The redox enzymes ErolL-alpha and Ero1L-beta play a central role in the maintenance of the Endoplasmic Reticulum (ER) by catalysing disulphide bond formation. Ero family proteins probably act byoxidising Protein Disulphide Isomerase (PDI), which in turnoxidises substrate proteins, enabling substrates to form disulphide bonds. Ero proteins are required for disulphide bond formation, however the substrate specificity of Ero proteins has not been addressed up until now. My project will ask the question:
What is the substrate specificity of human Ero proteins and how is this specificity achieved?
I would like to study the ER redox machinery by focusing on the MHC class I and II antigen presentation molecules. The components required for the oxidation of MHC molecules are largely unknown. Understanding the oxidation process of MHC molecules is very relevant as the binding of antigenic peptides to these important mediators of immunity requires properly folded molecules. In addition, I will address a number of specific questions:
- Are Ero proteins involved in the oxidation of MHC molecules? If so, how do they catalyze this process?
- What other (unknown) oxidoreductases or intermediates are involved in MHC class I and II oxidation ?
- How does MHC class I oxidation influence peptide loading?
- How is MHC class II and Ii oxidation coordinated in relation to Iiassociation? These experiments should result in an increased understanding of how oxidation of MHC class I and II molecules is regulated and coordinated. I hope to determine what proteins are involved and via what intermediates the formation of disulphide bonds proceeds. I will extend my expertise by coming to terms with novel techniques, especially proteomics, ultra-short pulse chase experiments and by establishing novel methods for studying redox proteins. The expertise of Dr. Benham in the field of ER oxidation, combined with the reagents and facilities available in his lab to study redox proteins, will provide an excellent setting to address the questions outlined above. My proposal will couple the established techniques of Dr. Benham with my experience with MHC class II peptide loading and recombinant DNA technology. All the facilities required for the proposed experiments are available at the department and are supported by well-trained technical staff.