Endoplasmic reticulum H₂O₂ : Ero1-driven generation and GPx-mediated detoxification

Endoplasmic reticulum (ER) oxidoreductin 1 alpha (Ero1alpha) is an ER-resident oxidase, which utilizes molecular oxygen (O2) as terminal electron acceptor to produce disulfide bonds and hydrogen peroxide (H2O2). The major target for Ero1alpha-derived disulfides is protein disulfide isomerase (PDI), which transfers them onto substrate proteins and plays an additional role as homeostatic regulator of Ero1alpha.
In this thesis, I demonstrated that PDI-mediated activation of Ero1alpha extends beyond the reduction of the known inhibitory disulfides Cys94-Cys131 and Cys99-Cys104 and involves an additional disulfide, Cys208-Cys241. Opening of this disulfide by PDI apparently enables diffusion of O2 towards and of H2O2 away from the catalytic flavin cofactor in Ero1alpha. Expression of a constitutively active Ero1alpha mutant, which is devoid of all three regulatory disulfides, compromises cell viability. Hence, redox regulation of the O2/H2O2 diffusion pathway in Ero1alpha emerges as critical determinant of ER homeostasis, in which PDI takes center stage by directly regulating O2 consumption.
I also elucidated the molecular basis for the specificity of glutathione peroxidase 8 (GPx8) to detoxify Ero1alpha-derived H2O2, as this enzyme binds to the site of H2O2 release in Ero1alpha. Only depletion of GPx8 but not of the abundant ER peroxidase peroxiredoxin IV (PrxIV) exhibited an additive effect with deregulated Ero1alpha on ER hyperoxidation and induction of unfolded protein response and antioxidant response target genes. Furthermore, only upon GPx8 knockdown I was able to detect leakage of Ero1alpha-derived H2O2 from ER to cytosol. Therefore, GPx8 acts as a specific molecular gatekeeper to protect the cytosol from Ero1alpha-derived H2O2. The exclusion of PrxIV from this process revealed a previously unappreciated compartmentalization of electron transport pathways in the ER.
Moreover, I successfully isolated mixed-disulfide interaction partners of the ER-resident peroxidase GPx7 and of PDI. The interactome of the latter was analyzed and found to be mainly comprised of other members of the PDI family, which, in conjunction with its function as Ero1alpha activator, places PDI as central regulator of ER disulfide homeostasis. With regard to GPx7 interaction partners I am confident that their identification will serve as basis for future elucidation of novel cellular functions of this peroxidase.