Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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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.
Mol Genet Metab 2001 Dec
PMID:Effect of PCCB gene mutations on the heteromeric and homomeric assembly of propionyl-CoA carboxylase. 1174 52

Propionic acidemia can result from mutations in the PCCA or PCCB genes encoding the alpha and beta subunits, respectively, of propionyl-CoA carboxylase. We have developed a method based on complementation of the enzyme defect using a lipid-mediated transient transfection of the normal human PCCA or PCCB cDNA into primary fibroblasts. We demonstrate the reliability of this method for identification of the defective PCC gene in order to unequivocally approach the mutational analysis in the corresponding PCCA and PCCB genes.
Mol Genet Metab 2002 Mar
PMID:Transfection screening for defects in the PCCA and PCCB genes encoding propionyl-CoA carboxylase subunits. 1191 40

Propionic acidemia (PA) is an autosomal recessive inborn error in the catabolism of methionine, isoleucine, threonine, and valine, odd-numbered chain length fatty acids and cholesterol. Clinical symptoms are very heterogeneous and present as a severe neonatal-onset or a late-onset form. It is caused by a deficiency of propionyl-CoA carboxylase (PCC, EC 6.4.1.3), a biotin-dependent enzyme that catalyzes the carboxylation of propionyl-CoA to D-methylmalonyl-CoA. PCC is a heteropolymeric enzyme composed of alpha- and beta-subunits. A greater heterogeneity is observed in the PCCA gene, while for the PCCB gene, a limited number of mutations is responsible for the majority of the alleles characterized in both Caucasian and Oriental populations. We identified eight Korean patients with PA by organic acid analysis confirmed in five patients by the PCC enzyme assay in the lymphoblasts. Two neonatal-onset patients showed undetectable PCC activities while three cases with residual enzyme activities had relatively late manifestations. In the molecular analysis, we identified five novel mutations, Y439C, 1527del3, 1357insT, IVS12-8T-->A, and 31del10, and one known mutation, T428I in PCCB gene. Alleleic frequency of T428I in Korean patients with PA was 56.3% in this study. Two neonatal-onset patients with null enzyme activities were homozygotes with 1527del3 and T428I, respectively. This finding implies that T428I and 1527del3 mutation could be responsible for their severe clinical courses and null enzyme activities. The mRNA of PCCB gene in T428I and 1527del3 homozygotes were normal but in Western blot analysis, the betaPCC-subunit was only absent in 1527del3 homozygote patient suggesting different molecular pathology.
Mol Genet Metab 2002 Nov
PMID:Molecular analysis of PCCB gene in Korean patients with propionic acidemia. 1240 68

Propionic acidemia is an inherited metabolic disease caused by the deficiency of the mitochondrial protein propionyl-CoA carboxylase (PCC), one of the four biotin-dependent enzymes. PCC is a multimeric protein composed of two different alpha- and beta-PCC subunits, nuclearly encoded by the PCCA and PCCB genes, respectively. Mutations in either gene cause the clinically heterogeneous disease propionic acidemia. In this work we describe the mutational analysis of PCCA and PCCB deficient patients from different European countries (Spain, Italy, Belgium, Croatia, and Austria) and from America (mainly USA). We report 24 novel PA mutations, nine affecting the PCCA gene and 15 affecting the PCCB gene. They include six missense mutations, one nonsense mutation, one point exonic mutation affecting splicing, seven splicing mutations affecting splice sequences, and nine short insertions or deletions, only two in-frame. We have found a highly heterogenous spectrum of PCCA mutations, most of the PCCA deficient patients are homozygous carrying a unique genotype. The PCCA mutational spectrum includes a high proportion of short insertions or deletions affecting one nucleotide. In the PCCA mutant alleles analyzed we have also found one single nucleotide change, a novel nonsynonymous SNP. On the other hand, the PCCB deficient patients carry a more reduced spectrum of mutations, 50% of them are missense. This work represents an extensive update of the mutational study of propionic acidemia providing important information about the worldwide distribution of PA mutations and representing another essential part in the study of the phenotype-genotype correlations for the prediction of the metabolic outcome and for the implementation of treatments tailored to each PA patient.
Mol Genet Metab 2003 Jan
PMID:Propionic acidemia: identification of twenty-four novel mutations in Europe and North America. 1255 49

