The effect of homocysteine on the oxidative folding pathway in the ER

Abstract

Upon translation into the endoplasmic reticulum (ER), ER resident proteins, and those destined for the secretory pathway, must be correctly folded into their native structure to be functional. During folding, many proteins must form disulphide bonds, a process facilitated by protein disulphide isomerase (PDI). Endoplasmic reticulum oxidoreductase 1 (Ero1) catalyses the formation of disulphides, using molecular oxygen as a terminal electron acceptor to drive the reoxidation of PDI. Ero1 must be tightly regulated to maintain the redox balance of the ER. One way in which this is achieved is by switching between an active or inactive state. Homocysteine, a precursor in the metabolism of cysteine and methionine, has been shown by the laboratory to induce the active form of Ero1α (OX1) in oesophageal cells. This finding was further investigated in this thesis using conformation-specific and anti-tag antibodies. Homocysteine is shown to induce an OX1 form in both transfected Ero1α and Ero1β. Ero1 OX1 was seen in multiple cell lines and in liver, but not pancreas tissue. This highlights the possibility that oxidative protein folding may be subjected to different regulation between tissues, depending on the redox poise. The site of homocysteine action, was investigated using Ero1α cysteine-to-alanine and Ero1β structural mutants. The data suggest that Ero1α is indirectly post-translationally modified by homocysteine at an antibody epitope site and not the CxxC redox-active cysteine residues.

The consequences of manipulating the oxidative folding environment were also examined by analysing a client protein, namely the MHC class II complex. These proteins are essential for the presentation of externally derived antigens to helper T cells. The disulphide containing MHC II molecules are folded in the ER, and the laboratory have previously shown that there are differences between the 3 isotypes (HLA-DR, DP and DQ) in their requirements to form stable dimers. Work in this thesis shows that the thermal stability of DPheterodimers is greater than that of DR heterodimers. However, homocysteine does not influence the ability of MHC class II molecules to form stable dimers.

The studies presented in this thesis have implications for diseases such as cancer, where ER and oxidative stress may involve dysregulation of the oxidative folding machinery. High expression of Ero1α has recently been associated with cancers of the oesophagus and stomach, and low expression of Ero1 has been associated with poor prognosis of cancers such as osteosarcoma. Thus manipulating the ER redox poise through small molecules such as homocysteine may be worthy of further exploration to limit Ero1α activation during pathogenesis.