PPCDC Human

What is PPCDC and what is its role in human metabolism?

PPCDC (phosphopantothenoylcysteine decarboxylase, EC 4.1.1.36) is an essential enzyme that catalyzes the third step in the biosynthesis of Coenzyme A (CoA), a critical cofactor involved in approximately 9% of all metabolic reactions . The enzyme specifically catalyzes the decarboxylation of 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine, using flavin mononucleotide (FMN) as a cofactor .

The CoA biosynthesis pathway consists of five enzymatic steps catalyzed by four enzymes:

• Pantothenate kinase (PANK, with isoforms 1-4)

• 4'-phosphopantothenoylcysteine synthetase (PPCS)

• Phosphopantothenoylcysteine decarboxylase (PPCDC)

• CoA synthase (COASY) - a bifunctional enzyme with 4'-phosphopantetheine adenylyltransferase (PPAT) and dephosphoCoA kinase (DPCK) activities

This pathway is highly conserved across prokaryotes and eukaryotes, underscoring the fundamental importance of CoA in cellular metabolism . CoA plays crucial roles in numerous metabolic pathways, including the activation of long-chain fatty acids for catabolism, the citric acid cycle, and various biosynthetic processes.

What is the structure and cellular localization of human PPCDC?

Human PPCDC functions as a homotrimer composed of 204 amino acid monomers . The enzyme's quaternary structure is critical for its function, with the active site formed at the interface between monomeric units. Key structural features include:

• FMN binding site: The enzyme uses flavin mononucleotide as a cofactor, with Thr53 being a crucial residue involved in FMN binding

• Conserved catalytic residues: Several amino acids are highly conserved across species, including Ala95, which appears to be important for protein stability

• Oligomerization domains: Responsible for the formation of the functional homotrimer

Regarding cellular localization, PPCDC is predominantly cytosolic in human cells . This cytosolic localization is consistent with its role in the CoA biosynthesis pathway, as the early steps of this pathway occur in the cytosol before CoA is transported to various cellular compartments, including mitochondria, where it is heavily utilized in energy metabolism pathways.

How does PPCDC function in the CoA biosynthesis pathway?

PPCDC catalyzes the third step in the five-step CoA biosynthesis pathway, performing the decarboxylation of 4'-phosphopantothenoylcysteine to form 4'-phosphopantetheine, with the release of carbon dioxide . This reaction is essential for the progression of the pathway that ultimately leads to the formation of CoA.

The reaction can be represented as:
4'-phosphopantothenoylcysteine → 4'-phosphopantetheine + CO₂

The complete CoA biosynthesis pathway proceeds as follows:

• Vitamin B5 (pantothenate) → 4'-phosphopantothenate (catalyzed by PANK)

• 4'-phosphopantothenate → 4'-phosphopantothenoylcysteine (catalyzed by PPCS)

• 4'-phosphopantothenoylcysteine → 4'-phosphopantetheine (catalyzed by PPCDC)

• 4'-phosphopantetheine → dephospho-CoA (catalyzed by PPAT domain of COASY)

• Dephospho-CoA → Coenzyme A (catalyzed by DPCK domain of COASY)

The PPCDC reaction requires FMN as a cofactor for the decarboxylation mechanism. Interestingly, there is also an alternative route for the incorporation of 4'-phosphopantetheine into the pathway, potentially providing a bypass mechanism under certain conditions .

What distinguishes PPCDC from other enzymes in the CoA biosynthesis pathway?

PPCDC has several distinctive features compared to other enzymes in the CoA biosynthesis pathway:

• Unique cofactor requirement: PPCDC specifically requires FMN for its catalytic activity, unlike other enzymes in the pathway

• Evolutionary conservation pattern: While the entire pathway is conserved, PPCDC shows interesting structural differences across species. In yeast, for example, PPCDC exists as a heterotrimer rather than a homotrimer

• Disease association: Until recently, PPCDC was the only enzyme in the CoA synthesis pathway not linked to human disease. Now, pathogenic variants have been identified with a distinctive cardiac phenotype, differing from the neurological presentations seen with defects in PANK2 and COASY

• Single isoform: Unlike PANK, which has four isoforms (PANK1-4), PPCDC is encoded by a single gene in humans without known tissue-specific isoforms

• Reaction mechanism: PPCDC performs a decarboxylation reaction that is mechanistically distinct from the phosphorylation, condensation, or adenylylation reactions catalyzed by other enzymes in the pathway

What pathogenic variants of PPCDC have been identified and what is their clinical significance?

