Congenital Adrenal Hyperplasia (CAH) ranks amongst the most common inherited metabolic diseases. It comprises a group of autosomal recessive disorders caused by mutations in genes encoding enzymes involved in steroid synthesis. Two key enzymes are the cytochrome P450 (CYP) type II enzymes 21-hydroxylase (CYP21A2) and 17α-hydroxylase (CYP17A1), both requiring electron transfer from the electron donor enzyme P450 oxidoreductase (POR). Functional analysis of CAH-causing mutations currently consists only of in vitro enzyme activity assays that do not address other and potentially very important molecular aspects such as protein folding and enzyme-cofactor dimerisation. Incorrect protein folding may lead to endoplasmic reticulum (ER) stress, which plays an important role in the pathogenesis of various diseases, e.g. diabetes mellitus. The first aim of the proposed project is to systematically analyse mutations in CYP21A2, CYP17A1 and POR by not only studying residual in vitro activity, but also by dissecting the mutations’ effects on protein stability and protein degradation. Subsequent studies will establish the potentially therapeutic role of small molecules (pharmacological chaperones, proteostasis regulators) in refolding and restoration of enzyme function of CYP21A2, CYP17A1 and POR enzymes. Secondly, this project will characterise the ER localisation and interactions of the CYP21A2, CYP17A1 and POR proteins, including dimer-formation. These data will provide essential insights into functional and structural in vivo topology of steroidogenic enzymes. This will lead to a better understanding of molecular mechanisms involved in steroidogenesis. In conclusion, this highly innovative project will apply novel strategies to study and to potentially restore steroidogenic enzyme function. The proposed studies will hopefully pave for a novel molecular tailored therapy and in addition they provide an ideal advanced training platform.