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Functional analysis of PCCB mutations causing propionic acidemia based on expression studies in deficient human skin
C Pérez-Cerdá1, S Clavero, B Pérez
1Dpto. de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Cantoblanco, 28049, Madrid, Spain.
Insights
Propionic acidemia (PA) is a genetic disorder affecting propionyl-CoA carboxylase (PCC). This study analyzed 18 PCCB gene mutations, identifying some with normal function and others causing severe enzyme deficiency or instability, aiding disease understanding.
Area of Science:
- Biochemistry
- Genetics
- Metabolic Disorders
Background:
- Propionic acidemia (PA) is a rare autosomal recessive metabolic disorder.
- PA results from deficient propionyl-CoA carboxylase (PCC) activity, an enzyme crucial for amino acid metabolism.
- PCC is a dodecameric enzyme comprising alpha-PCC and beta-PCC subunits, encoded by PCCA and PCCB genes, respectively.
Purpose of the Study:
- To investigate the functional consequences of 18 different mutations in the PCCB gene.
- To determine the impact of these mutations on PCC enzyme activity and protein stability.
- To enhance the understanding of genotype-phenotype correlations in propionic acidemia.
Main Methods:
- Expression studies of 18 PCCB sequence variants in a PCCB-deficient fibroblast cell line.
- Assay of propionyl-CoA carboxylase (PCC) activity for wild-type and mutant proteins.
- Western blot analysis to assess protein stability of mutant PCCB proteins.
Main Results:
- Two PCCB mutations (L17M, A497V) showed wild-type activity, indicating no functional impact.
- Three mutations (K218R, R410W, N536D) retained substantial PCC activity.
- Thirteen mutations resulted in null or very low PCC activity, with L519P, R512C, and G112D demonstrating protein instability.
Conclusions:
- The study functionally characterized 18 PCCB mutations, revealing diverse effects on PCC activity and stability.
- Pathogenicity of R67S, R165Q, and G112D mutations was confirmed.
- These findings contribute to a better understanding of propionic acidemia's genetic basis and clinical variability.
Abstract:
Propionic acidemia (PA) is a recessive disorder caused by a deficiency of propionyl-CoA carboxylase (PCC), a dodecameric enzyme composed of two different proteins alpha-PCC and beta-PCC, nuclear encoded by the PCCA and PCCB genes, respectively. Mutations in either gene cause PA and to date, up to 47 different allelic variations in the PCCB gene have been identified in different populations. In this work, we describe the expression studies of 18 PCCB sequence changes in order to elucidate their functional consequences. We have used a PCCB-deficient transformed fibroblast cell line to target the wild-type and mutant proteins to their physiological situation, analysing the effect of the mutations on PCC activity and protein stability. Of the 18 mutant proteins tested for activity, those carrying the L17M and A497V substitutions showed an activity similar to the wild-type one, which proves that these changes do not have any effect on protein activity. The other 16 mutant proteins exhibited two different functional behaviours, 3 retained substantial activity (K218R, R410W and N536D), and the remaining 13 proteins showed null or very low activity. Western blot analysis demonstrated instability only for the L519P, R512C and G112D mutant proteins. We have proved the pathogenicity of R67S, R165Q and G112D mutation in PCCB gene, expressed for the first time in this work. The information derived from the expression analysis is discussed in the phenotype and genotype context in order to improve the knowledge of this complex disease.