Recent research has identified biallelic pathogenic variants of PPCDC in two sisters from a non-consanguineous family who presented with a fatal cardiac phenotype . The specific variants identified were:

• p.Thr53Pro: This variant affects a residue directly involved in binding the FMN cofactor, which is essential for the catalytic activity of PPCDC

• p.Ala95Val: This variant affects another highly conserved residue and is likely a destabilizing mutation

The clinical presentation in both affected individuals was characterized by:

• Dilated cardiomyopathy (HP:0001644)

• Metabolic decompensation with hypoglycemia (HP:0001943)

• Ketonuria (HP:0002919)

• Elevated alanine levels (HP:0003348)

• Urinary excretion of dicarboxylic acids (HP:0003215)

• Increased levels of long-chain acylcarnitine in plasma (HP:0045045)

• Hypotonia (HP:0001319) and lethargy (HP:0001254)

The phenotype was progressive and fatal, with both patients dying in early infancy (around 4 months of age) from cardiac and respiratory failure . Notably, this predominantly cardiac presentation differs from the neurodegeneration with brain iron accumulation (NBIA) seen in PANK2 and COASY deficiencies but resembles the cardiac phenotype reported in PPCS deficiency .

How do mutations in PPCDC affect CoA levels and cellular metabolism?

Functional studies on patient-derived fibroblasts with pathogenic PPCDC variants revealed several significant metabolic alterations:

• CoA level reduction: Cells showed a nearly 50% reduction in intracellular CoA levels compared to control cells

• PPCDC protein absence: Western blot analysis revealed an absence of detectable PPCDC protein

• Energy metabolism dysfunction:

  • Defects in mitochondrial respiration

  • Predominantly glycolytic ATP synthesis (rather than oxidative phosphorylation)

  • Elevated levels of Krebs cycle intermediates (succinate, fumarate, and 2-oxoglutarate)

• Fatty acid oxidation abnormalities:

  • Increased levels of long-chain acylcarnitines (C12-C18)

  • Presence of long-chain dicarboxylic acids in urine

These findings suggest that PPCDC deficiency leads to a significant impairment in energy metabolism, particularly affecting tissues with high energy demands such as the heart . The metabolic profile resembles that seen in fatty acid oxidation disorders or mitochondrial diseases, consistent with the critical role of CoA in these pathways.

Treatment with medium-chain fatty acids and carnitine supplementation showed some improvement in the biochemical phenotype of one patient, restoring normal levels of acylcarnitine and amino acids, although some Krebs cycle intermediates remained elevated .

What experimental models are used to study PPCDC deficiency?

Several experimental models have been developed to study PPCDC deficiency:

• Patient-derived fibroblasts:

  • Primary fibroblasts from affected individuals provide a direct cellular model of the disease

  • These cells exhibited absent PPCDC protein, reduced CoA levels, and impaired mitochondrial respiration

• Yeast models (Saccharomyces cerevisiae):

  • In S. cerevisiae, PPCDC exists as a heterotrimer composed of a necessary subunit, Cab3 (Ykl088W), plus two Hal3 or Vhs3 subunits

  • The catalytic site is formed at the interface of the Hal3/Vhs3 and Cab3 monomers

  • Functional studies in yeast confirmed the pathogenicity of the p.Thr53Pro and p.Ala95Val mutations

• Gene silencing approaches:

  • PPCDC-interfering shRNA can create cellular models of PPCDC deficiency

  • Multiple shRNA constructs targeting PPCDC (TRCN0000155649, TRCN0000152453, TRCN0000154478, TRCN0000155901, and TRCN0000155902) have been described

  • Lentiviral particles can deliver these constructs for efficient transduction

• Overexpression systems:

  • Expression vectors containing wild-type or mutant PPCDC cDNA allow functional studies

  • The pEZ-EX-I1642-Lv205 and pEZ-EX-I1642-M55 vectors have been used for transient expression of PPCDC

  • Site-directed mutagenesis enables introduction of specific mutations for comparative analyses

Each model system offers unique advantages for investigating different aspects of PPCDC function and pathology, from basic biochemical mechanisms to cellular consequences of deficiency.