Propionic acidemia (PA) is an inborn error of organic acid metabolism caused by a deficiency of propionyl-CoA carboxylase. This enzyme is composed of two non-identical subunits, alpha and beta, which are encoded by the PCCA and PCCB genes, respectively. An enzyme deficiency can result from mutations in either PCCA or PCCB. To elucidate the mutation spectrum in Japanese patients, we have performed a mutation analysis of 30 patients with PA, which included nine previously reported patients. The study revealed that 15 patients were alpha-subunit deficient and 15 patients were beta-subunit deficient. Seven novel mutations were found (IVS18-6C >G, 1746G >A, C398R, G197E and IVS18+1G >A in the PCCA; A153P and IVS9+1G >T in the PCCB). Among these Japanese patients with alpha-subunit deficiencies, 923-924insT, IVS18-6C >G, and R399Q mutations were frequent and the total allelic frequency of these three mutations combined was 56% (17/30). This is in sharp contrast to the mutation spectrum found in Caucasian patients, where no prevalent mutations have been identified. Among the beta-subunit deficiencies, there were three frequent mutations; R410W, T428I, and A153P, whose allelic frequencies were 30, 26.7, and 13.3%, respectively. In conclusion, a limited number of mutations are predominant in both PCCA and PCCB genes among Japanese patients with propionic acidemia.
Mol Genet Metab 2004 Apr
PMID:Mutation spectrum of the PCCA and PCCB genes in Japanese patients with propionic acidemia. 1505 21

Mutations in the PCCA or PCCB genes, encoding both subunits of propionyl-CoA carboxylase, result in propionic acidemia, a life-threatening inborn error of metabolism with autosomal recessive inheritance. To date, 41 mutations in the PCCA gene and 54 in the PCCB gene have been reported, most of them single base substitutions causing amino acid replacements, and a variety of small insertions and deletions and splicing defects. A greater heterogeneity is observed in the PCCA gene, specially in Caucasians, with no prevalent mutations, while in the Japanese population three mutations account for more than half of the alleles studied. For the PCCB gene a limited number of mutations is responsible for the majority of the alleles characterized in both Caucasian and Oriental populations. These two populations show a different mutational spectrum, only sharing some involving CpG dinucleotides probably as recurrent mutational events. Functional characterization of the mutant missense alleles has been accomplished using different prokaryotic and eukaryotic systems, and the structural consequences have been analyzed in the available crystal models. For the PCCA gene, the main molecular effect of the expressed mutations is related to protein instability, except two mutations in the active site predictably affecting ATP binding. In the PCCB gene the majority of the analyzed mutations are predicted to alter the active site conformation resulting in diminished activity. A few carboxy-terminal PCCB mutations affect the interaction between subunits and the assembly with PCCA to form a functional PCC oligomer. The amount of normal transcripts resulting from some PCCA and PCCB splicing mutations has also been analyzed. Overall, the data generated from the expression analysis reveal potential genotype-phenotype correlations for this clinically heterogeneous disorder.
Mol Genet Metab
PMID:Propionic acidemia: mutation update and functional and structural effects of the variant alleles. 1546 17

Methylmalonyl-CoA epimerase (MCE) catalyzes the interconversion of D- and L-methylmalonyl-CoA in the pathway responsible for the degradation of branched chain amino acids, odd chain-length fatty acids, and other metabolites. Despite the occurrence of metabolic disorders in the enzymatic step occurring immediately upstream of MCE (propionyl-CoA carboxylase) and downstream of MCE (adenosylcobalamin-dependent methylmalonyl-CoA mutase), no disease-causing mutations have been described affecting MCE itself. A patient, formerly identified as belonging to the cblA complementation group of vitamin B12 disorders but lacking mutations in the affected gene, MMAA, was tested for mutations in the MCEE gene. The patient's fibroblasts had normal levels of adenosylcobalamin compared to controls, whereas other cblA cell lines typically had reduced levels of the cofactor. As well, this patient had a milder form of methylmalonic aciduria than usually observed in cblA patients. The patient was found to be homozygous for a c.139C>T (p.R47X) mutation in MCEE by sequence analysis that was confirmed by restriction digestion of PCR products. One sibling, also with mild methylmalonic aciduria, was homozygous for the mutation. Both parents and one other sibling were heterozygous. A nearby insertion polymorphism, c.41-160_161insT, heterozygous in both parents, showed the wild-type configuration on the mutant alleles. To assess the impact of isolated MCE deficiency in cultured cells, HeLa cells were transfected with a selectable vector containing MCEE-specific small interfering RNA (siRNA) to suppress gene expression. The reduced level of MCEE mRNA resulted in the reduction of [14C]-propionate incorporation into cellular macromolecules. However, siRNA only led to a small reduction in pathway activity, suggesting that previously postulated non-enzymatic conversion of D- to L-methylmalonyl-CoA may contribute to some flux through the pathway. We conclude that the patient's MCEE defect was responsible for the mild methylmalonic aciduria, confirming a partial requirement for the enzymatic activity in humans.
Mol Genet Metab 2006 Aug
PMID:Homozygous nonsense mutation in the MCEE gene and siRNA suppression of methylmalonyl-CoA epimerase expression: a novel cause of mild methylmalonic aciduria. 1669 27

