University of Oulu
Supervisor: Lloyd Ruddock
Formation of disulphide bonds within the mammalian endoplasmic reticulum (ER) is a complex and highly regulated process that is essential for roughly one-third of all human proteins. It can take place via multiple pathways but the primary route is thought to occur via the action of the disulphide-generating oxidases of the Ero1 family on protein disulphide isomerase (PDI). In human two Ero1 isoforms, Ero1α and Ero1β, exist. Ero1 oxidizes PDI and the disulfide bond formed between the active site cysteines is transferred directly to folding proteins. Reduced Ero1 becomes reoxidized by molecular oxygen via its flavin cofactor in a reaction which results in the formation of one molecule of hydrogen peroxide per disulfide bond made. Formation of hydrogen peroxide and the fact that the correct redox environment has to be maintained in the ER for native disulfide bond formation to occur, implies that the action of Ero1 must be highly regulated by a mechanism which senses the redox state of the ER and the production levels of disulfide bonded proteins being formed in the ER. Intricate feedback mechanisms have evolved to regulate Ero1 activity and central to these mechanisms are non-catalytic cysteines which form regulatory disulphides and influence catalytic activity of Ero1 in relation to local redox conditions. Some details of the regulatory disulphides of Ero1α are known, but still there is much that requires to be explicated on the mechanism of action of these essential enzymes and especially on their regulation.
This project will undertake detailed mechanistic studies on the human Ero1α and Ero1β and will include rapid-reaction kinetics in order to examine individual steps of the mechanism of action, analysis of the regulatory mechanisms in vitro and in vivo and parallel structural studies.