How does PPCDC deficiency compare to other defects in the CoA biosynthesis pathway?

Defects in the CoA biosynthesis pathway present with distinct clinical phenotypes depending on which enzyme is affected:

It is notable that defects in two enzymes (PANK2 and COASY) result in similar neurological phenotypes with brain iron accumulation, while defects in the other two enzymes (PPCS and PPCDC) present primarily with cardiac manifestations . This tissue-specific vulnerability suggests potential differences in:

• Tissue-specific expression patterns of different isoforms

• Varying demands for CoA in different tissues

• Possible alternative pathways that may compensate in some tissues but not others

• Differential regulation of the CoA biosynthesis pathway across tissues

These differences highlight the complexity of CoA metabolism in humans and suggest that further research into tissue-specific aspects of the pathway could provide important insights into the pathophysiology of these disorders .

What techniques are used to identify novel PPCDC variants in patients?

The identification of novel PPCDC variants typically involves a combination of genetic and functional approaches:

• Next-Generation Sequencing (NGS):

  • Whole Exome Sequencing (WES): The initial cases of PPCDC deficiency were identified using the Nextera DNA Exome Kit (Illumina)

  • Targeted gene panels: These can focus on known genes associated with metabolic disorders or cardiomyopathies

  • Whole Genome Sequencing (WGS): Provides comprehensive coverage including non-coding regions

• Variant filtering and prioritization:

  • Minor allele frequency filtering to exclude common variants

  • Inheritance pattern analysis (typically autosomal recessive)

  • In silico prediction tools to assess potential functional impact

  • Conservation analysis of affected residues across species

• Variant validation:

  • Sanger sequencing to confirm NGS findings and perform segregation analysis

  • MLPA or arrayCGH to detect large deletions or duplications

• Functional validation:

  • Expression analysis to measure PPCDC mRNA and protein levels

  • Enzymatic assays to assess PPCDC activity directly or indirectly through CoA levels

  • Cell-based functional studies to evaluate the impact on cellular metabolism

This multi-tiered approach allows for robust identification and validation of pathogenic PPCDC variants, combining genomic analysis with biochemical and cellular functional studies.

How are functional studies of PPCDC mutations conducted in cellular models?

Functional studies of PPCDC mutations in cellular models employ several complementary approaches:

• Analysis of patient-derived fibroblasts:

  • Western blotting to detect PPCDC protein levels using anti-PPCDC antibodies (e.g., PA5-61065, Thermo Fisher Scientific)

  • Native-PAGE to assess oligomerization/trimer formation

  • Immunofluorescence to determine subcellular localization

• Energy metabolism assessment:

  • ATP measurements under different conditions:

    • Basal conditions

    • After glycolysis inhibition with 2-deoxy-D-glucose (2-DG)

    • After mitochondrial ATP synthesis inhibition with oligomycin

  • Analysis of OxPhos complexes in mitochondrial extracts using antibodies against respiratory chain components (CI-NDUFB8, CII-SDHB, CIII-UQCRC2, CIV-MTCOI, and CV-ATP5A)

• Complementation studies:

  • Transfection with wild-type PPCDC cDNA using expression vectors like pEZ-EX-I1642-Lv205

  • Assessment of phenotypic rescue through CoA levels, protein expression, and energy metabolism markers

• Mutagenesis studies:

  • Introduction of specific mutations using site-directed mutagenesis (e.g., QuickchangeTM Lightning Kit)

  • Expression of mutant proteins in control cells

  • Comparison of wild-type and mutant protein functionality and localization

These methods allow comprehensive characterization of the cellular consequences of PPCDC mutations and provide insights into the pathophysiology of PPCDC deficiency.

