Effect of PCCB gene mutations on the heteromeric and homomeric assembly of propionyl-CoA carboxylase.

Propionic acidemia is an inherited metabolic disorder caused by deficiency of propionyl-CoA carboxylase, a dodecameric enzyme composed of alpha-PCC and beta-PCC subunits (encoded by genes PCCA and PCCB) that have been associated with a number of mutations responsible for this disease. To clarify the molecular effect associated with gene alterations causing propionic acidemia, 12 different mutations affecting the PCCB gene (R67S, S106R, G131R, R165W, R165Q, E168K, G198D, A497V, R512C, L519P, W531X, and N536D) were analyzed for their involvement in alpha-beta heteromeric and beta-beta homomeric assembly. The experiments were performed using the mammalian two-hybrid system, which was assayed at two different temperatures to distinguish between mutations directly involved in interaction and those probably affecting polypeptide folding, thus indirectly affecting the correct assembly. Mutations R512C, L519P, W531X, and N536D, located at the carboxyl-terminal end of the PCCB gene, were found to inhibit alpha-beta heteromeric and/or the beta-beta homomeric interaction independently of the cultivation temperature, reflecting their primary effect on the assembly. Two mutations A497V and R165Q did not affect either heteromeric or homomeric assembly. The remaining mutations (R67S, S106R, G131D, R165W, E168K, and G198D), located in the amino-terminal region of the beta-polypeptide, resulted in normal interaction levels only when expressed at the lower temperature, suggesting that these changes could be considered as folding defects. From these results and the clinical manifestations associated with patients bearing the mutations described above, several genotype-phenotype correlations may be established. In general, the temperature-sensitive mutations are associated with a less severe, although variable phenotype. This could correlate with the recent hypothesis that the effect of folding mutations can be influenced by the capacity of the cellular protein quality control machinery, which provides clues to our understanding of the variability of the clinical symptoms observed among the patients bearing these mutations.