We have utilized Caenorhabditis elegans to study human methylmalonic acidemia. Using bioinformatics, a full complement of mammalian homologues for the conversion of propionyl-CoA to succinyl-CoA in the genome of C. elegans, including propionyl-CoA carboxylase subunits A and B (pcca-1, pccb-1), methylmalonic acidemia cobalamin A complementation group (mmaa-1), co(I)balamin adenosyltransferase (mmab-1), MMACHC (cblc-1), methylmalonyl-CoA epimerase (mce-1) and methylmalonyl-CoA mutase (mmcm-1) were identified. To verify predictions that the entire intracellular adenosylcobalamin metabolic pathway existed and was functional, the kinetic properties of the C. elegans mmcm-1 were examined. RNA interference against mmcm-1, mmab-1, mmaa-1 in the presence of propionic acid revealed a chemical phenotype of increased methylmalonic acid; deletion mutants of mmcm-1, mmab-1 and mce-1 displayed reduced 1-[(14)C]-propionate incorporation into macromolecules. The mutants produced increased amounts of methylmalonic acid in the culture medium, proving that a functional block in the pathway caused metabolite accumulation. Lentiviral delivery of the C. elegans mmcm-1 into fibroblasts derived from a patient with mut(o) class methylmalonic acidemia could partially restore propionate flux. The C. elegans mce-1 deletion mutant demonstrates for the first time that a lesion at the epimerase step of methylmalonyl-CoA metabolism can functionally impair flux through the methylmalonyl-CoA mutase pathway and suggests that malfunction of MCEE may cause methylmalonic acidemia in humans. The C. elegans system we describe represents the first lower metazoan model organism of mammalian propionate spectrum disorders and demonstrates that mass spectrometry can be employed to study a small molecule chemical phenotype in C. elegans RNAi and deletion mutants.
Mol Genet Metab
PMID:Propionyl-CoA and adenosylcobalamin metabolism in Caenorhabditis elegans: evidence for a role of methylmalonyl-CoA epimerase in intermediary metabolism. 1684 92

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that plays a crucial role in gluconeogenesis, lipogenesis, Krebs cycle anaplerosis and amino acid catabolism. Biotin deficiency reduces its mass besides its activity. Enzyme mass is the result of its cellular turnover, i.e., its rates of synthesis and degradation. We have now investigated, by a pulse and chase approach in cultured primary hepatocytes, the effects of biotin deficiency on these rates. Wistar rats were fed a biotin-deficient diet and the controls were fed the same diet supplemented with biotin; their biotin status was monitored measuring lymphocytes propionyl-CoA carboxylase activity and urinary 3-hydroxyisovaleric acid. After 6-7 weeks primary hepatocytes were cultured in biotin-deficient or complete DMEM. PC activity was determined by measuring the incorporation of (14)C-bicarbonate into acid-non-volatile products, and its mass by streptavidin Western blots. Its synthesis rate was estimated from [(35)S] methionine incorporation into anti-PC antibody immunoprecipitate. Its degradation rate was calculated from the loss of radioactivity from previously labeled hepatocytes, in a medium containing an excess of non-radioactive methionine. PC synthesis rate in biotin-deficient hepatocytes was approximately 4.5-fold lower than in the controls, and its degradation rate was 5.1-fold higher. Therefore, the decrement of PC mass during biotin deficiency results both from a decrease in its synthesis and an increase in its degradation rates. To our knowledge, this is the first instance where a mammalian enzyme cofactor is necessary to sustain both processes.
Mol Genet Metab 2007 Nov
PMID:Biotin deficiency affects both synthesis and degradation of pyruvate carboxylase in rat primary hepatocyte cultures. 1772 May 79

Holocarboxylase synthetase (HCS) catalyzes the binding of the vitamin biotin to carboxylases and histones. Carboxylases mediate essential steps in macronutrient metabolism. For example, propionyl-CoA carboxylase (PCC) catalyzes the carboxylation of propionyl-CoA in the metabolism of odd-chain fatty acids. HCS comprises four putative domains, i.e., the N-terminus, the biotin transfer/ATP-binding domain, a putative linker domain, and the C-terminus. Both N- and C-termini are essential for biotinylation of carboxylases by HCS, but the exact functions of these two domains in enzyme catalysis are unknown. Here we tested the hypothesis that N- and C-termini play roles in substrate recognition by HCS. Yeast-two-hybrid (Y2H) assays were used to study interactions between the four domains of human HCS with p67, a PCC-based polypeptide and HCS substrate. Both N- and C-termini interacted with p67 in Y2H assays, whereas the biotin transfer/ATP-binding and the linker domains did not interact with p67. The essentiality of N- and C-termini for interactions with carboxylases was confirmed in rescue experiments with mutant Saccharomyces cerevisiae, using constructs of truncated human HCS. Finally, a computational biology approach was used to model the 3D structure of human HCS and identify amino acid residues that interact with p67. In silico predictions were consistent with observations from Y2H assays and yeast rescue experiments, and suggested docking of p67 near Arg508 and Ser515 within the central domain of HCS.
Mol Genet Metab 2009 Apr
PMID:N- and C-terminal domains in human holocarboxylase synthetase participate in substrate recognition. 1915 41


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