What yeast models can be used to validate PPCDC variants?

• Yeast PPCDC structure and organization:

  • In S. cerevisiae, PPCDC exists as a heterotrimer composed of:

    • One Cab3 (Ykl088W) subunit, which is necessary for function

    • Two additional subunits that can be either Hal3, Vhs3, or a combination of both

  • The catalytic site is formed at the interface between subunits:

    • Cab3 provides the catalytic Cys478 residue

    • Hal3 (or Vhs3) provides the crucial His378 (or His459 in Vhs3) residue

  • CAB3 is an essential gene, and concurrent mutations in HAL3 and VHS3 result in a lethal phenotype

• Functional validation approaches:

  • Complementation studies with human PPCDC variants

  • Assessment of growth rescue under various conditions

  • Analysis of protein-protein interactions and complex formation

• Advantages of yeast models:

  • Rapid growth and ease of genetic manipulation

  • Well-characterized genetics and metabolism

  • Ability to perform high-throughput screens

  • Complementary insights to mammalian cell studies

In the first identified cases of PPCDC deficiency, yeast studies confirmed the functional relevance of the p.Thr53Pro and p.Ala95Val mutations, supporting their pathogenic role .

How is PPCDC protein expression and localization analyzed in patient samples?

Analysis of PPCDC protein expression and localization in patient samples involves several techniques:

• Western blot analysis:

  • Sample preparation from patient-derived fibroblasts

  • SDS-PAGE for protein expression analysis

  • Native-PAGE for oligomerization assessment

  • Immunodetection with anti-PPCDC antibodies (1:250; PA5-61065, Thermo Fisher Scientific)

  • Quantification relative to loading controls like anti-tubulin (1:1000; T9026, Sigma-Aldrich)

• Immunofluorescence microscopy:

  • Fixation of fibroblasts with 10% formalin (20 min at room temperature)

  • Permeabilization with 0.1% Triton X-100 in TBS (5 min)

  • Blocking with 0.3% donkey serum, 0.3% Triton X-100 in TBS

  • Primary antibody staining with anti-PPCDC (1:50)

  • Secondary antibody staining with Alexa 488 or Alexa 555 conjugates (1:200)

  • Nuclear counterstaining with DAPI (1:10,000)

  • Imaging with confocal microscopy (e.g., LSM710 confocal microscope)

• Expression of tagged PPCDC:

  • Transfection with vectors expressing wild-type or mutant PPCDC

  • PPCDC fused to fluorescent proteins (e.g., mCherry)

  • Live cell imaging or fixed-cell analysis

  • Co-localization with mitochondrial markers (e.g., anti-cytochrome C, 1:500, ab173529, Abcam)

These approaches allow comprehensive characterization of PPCDC protein expression, stability, oligomerization, and subcellular localization in patient samples.

What approaches are used to measure CoA levels in PPCDC-deficient cells?

Accurate measurement of CoA levels is critical for understanding the functional consequences of PPCDC deficiency. Several approaches can be employed:

• Total cellular CoA measurements:

  • Commercial CoA assay kits based on enzymatic cycling assays

  • Results normalized to total protein content determined by the Bradford method

• High-Performance Liquid Chromatography (HPLC):

  • Separation of CoA from other cellular components

  • UV detection at characteristic wavelengths

  • Quantification based on comparison to standards

• Liquid Chromatography-Mass Spectrometry (LC-MS/MS):

  • Highly sensitive and specific method for CoA detection

  • Ability to distinguish between free CoA and acyl-CoA species

  • Can provide information on the entire CoA profile

• Indirect assessment through metabolic profiling:

  • Analysis of downstream metabolites affected by CoA deficiency:

    • Acylcarnitine profiles in cell extracts

    • Organic acid analysis

    • Intermediates of the Krebs cycle (succinate, fumarate, 2-oxoglutarate)

In PPCDC-deficient patient fibroblasts, total CoA levels were found to be reduced by nearly 50% compared to control cells, consistent with the critical role of PPCDC in the CoA biosynthesis pathway .