Propionyl-coenzyme A Carboxylase Research Articles

The mitochondrial carboxylase PCCA interacts with Listeria monocytogenes phospholipase PlcB to modulate bacterial survival.

Mitochondria represent attractive targets for pathogenic bacteria seeking to modulate host cellular processes to promote their survival and replication. Our current study has uncovered mitochondrial carboxylase propionyl-coenzyme A carboxylase (PCCA) as a novel host cell protein that interacts with L. monocytogenes PlcB. The results demonstrate that PCCA plays a negative regulatory role in L. monocytogenes infection, as heightened PCCA levels are associated with reduced bacterial survival and persistence. However, L. monocytogenes may exploit the PlcB-PCCA interaction to maintain stable PCCA expression and establish a favorable intracellular milieu for bacterial infection. Our findings shed new light on the intricate interplay between bacterial pathogens and host cell mitochondria, while also highlighting the potential of mitochondrial metabolic enzymes as antimicrobial agents.

Multidirectional pathway engineering of Mycobacterium neoaurum LY-2 for the enhancement of androstenedione production from phytosterols

Androstenedione (ADD), an important precursor for steroid hormone drug synthesis, can be obtained through the transformation of phytosterols by members of the Mycobacteria genus. In this study, multidirectional pathway engineering of Mycobacterium neoaurum LY-2 was applied to improve ADD production from phytosterols. Initially, the nonessential gene KasB encoding a cell wall β-ketoacyl acyl carrier protein synthase in Mycobacterium neoaurum LY-2 was knocked out, and the production of ADD was increased by 21.0%. Then, the key enzymes involved in ADD synthetic metabolism were systematically explored. These assays revealed that propionyl coenzyme A carboxylase (PCC) and Hsd4A played important roles in the synthesis of ADD. In addition, the heterologous expression of Vitreoscilla hemoglobin (VHb), a protein with oxygen-binding capacity, was successfully realized in Mycobacterium neoaurum LY-2, and the ADD concentration reached 4.42 g/L. Finally, the recombinant strain LY-2-ΔKasB-hsd4A-pcc-vgb with high ADD production was constructed by combining the above strategies. Through optimization of conversion conditions, the recombinant strain LY-2-ΔKasB-hsd4A-pcc-vgb produced an ADD concentration of 11.12 g/L when adding 20 g/L phytosterols.

Import of TAT-Conjugated Propionyl Coenzyme A Carboxylase Using Models of Propionic Acidemia.

Propionic acidemia is caused by a deficiency of the enzyme propionyl coenzyme A carboxylase (PCC) located in the mitochondrial matrix. Cell-penetrating peptides, including transactivator of transcription (TAT), offer a potential to deliver a cargo into the mitochondrion. Here, we investigated the delivery of an α6β6 PCC enzyme into mitochondria using the HIV TAT peptide at several levels: into isolated mitochondria, in patient fibroblast cells, and in a mouse model. Results from Western blots and enzyme activity assays confirmed the import of TAT-PCC into mitochondria, as well as into patient fibroblasts, where the colocalization of imported TAT-PCC and mitochondria was also confirmed by confocal fluorescence microscopy. Furthermore, a single-dose intraperitoneal injection into PCC-deficient mice decreased the propionylcarnitine/acetylcarnitine (C3/C2) ratio toward the normal level. These results show that a cell-penetrating peptide can deliver active multimeric enzyme into mitochondria in vitro, in situ, and in vivo and push the size limit of intracellular delivery achieved so far. Our results are promising for other mitochondrion-specific deficiencies.

