ECE2016 Guided Posters Adrenal (1) (10 abstracts)
Reduced temperature and the chemical chaperone 4-phenylbutyrate enhance stability of CYP21A2 mutations
Yiqing Chen 1 , Kerstin Schaefer 1 , Maria Gasteiger 1 , Angela Taylor 2 , Wiebke Arlt 2 , Nils Krone 3 , Soeren W. Gersting 4 & Nicole Reisch 1
1Medizinische Klinik und Poliklinik IV; Klinikum der Universität München, Munich, Bavaria, Germany; 2Institute of Metabolism and Systems Research; University of Birmingham, Birmingham, West Midlands, UK; 3Academic Unit of Child Health, Department of Oncology and Metabolism; University of Sheffield, Sheffield, Yorkshire and the Humber, UK; 4Melokulare Pädiatrie Dr. von Haunersches Kinderspital; Kinderspital der Ludwig-Maximilians-Universität München, Munich, Bavaria, Germany.
Background: Mutations of 21-hydroxylase (CYP21A2) cause congenital adrenal hyperplasia. Its severe (classic) form constitutes a life-threatening disease. Patients suffer a significant disease burden due to co-morbidities that are often treatment-related. The current therapeutic situation is unsatisfying and demands novel treatment approaches. In silico modelling suggests protein misfolding and intracellular retention to play a significant role in the pathogenesis of CAH.
Objective: To investigate protein misfolding and intracellular retention due to CYP21A2 mutations and explore pharmacological chaperones as potential therapeutic tools.
Methods: Clinically relevant CYP21A2 mutations where in silico modelling suggested protein misfolding to play a role in the pathogenesis of CAH were selected and subcloned into pcDNA6-V5/His expressing vectors. Residual activity of variant CYP21A2 proteins was determined in living COS-7 cells using an enzyme activity assay with LC/MSMS based analysis of steroids. The effect of mutations on protein half-life (susceptibility to proteinase K) was measured comparing wild-type and variant CYP21A2. The influence of 4-PBA and temperature on protein half-life was investigated.
Results: Our preliminary results show that with decreased temperature, susceptibility of selected mutations to proteolysis was significantly attenuated (protein half-life increased from 0.9 min±0.4 to 5.9 min±0.8 for G90V; from 1.4 min±0.1 to 5.5 min±0.9 for P30L; from 1.0 min±0.1 to 8.0 min±1.3 for P30Q; from 0.6 min±0.1 to 9.8 min±1.0 for R356W). The use of the chemical chaperone 4-PBA enhanced CYP21A2 half-life (3.8 min±0.5 to 11.0 min±1.6 for G375S). The same applied when the temperature was reduced to 30°C resulting in partial rescue of the CYP21A2 loss-of-function phenotype.
Conclusions: Our preliminary data substantiate the hypothesis of protein misfolding with loss-of-function as a relevant molecular mechanism in CAH that can be addressed by structural stabilisation of CYP21A2.
Article tools
My recent searches
My recently viewed abstracts
Authors
Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
© Bioscientifica 2024 |
Privacy policy |
Cookie settings
BiosciAbstracts
Bioscientifica Abstracts is the gateway to a series of products that provide a permanent, citable record of abstracts for biomedical and life science conferences.
Find out more