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Thesis (Ph. D.)--Columbia University, 1925. / Vita. Bibliography: p. 27-28.

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Abstract Protein folding of outer membrane and secreted proteins, including receptors, cytokines and antibodies is often linked to disulphide bond formation. Native disulphide bond formation is complex and is usually the rate limiting step in the folding of such proteins. The enzymes which catalyse the slow steps in disulphide bond formation belong to the protein disulphide isomerase (PDI) family. PDI catalyses formation, reduction and isomerization of newly synthesized disulphide bonds. The mechanisms of action of the PDIs are currently poorly understood and this not only inhibits our understanding of the biogenesis of a range of medically important proteins, and hence associated disease states, but also prevents the effective manipulation of the cellular environment by the biotechnology industry for the production of high value therapeutic proteins. Hence, understanding the mechanism of action of these enzymes is vital for a wide range of medically important processes and therapies. In this study the role of a conserved arginine residue in the catalytic activity of PDI was shown. The movement of this residue into and out of the active site locale of PDI was shown to modulate the pKa of the C-terminal active site cysteine of PDI and by that way to allow the enzyme to act efficiently as catalyst both of oxidation and isomerization reactions. The possible role of hydrogen peroxide produced by sulphydryl oxidases during disulphide bond formation was studied in an oxidative protein refolding assay. Analysis showed that hydrogen peroxide can be used productively to make native disulphide bonds in folding proteins with minimal side reactions. In addition, the kinetics of oxidation and reduction of the <b>a</b> domains of PDI and Pdi1p by glutathione was studied in this thesis. The kinetics obtained with stopped-flow and quenched-flow experiments showed the reactions to be more rapid and complex than previously thought. Significant differences exist between the kinetics of PDI and Pdi1p. This implies that the use of yeast systems to predict physiological roles for mammalian PDI family members should be treated cautiously.

Currently the mammalian endoplasmic reticulum (ER) is known to contain at least 20 different protein disulphide isomerase (PDI) family members. The oxidoreductases in the PDI family are thought to catalyse the formation and rearrangement of disulphide bonds in newly synthesised proteins. The focus of this work was to characterise two of the PDI family members: PDI and ERp46. In vitro translation reactions of major histocompatibility complex (MHC), β1-integrin (β1-I), haemagglutinin (HA), procollagen α1(III) and preprolaction (pPL) were carried out in untreated or PDI-depleted cells. The depletion of PDI decreased the rate of folding of MHC and β1-I and also prevented the oligomerisation of HA, suggesting a role for PDI in folding these putative substrates. However, when PDI was depleted neither the folding of pPL or HA was affected, implying that they may not be substrates for PDI. To determine the role of ERp46 in the cell, a substrate-trapping approach was used. Here substrates interacting with ERp46 were trapped as mixed disulphides isolated by immunoprecipitation, separated by 2D SDS-PAGE and identified by mass spectrometry. It was demonstrated that ERp46 forms mixed disulphides with at least 23 proteins, including heavily secreted proteins such as laminins, integrins and collagens. In particular, interactions with Ero1, Prx IV, EDEM3 and ERAP2 were found and confirmed by immunoprecipitation of radiolabelled in vitro translated protein. Notably nine of these clients of ERp46 have previously been identified as substrates of ERp57 (Jessop, Watkins et al. 2009). This would support the hypothesis that several different oxidoreductases, working in concert, are required to fold certain substrate proteins. Also, it was confirmed that Prx IV and Ero1 each form a mixed disulphide with PDI. These results highlight the importance of PDI family members in recruiting co-factors to substrates. Additionally, the over-expression of ERp46 led to increased cell survival following DTT treatment, yet after depletion of ERp46, cells were less able to grow, perhaps suggesting a role for ERp46 in maintaining ER redox homeostasis and cell survival. This suggestion was supported by the finding that ERp46 is able to catalyse the reduction of Prx IV in the presence of glutathione. These results suggest that Prx IV provides a novel mechanism for the transfer of disulphide bonds to nascent proteins in the ER via PDI family members such as ERp46 and PDI.