Towards the development of an enzyme replacement therapy for the metabolic disorder propionic acidemia

Propionic acidemia (PA) is a life-threatening disease caused by the deficiency of a mitochondrial biotin-dependent enzyme known as propionyl coenzyme-A carboxylase (PCC). This enzyme is responsible for degrading the metabolic intermediate, propionyl coenzyme-A (PP-CoA), derived from multiple metabolic pathways. Currently, except for drastic surgical and dietary intervention that can only provide partial symptomatic relief, no other form of therapeutic option is available for this genetic disorder. Here, we examine a novel approach in protein delivery by specifically targeting and localizing our protein candidate of interest into the mitochondrial matrix of the cells. In order to test this concept of delivery, we have utilized cell penetrating peptides (CPPs) and mitochondria targeting sequences (MTS) to form specific fusion PCC protein, capable of translocating and localizing across cell membranes. In vitro delivery of our candidate fusion proteins, evaluated by confocal images and enzymatic activity assay, indicated effectiveness of this strategy. Therefore, it holds immense potential in creating a new paradigm in site-specific protein delivery and enzyme replacement therapeutic for PA.

Development of an UPLC–MS/MS Method for Assaying The Enzymatic Activity of Propionyl Coenzyme-A Carboxylase

Propionyl coenzyme-A carboxylase (PCC) is a mitochondrial enzyme previously quantifiable only by radiometric assay. Herein, we report a UPLC-MS/MS method as a superior alternative method for assaying PCC's activity. METHODOLOGY & RESULTS: For the development of the UPLC-MS/MS method, the mass spectra of propionyl coenzyme-A and methyl malonyl coenzyme-A precursor ions, and their full scan product ions were determined. MRM was used for the quantification of the analytes. The method showed good linearity and selectivity for further bioanalytical study. The developed UPLC-MS/MS method is capable of rapidly quantifying PCC's enzymatic activity and demonstrated suitability for assaying PCC's activity in complex biological samples. Thus, the method will be useful in validating recombinant expression of PCC, and potentially for routine quantification of mitochondrial PCC's activity level in patient cells.

Adeno-Associated Virus Serotype 8 Gene Transfer Rescues a Neonatal Lethal Murine Model of Propionic Acidemia

Propionic acidemia (PA) is an autosomal recessive disorder of metabolism caused by a deficiency of propionyl-coenzyme A carboxylase (PCC). Despite optimal dietary and cofactor therapy, PA patients still suffer from lethal metabolic instability and experience multisystemic complications. A murine model of PA (Pcca(-/-)) of animals that uniformly die within the first 48 hr of life was used to determine the efficacy of adeno-associated viral (AAV) gene transfer as a potential therapy for PA. An AAV serotype 8 (AAV8) vector was engineered to express the human PCCA cDNA and delivered to newborn mice via an intrahepatic injection. Greater than 64% of the Pcca(-/-) mice were rescued after AAV8-mediated gene transfer and survived until day of life 16 or beyond. Western analysis of liver extracts showed that PCC was completely absent from Pcca(-/-) mice but was restored to greater than wild-type levels after AAV gene therapy. The treated Pcca(-/-) mice also exhibited markedly reduced plasma levels of 2-methylcitrate compared with the untreated Pcca(-/-) mice, which indicates significant PCC enzymatic activity was provided by gene transfer. At the time of this report, the oldest treated Pcca(-/-) mice are over 6 months of age. In summary, AAV gene delivery of PCCA effectively rescues Pcca(-/-) mice from neonatal lethality and substantially ameliorates metabolic markers of the disease. These experiments demonstrate a gene transfer approach using AAV8 that might be used as a treatment for PA, a devastating and often lethal disorder desperately in need of new therapeutic options.

Plasma concentration of 3-hydroxyisovaleryl carnitine is an early and sensitive indicator of marginal biotin deficiency in humans

