Year

1999

Degree Name

Doctor of Philosophy

Department

Department of Biological Sciences

Abstract

Clusterin is a highly conserved protein that has been found in every mammalian species examined and in two species of aves. Clusterin is distributed widely throughout the mammalian body and interacts with a diverse array of proteins. These many binding interactions have led to suggestions that clusterin is involved in an equally diverse array of functions. Clusterin is also widely reported to be dramatically upregulated in times of stress and during apoptosis indicating that it may regulate cell death. However, no genuine physiological function for clusterin has yet been established.

One of the two main goals of this project was to determine whether clusterin expression was involved in regulating cell death. This was investigated by transfecting two different expression systems containing human clusterin cDNA into L929 cells and determining if the upregulated expression of clusterin altered the rate of cell death induced by either TNFα, TNFα + actinomycin D, staurosporine, colchicine, or azide. Cells transfected with an expression system containing a cytomegalovirus (CMV) promotor were found to be resistant against TNFα-induced cell death. These transfected cells were not protected from any other cytotoxic agent tested. The other expression system (which resulted in lower levels of clusterin expression) did not provide cells with protection against TNFα or any of the other cytotoxic agents. Further studies suggested that clusterin, normally a secreted product, acted intracellularly to protect L929 cells from TNFa-induced death. However, a control L929 cell line that overexpressed green fluorescent protein was also shown to be protected from TNFα-induced death. Therefore it is possible that the cytoprotection associated with overexpression of clusterin is an artifact resulting from non-specific effects of high levels of protein expression.

It has been deduced from the amino acid sequence of clusterin that it contains three amphipathic helixes, which are required for hydrophobic interactions. It has also been demonstrated that clusterin has a high affinity for hydrophobic surfaces as it can be purified from ram rete testis fluid on the basis of it's strong hydrophobic interaction with Affi-blue gel. Other reports have suggested that clusterin's association with complement, apolipoprotein A-I and cell aggregation are due to hydrophobic interactions. Thus, it appears likely that many of clusterin's binding interactions involve binding to regions of exposed hydrophobicity, a similar property to that of chaperone proteins which bind to regions of exposed hydrophobicity on other proteins. It was also recently demonstrated that a conserved region in the clusterin promotor binds heat shock factor 1, a transcriptional activator of the heat shock protein chaperones. This element can also mediate heat-shock-induced transcription suggesting that expression of clusterin and heat shock proteins may be co-regulated. The second main goal of this project was to test the hypothesis that clusterin has chaperone-like activity.

Clusterin was shown to prevent catalase and GST from heat-induced precipitation as well as preventing BSA and α-lactalbumin from reduction-induced precipitation. It was demonstrated by ELISA that clusterin binds with higher affinity to the denatured form of these proteins. Futhermore, gel filtration analyses of solutions containing any one of these protein together with clusterin, following stress treatment, revealed that high molecular weight complexes had formed between the two proteins. It is proposed that clusterin protects proteins from stress-induced precipitation by binding to hydrophobic regions that become exposed to the solvent to form solubilized high molecular weight complexes. The ability of clusterin to non-specifically protect various proteins from stress-induced precipitation confirms that clusterin has chaperone-like activity and suggests that clusterin has a complementary role to heat shock proteins in vivo.

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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.