Functional characterization of PCCA mutations causing propionic acidemia

Clavero, Sonia; Martínez, María Ángeles; Pérez, Belén; Pérez‐Cerdá, Celia; Ugarte, Magdalena; Desviat, Lourdes R.

doi:10.1016/s0925-4439(02)00155-2

Cited by 37 publications

(40 citation statements)

References 31 publications

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“…In the initial analysis of the amplified cDNA visualized by ethidium bromide staining, no normal transcript could be detected, although the splicing score of the 5′-splice-donor site of exon 21 was reduced to only 75% according to Shapiro and Senapathy (1987), a value also found in natural splicing sites. Exon 21 skipping corresponds to an in-frame deletion of 54 bp (18 amino acids), which we later demonstrated to be associated with a total absence of enzyme activity and an accelerated turnover of the mutant protein in mitochondria (Clavero et al 2002). These results contrast sharply with the mild phenotypic expression of the disease (late onset and nearnormal development) in the patient.…”

Section: Resultsmentioning

confidence: 74%

“…In propionic acidemia several reports have explored the genotype-phenotype correlations based on the findings in a collection of patients and on the expression analysis of mutant alleles (Chloupkova et al 2002;Clavero et al 2002;Pérez-Cerdá et al 2003;Pérez-Cerdá et al 2000). The results consistently point to certain missense mutations retaining partial activity and which are associated with a less severe phenotype, while other missense mutations with no activity, along with nonsense and splicing mutations, out of frame insertions and deletions predictably resulting in truncated proteins, are associated with the most severe phenotypes.…”

Section: Discussionmentioning

confidence: 99%

“…The genotype-phenotype correlations have been broadly explored (Pérez-Cerdá et al 2000) and the functional analysis of missense alleles performed in deficient fibroblasts (Clavero et al 2002;Pérez-Cerdá et al 2003). Functionally null alleles, which include, apart from the ones tested in expression assays, nonsense and splicing mutations causing frameshift, are almost always associated with the most severe forms of the disease.…”

Section: Introductionmentioning

confidence: 99%

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Qualitative and quantitative analysis of the effect of splicing mutations in propionic acidemia underlying non-severe phenotypes

Clavero

Pérez²,

Rincón

et al. 2004

Hum Genet

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In this work we analyze splicing mutations identified in propionic acidemia patients to clarify their functional effects and their involvement in the disease phenotype. Two mutations in the PCCA gene detected in homozygous patients and involving consensus splice sequences (IVS21+3del4 and IVS22-2A>G) were shown to produce some normal splicing in patients' cells, at very low levels, which were quantitated by real-time PCR methods, and which presumably are sufficient to moderate the phenotype. We have also analysed the effect of mutations c.653A>G and IVS10-11del6 in the PCCB gene present in heterozygous patients with mild phenotype. The c.653A>G mutation is located in the last codon of exon 6 and interferes with the correct spliceosomal assembly activating a cryptic splice site within exon 6, which leads to an in-frame six-nucleotide deletion (delV217-K218). Minigene analysis and sequence-specific hybridization probes using real-time PCR methods showed that no normally spliced transcript is detectable in the patients' fibroblasts. The IVS10-11del6 mutation shortens the polypyrimidine tract of the 3'-splice site of exon 11, resulting in exon skipping. Some normal transcript is detectable by allele-specific hybridization probes. These analyses suggest that, in some cases, the regulation of gene splicing can potentially play an important role in human disease influencing phenotypic parameters.

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Section: Resultsmentioning

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Qualitative and quantitative analysis of the effect of splicing mutations in propionic acidemia underlying non-severe phenotypes

Clavero

Pérez²,

Rincón

et al. 2004

Hum Genet

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“…A large number of PCCB mutations have been studied in the bacterial expression system (14 -16), and in a SV40-transformed fibroblast expression system (17,18). In addition, the effect of some PCCB mutations on the ␣-␤ heteromeric and ␤-␤ homomeric subunit assembly have been studied by a mammalian twohybrid expression system (19,20).…”

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confidence: 99%

Characterization of Four Variant Forms of Human Propionyl-CoA Carboxylase Expressed in Escherichia coli