Blood-based indicators of biotin status in humans were shown to be useful tools in several clinical situations, including pregnancy. We previously validated the activity of the biotin-dependent enzyme propionyl-coenzyme A carboxylase (PCC) in lymphocytes as a sensitive and specific blood-based indicator of marginal degrees of biotin deficiency. However, the measurement of PCC activity in population studies presents substantial analytic challenges. 3-Hydroxyisovaleryl carnitine (3HIA-carnitine) increases in response to the decreased activity of the biotin-dependent enzyme methylcrotonyl-coenzyme A carboxylase and might reflect biotin status. We sought to determine whether the plasma concentration of 3HIA-carnitine increases significantly in marginal biotin deficiency. We experimentally induced marginal, asymptomatic biotin deficiency in 10 healthy adults (8 women) by having the subjects consume undenatured egg white for 28 d; biotin status was then repleted. Plasma concentrations of 3HIA-carnitine were measured on days 0, 14, 28, 35, and 50 by liquid chromatography-mass spectroscopy. The mean plasma 3HIA-carnitine concentration increased with depletion (P < 0.0001) and decreased with repletion (P < 0.0001). Plasma 3HIA-carnitine concentrations were greater than the upper limit of normal concentrations in 7 of 10 subjects by day 14 and in 9 of 10 subjects by day 28 and decreased to within normal limits in 9 of 10 subjects by day 50. These studies provide evidence that 3HIA-carnitine is an early and sensitive indicator of marginal biotin deficiency. The ease of sample collection, small sample volume requirement, and stability of 3HIA-carnitine during storage suggest that plasma 3HIA-carnitine concentration is likely to be a useful indicator of marginal biotin deficiency for larger population studies.

Crystal structure of the α6β6 holoenzyme of propionyl-coenzyme A carboxylase

Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol, and fatty acids with an odd number of carbon atoms. Deficiencies of PCC activity in humans are linked to the disease propionic acidemia (PA), an autosomal recessive disorder that can be fatal in infants 1–4. The holoenzyme of PCC is an α6β6 dodecamer, with a molecular weight of 750 kD. The α subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, while the β subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2 Å resolution of a bacterial PCC α6β6 holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstructionat 15 Å resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the α subunits are arranged as monomers in the holoenzyme, decorating a central β6 hexamer. A hitherto unrecognized domain in the α subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the β subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 Å, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the β subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC, and also has important relevance for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) 5–7 and eukaryotic acetyl-CoA carboxylase (ACC) 8,9.

Association of Novel Genetic Loci With Circulating Fibrinogen Levels

Background— Fibrinogen is both central to blood coagulation and an acute-phase reactant. We aimed to identify common variants influencing circulation fibrinogen levels. Methods and Results— We conducted a genome-wide association analysis on 6 population-based studies, the Rotterdam Study, the Framingham Heart Study, the Cardiovascular Health Study, the Atherosclerosis Risk in Communities Study, the Monitoring of Trends and Determinants in Cardiovascular Disease/KORA Augsburg Study, and the British 1958 Birth Cohort Study, including 22 096 participants of European ancestry. Four loci were marked by 1 or more single-nucleotide polymorphisms that demonstrated genome-wide significance ( P &lt;5.0�10 −8 ). These included a single-nucleotide polymorphism located in the fibrinogen β chain ( FGB ) gene and 3 single-nucleotide polymorphisms representing newly identified loci. The high-signal single-nucleotide polymorphisms were rs1800789 in exon 7 of FGB ( P =1.8�10 −30 ), rs2522056 downstream from the interferon regulatory factor 1 ( IRF1 ) gene ( P =1.3�10 −15 ), rs511154 within intron 1 of the propionyl coenzyme A carboxylase ( PCCB ) gene ( P =5.9�10 −10 ), and rs1539019 on the NLR family pyrin domain containing 3 isoforms ( NLRP3 ) gene ( P =1.04�10 −8 ). Conclusions— Our findings highlight biological pathways that may be important in regulation of inflammation underlying cardiovascular disease.

Effect of Biotin on Activity and Gene Expression of Biotin-Dependent Carboxylases in the Liver of Dairy Cows

Biotin is a cofactor of the gluconeogenic enzymes pyruvate carboxylase (PC) and propionyl-coenzyme A carboxylase (PCC). We hypothesized that biotin supplementation increases the activity and gene expression of PC and PCC and the gene expression of phosphoenol-pyruvate carboxykinase (PEPCK) in the liver of lactating dairy cows. Eight multiparous Holstein cows (40±2 kg/d of milk yield and 162±35 d in milk) were randomly assigned to 1 of 2 diet sequences in a crossover design with two 22-d periods. Treatments consisted of a basal diet (60% concentrate) containing 0 or 0.96 mg/kg of supplemental biotin. On d 21 of each period, liver tissue was collected by percutaneous liver biopsy. Activities of PC and PCC were determined by measuring the fixation of [14C]O2 in liver homogenates. Abundance of mRNA for PCC, PC, and PEPCK was determined by quantitative reverse-transcription PCR. Biotin supplementation did not affect milk production or composition. Biotin supplementation increased the activity of PC but had no effect on PCC activity. Biotin supplementation did not affect the gene expression of PC, PCC, and PEPCK. The increased activity of PC without changes in mRNA abundance may have been caused by increased activation of the apoenzymes by holocar-boxylase synthetase. In conclusion, biotin supplementation affected the activity of PC in the liver of lactating dairy cows, but whether biotin supplementation increases glucose production in the liver remains to be determined.

