works at: https://ro.uow.edu.au/theses1 University of Wollongong Copyright Warning You may print or download ONE copy of this document for the purpose of your own research or study. The University does not authorise you to copy, communicate or otherwise make available electronically to any other person any copyright material contained on this site. You are reminded of the following: This work is copyright. Apart from any use permitted under the Copyright Act 1968, no part of this work may be reproduced by any process, nor may any other exclusive right be exercised, without the permission of the author. Copyright owners are entitled to take legal action against persons who infringe their copyright. A reproduction of material that is protected by copyright may be a copyright infringement. A court may impose penalties and award damages in relation to offences and infringements relating to copyright material. Higher penalties may apply, and higher damages may be awarded, for offences and infringements involving the conversion of material into digital or electronic form. 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. Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: [email protected] Extracellular Chaperones and their Cell Surface Receptors By Jennifer Renee West Bachelor of Arts (Biological Sciences) – Hanover College Bachelor of Science (Honours) – University of Wollongong Supervised By Senior Professor Mark Wilson Co-supervisor Associate Professor Heath Ecroyd This thesis is presented as part of the requirements for the degree of Doctor of Philosophy School of Chemistry & Molecular Bioscience And The Illawarra Health & Medical Research Institute University of Wollongong Wollongong, Australia June 2020 Declaration of Authority This thesis is submitted in accordance with the regulations of the University of Wollongong in partial fulfilment of the Degree of Doctor of Philosophy. It does not include any material published by another person except when due reference is made in the text. The experimental work described in this thesis is original and has not been submitted for a degree to any other university. Some experimental work conducted for this project was completed with the assistance of a facilitator, provided by the University of Wollongong. Jennifer Renee West i Acknowledgments First and foremost, thank you to Mark for being not only an excellent supervisor, but also a patient teacher/mentor and someone who genuinely cares about your success as a student. I am so grateful to have had the opportunity to learn from and work along-side such an intelligent and inspiring person. Mark – also, thank you for pulling me out of my Debbie-downer thinking and providing many delicious baked goods! I will miss working in your lab very much! Facilitators: Daniel Whitten, Luke McAlary, Rebecca Brown, Kate Roberts, Rafaa Zeineddine, Jay Perry, & Sandeep Satapathy – thank you all so very much for dedicating your valuable time in assisting me with my experimental work – I literally could not have done this without you guys! Megan Kelly – thank you for all of your help with protein aggregation and cytotoxicity assays, and also your company, encouragement, and conversations in the lab. Lab 210 colleagues – thank you all for making my journey at UOW both memorable and fun. Thank you all for your advice, recommendations, and overall friendship. I will miss all of you very much! IHMRI Staff – thank you for making IHMRI such a safe, well-organised, and fantastic environment to work in. A huge thank you to Tom, for putting up with me when I was stressed and at times discouraged throughout this monstrous project. Thank you to my parents, Tom’s parents, and all of my extended family for your support and words of encouragement – it really meant a lot. I love you all! This research has been conducted with the combined support of the Australian Government Research Training Program Scholarship and a scholarship received from Samsung, to which I am very thankful to have received. ii Abstract Nearly all biological systems require proteins to achieve and maintain their native, three- dimensional conformation, however, various stresses and genetic mutations can cause proteins to misfold and aggregate. Both intracellular and extracellular protein aggregates are associated with protein deposition diseases (PDDs), such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). Although the processes involved in maintaining intracellular protein quality control are well established, much less is known regarding the maintenance of extracellular proteostasis. Extracellular chaperones (ECs) are a unique class of proteins that have been shown to inhibit the misfolding and aggregation of various client proteins in vitro by forming stable, soluble high molecular weight (HMW) complexes with them. Furthermore, ECs direct aggregates to (yet unknown) cell surface receptors, which internalise the complex by receptor-mediated endocytosis for degradation within lysosomes. Using in vitro protein aggregation assays, this study identified neuroserpin (NS), transthyretin (TTR), and thyroglobulin (Tg) as new ECs, with NS and TTR being the first ECs classified as amyloid-specific. Though less efficient compared to clusterin (CLU), NS did partially inhibit the aggregation of citrate synthase (CS), an amorphously- aggregating client protein, but had no inhibitory effect on the amorphous aggregation of chloride intracellular channel 1 (Clic1). Neither native tetrameric TTR (hTTR) nor a monomeric TTR mutant (mTTR) specifically inhibited the amorphous aggregation of bovine serum albumin (BSA), creatine phosphokinase (CPK), or CS. In contrast, NS and mTTR were efficient inhibitors of amyloid formation by both coiled-coil beta peptide (ccbw) and alpha-synuclein (a-syn); hTTR specifically inhibited a-syn aggregation only. Sequence comparisons between NS and TTR identified a contiguous 14-residue region in both proteins that is 64% identical and 71% similar. The majority (57% in NS and 71% in TTR) of the residues within this region corresponded to an a-helix in both NS and TTR and, in the case of TTR, residues within this helix are known to be involved in the binding of beta-amyloid (Ab) to TTR. Synthetic peptides were generated corresponding to the conserved sequences incorporating the NS and TTR a-helices for use in in vitro protein aggregation assays. Rather than having an inhibitory effect, the peptide corresponding to the NS sequence increased amyloid formation by ccbw in a concentration-dependent manner, suggesting that the peptide may be interacting with ccbw amyloid species. Tg appears to have a more broad-spectrum chaperone action similar to that of CLU and alpha-2- macroglobulin (a2M). Tg concentration-dependently inhibited amorphous protein aggregation by iii CS and ovotransferrin (OT) and amyloid formation by Ab and calcitonin (Cal). Like CLU, Tg forms stable, soluble HMW complexes with CS, which have an average particle size of 82.5 nm according to DLS analyses. The Tg-CS complexes were significantly larger than both Tg (average particle size of 45.6 nm) and CS (average particle size of 8.5 nm). It was also shown that the Tg dimer dissociates into monomers when heated to 60°C for 5 min, and that heat-treated Tg has a significantly higher surface hydrophobicity compared to untreated Tg. Attempts were made to identify the cell surface receptors involved in the endocytosis of EC-client complexes, however, these initial experiments used biotinylated EC-client complexes. Subsequent results showed that biotinylation of HMW CLU-client complexes induced structural changes leading to differences in (i) size (increased), (ii) their ability to bind to cells, (iii) dissociation of the complex into its constituent individual proteins under non-reducing and reducing conditions and (iv) antibody recognition of the complex. The work presented here demonstrates that the cell lines HepG2 and EOC13.31 and two receptors (apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VLDLR)) may bind CLU-GST complexes specifically, however, further testing would be required to definitively establish this. Collectively, the work presented here suggests that NS, TTR, and Tg likely have important protective roles as ECs in vivo. Determining whether the conserved a-helix shared between NS and TTR plays a role in the proteins’ chaperone action may lead to a better understanding of the structure or biophysical properties required for amyloid-interaction and lead to the future development of therapies for diseases associated with amyloid deposition. Like CLU, Tg appears to have a promiscuous chaperone activity and can form soluble HMW complexes with amorphously-aggregating client proteins. However, whether Tg plays a role in the cellular uptake of Tg-client complexes for intracellular degradation remains unknown. Future work is required to (i) identify the particular biophysical properties and/or binding site on EC-client complexes that mediate their uptake and (ii) the specific receptors involved
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