Jiang

Rao

Yee

et al. 2005

Journal of Biological Chemistry

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Propionyl-CoA carboxylase (PCC) is a biotin-dependent mitochondrial enzyme that catalyzes the conversion of propionyl-CoA to D-methylmalonyl-CoA. PCC consists of two heterologous subunits, ␣ PCC and ␤ PCC, which are encoded by the nuclear PCCA and PCCB genes, respectively. Deficiency of PCC results in a metabolic disorder, propionic acidemia, which is sufficiently severe to cause neonatal death. We have purified three PCCs containing pathogenic mutations in the ␤ subunit (R165W, E168K, and R410W) and one PCCB polymorphism (A497V) to homogeneity to elucidate the potential structural and functional effects of these substitutions. We observed no significant difference in K m values for propionyl-CoA between wild-type and the variant enzymes, which indicated that these substitutions had no effect on the affinity of the enzyme for this substrate. Furthermore, the kinetic studies indicated that mutation R410W was not involved in propionyl-CoA binding in contrast to a previous report. The three mutant PCCs had half the catalytic efficiency of wild-type PCC as judged by the k cat /K m ratios. No significant differences have been observed in molecular mass or secondary structure among these enzymes. However, the variant PCCs were less thermostable than the wild-type. Following incubation at 47°C, blue native-PAGE revealed a lower oligomeric form (␣ 2 ␤ 2 ) in the three mutants not detectable in wildtype and the polymorphism. Interestingly, the lower oligomeric form was also observed in the corresponding crude Escherichia coli extracts. Our biochemical data and the structural analysis using a ␤ PCC homology model indicate that the pathogenic nature of these mutations is more likely to be due to a lack of assembly rather than disruption of catalysis. The strong favorable effect of the co-expressed chaperone proteins on PCC folding, assembly, and activity suggest that propionic acidemia may be amenable to chaperone therapy.Four different biotin-dependent carboxylases are known to play a central role in mammalian metabolic pathways, such as oxidation of odd-chain fatty acids, catabolism of branched amino acids, fatty acid synthesis, and gluconeogenesis. One of these is propionyl-CoA carboxylase (PCC, 1 EC 6.4.1.3), which catalyzes the conversion of propionyl-CoA to D-methylmalonylCoA in the mitochondrial matrix (1). This enzyme consists of two nonidentical subunits, ␣ and ␤, encoded by two different nuclear genes, designated PCCA and PCCB, respectively. Structural studies of the human enzyme have indicated that ␣ PCC is 72 kDa in size, whereas ␤ PCC is 54 kDa. Overall, PCC has an ␣ 6 ␤ 6 structure (1, 2). Mutations in either gene result in an autosomal recessive disease, propionic acidemia (MIM 606054), which usually presents as a life-threatening ketoacidosis in the neonatal period with protein intolerance, vomiting, failure to thrive, lethargy, and profound metabolic acidosis symptoms. This disease can result in mental retardation and can be sufficiently severe to cause neonatal death (1).The cDNAs for ␣ and ␤ subunits hav...

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“…Several disease-causing missense mutations have been identified in human propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase (5-7), revealing that disease phenotypes can arise from mutations in either the ␣ or ␤ subunit. To date, characterization of the mutant proteins has included specific activity measurements of fibroblast extracts (8,9) or of purified recombinant PCC␤ subunit (10). A useful model system for detailed analysis of mutations in the ␤ subunit is provided by trans-carboxylase found in Propionibacterium shermanii.…”

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Kinetic Characterization of Mutations Found in Propionic Acidemia and Methylcrotonylglycinuria

Sloane

Waldrop

2004

Journal of Biological Chemistry

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Acetyl-CoA carboxylase catalyzes the committed step in fatty acid synthesis in all plants, animals, and bacteria. The Escherichia coli form is a multifunctional enzyme consisting of three separate proteins: biotin carboxylase, carboxyltransferase, and the biotin carboxyl carrier protein. The biotin carboxylase component, which catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the carboxylate source, has a homologous functionally identical subunit in the mammalian biotin-dependent enzymes propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase. In humans, mutations in either of these enzymes result in the metabolic deficiency propionic acidemia or methylcrotonylglycinuria. The lack of a system for structurefunction studies of these two biotin-dependent carboxylases has prevented a detailed analysis of the diseasecausing mutations. However, structural data are available for E. coli biotin carboxylase as is a system for its overexpression and purification. Thus, we have constructed three site-directed mutants of biotin carboxylase that are homologous to three missense mutations found in propionic acidemia or methylcrotonylglycinuria patients. The mutants M169K, R338Q, and R338S of E. coli biotin carboxylase were selected for study to mimic the disease-causing mutations M204K and R374Q of propionyl-CoA carboxylase and R385S of 3-methylcrotonyl-CoA carboxylase. These three mutants were subjected to a rigorous kinetic analysis to determine the function of the residues in the catalytic mechanism of biotin carboxylase as well as to establish a molecular basis for the two diseases. The results of the kinetic studies have revealed the first evidence for negative cooperativity with respect to bicarbonate and suggest that Arg-338 serves to orient the carboxyphosphate intermediate for optimal carboxylation of biotin.For over 20 years, site-directed mutagenesis has been used for determining the roles of active site residues in enzymecatalyzed reactions. In designing such experiments, one is invariably faced with the question of which residue to mutate and to what to mutate this residue. Residues targeted for mutagenesis are usually identified using three-dimensional structural information or sequence alignments with homologous proteins, indicating which residues are highly conserved. Once the residues for mutagenesis are identified, they are commonly replaced with an isosteric residue or alanine (i.e. alanine-scanning mutagenesis) to minimize disruption of the protein structure. However, prior to the advent of site-directed mutagenesis, mutant human hemoglobins provided the only opportunity to study structure-function relationships in proteins.

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Functional characterization of PCCA mutations causing propionic acidemia

Cited by 37 publications

References 31 publications

Qualitative and quantitative analysis of the effect of splicing mutations in propionic acidemia underlying non-severe phenotypes

Qualitative and quantitative analysis of the effect of splicing mutations in propionic acidemia underlying non-severe phenotypes

Characterization of Four Variant Forms of Human Propionyl-CoA Carboxylase Expressed in Escherichia coli

Kinetic Characterization of Mutations Found in Propionic Acidemia and Methylcrotonylglycinuria

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