Lymphocyte propionyl-CoA carboxylase and its activation by biotin are sensitive indicators of marginal biotin deficiency in humans.

Marginal biotin deficiency may be a human teratogen. A biotin status indicator that is not dependent on renal function may be useful in studies of biotin status during pregnancy. A previous study of experimental biotin deficiency suggested that propionyl-coenzyme A carboxylase (PCC) activity in peripheral blood lymphocytes (PBLs) is a sensitive indicator of biotin status. We examined the utility of measuring PCC activity and the activation of PCC by biotin in detecting marginal biotin deficiency. Marginal biotin deficiency was induced in 7 adults (3 women) by egg-white feeding for 28 d. Blood and urine were obtained on days 0, 14, and 28 (depletion phase) and 44 and 65 (repletion phase). PBLs were incubated with (activated) or without (control) biotin before PCC assay. The activation coefficient of PCC is the ratio of PCC activity in activated PBLs to that in control PBLs. The significance of differences for all measurements was tested by repeated-measures analysis of variance with Fisher's post hoc test and Bonferroni correction. Changes in the urinary excretion of biotin and of 3-hydroxyisovaleric acid confirmed that marginal biotin deficiency was successfully induced. By day 14, PCC activity had decreased (P < 0.0001) to below the lower limit of normal in all subjects. By day 28, the activation coefficient of PCC had increased significantly (P = 0.003) and was above the upper limit of normal in 6 of 7 subjects. PCC activity is the most sensitive indicator of biotin status tested to date. In future pregnancy studies, the use of lymphocyte PCC activity data should prove valuable in the assessment of biotin status.

Lymphocyte propionyl-CoA carboxylase and its activation by biotin are sensitive indicators of marginal biotin deficiency in humans

Background: Marginal biotin deficiency may be a human teratogen. A biotin status indicator that is not dependent on renal function may be useful in studies of biotin status during pregnancy. A previous study of experimental biotin deficiency suggested that propionyl-coenzyme A carboxylase (PCC) activity in peripheral blood lymphocytes (PBLs) is a sensitive indicator of biotin status. Objective: We examined the utility of measuring PCC activity and the activation of PCC by biotin in detecting marginal biotin deficiency. Design: Marginal biotin deficiency was induced in 7 adults (3 women) by egg-white feeding for 28 d. Blood and urine were obtained on days 0, 14, and 28 (depletion phase) and 44 and 65 (repletion phase). PBLs were incubated with (activated) or without (control) biotin before PCC assay. The activation coefficient of PCC is the ratio of PCC activity in activated PBLs to that in control PBLs. The significance of differences for all measurements was tested by repeated-measures analysis of variance with Fisher’s post hoc test and Bonferroni correction. Results: Changes in the urinary excretion of biotin and of 3-hydroxyisovaleric acid confirmed that marginal biotin deficiency was successfully induced. By day 14, PCC activity had decreased (P < 0.0001) to below the lower limit of normal in all subjects. By day 28, the activation coefficient of PCC had increased significantly (P = 0.003) and was above the upper limit of normal in 6 of 7 subjects. Conclusion: PCC activity is the most sensitive indicator of biotin status tested to date. In future pregnancy studies, the use of lymphocyte PCC activity data should prove valuable in the assessment of biotin status.

