114 - Two structures from a single gene: investigating the differential folding of SOD3

Abstract

Extracellular superoxide dismutase (SOD3) is a zinc and copper-containing enzyme that is responsible for the dismutation of the reactive oxygen species superoxide. SOD3 has previously been shown to exist in two distinct folding variants. These variants are characterized by the connectivity of their disulfide bridges and by their enzymatic activity. One folding variant presents enzymatic activity (aSOD3), while the other, iSOD3, is enzymatically inactive. It has previously been established, that the folding of these variants is an intracellular process and not a result of extracellular regulation. However, the mechanisms involved in the folding and maturation processes have yet to be described. Therefore we aim to describe the process by which this differential folding is achieved. In order to monitor the disulfide isomerization, we have developed a mass spectrometry-based assay that will allow us to determine the ratio between iSOD3 and aSOD3 both in the secreted SOD3 pool and in SOD3 present in the intracellular space. In addition, we are currently aiming to establishing an expression assay, which supports the expression of SOD3 engineered with a specific cleavage site that will enable the differentiation between aSOD3 and iSOD3. By the use of these assays, we aim to quantify the ratio of SOD3 folding variants in cells expressing different SOD3 species as well as in cells exposed to, e.g., oxidative stress conditions.