Propionic acidemia: unusual course with late onset and fatal outcome

A 4 1/2-year-old girl with a so far unremarkable medical history became comatose during a simple infection. She showed severe metabolic acidosis without elevation of lactate. In blood the branched-chain amino acids were increased. In urine ketone-bodies, increased 3-OH-isovaleric and 3-OH propionic acid excretion were detected, while methylmalonate was not found. The profile of acylcarnitines revealed increased propionylcarnitine. Despite restriction of protein supply, high-caloric nutrition, correction of acidosis, and supplementation of biotin and carnitine, the girl died 2 days after admission due to arrhythmia of the heart. In skin fibroblasts the activity of propionyl—coenzyme A carboxylase (PCC) was markedly decreased. Mutation analysis confirmed the diagnosis of propionic acidemia (PA) with compound heterozygosity for 2 new missense mutations L417W/Q293E in the PCCA gene, with the mother carrying the Q293E and the father the L417W mutation. Late-onset PA should be included in the differential diagnosis of unclear coma. Determination of the acylcarnitines using tandem mass spectrometry as well as organic acids in urine is recommended.

Valproate in the Treatment of Seizures Associated with Propionic Acidemia

MacDermont et al1 (Pediatrics 65:624, 1980) have described the uncomplicated efficacy of valproate in treating the seizures of two patients with nonketotic hyperglycinemia even though valproate elevates urinary and serum glycine concentrations in individuals with convulsive disorders.2,3 Recent observations of increased urinary propionate excretion as a metabolic consequence of valproate therapy in two patients with seizures seem to contraindicate its use in children with disorders of propionate metabolism.4 However, we have successfully and safely used valproate to control seizures in a patient with propionyl coenzyme A carboxylase (PCC) deficiency, a disorder characterized by both hyperglycinemia and propionic acidemia.

Dysautonomia in an Infant with Secondary Hyperammonemia Due to Propionyl Coenzyme A Carboxylase Deficiency

A male infant who had vomiting and coma in the absence of ketoacidosis was initially thought to have dysautonomia because of abnormal responses to methacholine and histamine, as well as abnormal urinary catecholamine excretion. Following an episode of hyperammonemia, a liver biopsy was performed which revealed a partial deficiency of carbamyl phosphate synthetase activity. The patient was treated with a protein-restricted diet supplemented with a mixture of ketoacid analogues of the essential amino acids, which precipitated ketosis and acidosis. A primary deficiency of propionyl coenzyme A (CoA) carboxylase was subsequently demonstrated. Because disorders of propionate metabolism may not initially present with ketoacidosis, we recommend examination of both plasma and urine for metabolites of this pathway, as well as direct measurement of propionyl CoA carboxylase activity in peripheral blood leukocytes, before performing a liver biopsy to evaluate urea cycle enzyme activities, and particularly before adding keto acid/amino acid mixtures to a protein-restricted diet.

Heterozygote expression in propionyl coenzyme A carboxylase deficiency. Differences between major complementation groups.

We measured propionyl coenzyme A carboxylase (PCC) activity in extracts of skin fibroblasts and peripheral blood leukocytes from controls and obligate heterozygotes for PCC deficiency. 6 heterozygotes were from the pcc A complementation group; 12 were from the other major complementation group, designated pcc C. Mean PCC activity in fibroblast extracts from pcc A heterozygotes was 52% of that in controls, whereas mean PCC activity in pcc C heterozygotes was indistinguishable from that of controls. Similar results were obtained with extracts of peripheral blood leukocytes. In none of eight families (three pcc A and five pcc C) in which PCC activity was studied in both parents of an affected child were significant intrafamilial differences observed. The activities of two other mitochondrial enzymes (beta-methyl-crotonyl CoA carboxylase and glutamate dehydrogenase) were comparable in controls and both groups of heterozygotes. Whereas the data from pcc A heterozygotes are consistent with expected gene dosage effects, those from pcc C heterozygotes are not. Inasmuch as mammalian PCC is a large molecular weight tetramer, each protomer of which is probably composed of two nonidentical subunits, the latter results are most consistent with unbalanced rates of synthesis and(or) degradation of the two subunits in normal cells with compensatory balancing in pcc C heterozygotes.