ATP5D Human

What is ATP5D and what is its role in cellular bioenergetics?

ATP5D (ATP5F1D) is a subunit of mitochondrial ATP synthase, specifically part of the F1 complex (δ subunit). It plays a crucial role in coupling proton translocation across the inner mitochondrial membrane to ATP production through oxidative phosphorylation. This subunit is essential for proper assembly and function of ATP synthase (Complex V), the final enzyme in the electron transport chain responsible for generating ATP . Research methodologies to study ATP5D function include blue native PAGE for complex assembly analysis, respirometry for measuring ATP production capacity, and structural biology approaches to determine its position within the ATP synthase complex.

What are the characterized pathogenic variants of ATP5D in humans?

Two significant pathogenic homozygous missense variants in ATP5F1D have been well-characterized: c.245C>T (p.Pro82Leu) and c.317T>G (p.Val106Gly) . These mutations affect conserved residues and result in impaired assembly of the F1FO ATP synthase complex. Researchers investigating ATP5D variations should employ whole exome sequencing or targeted gene panels with appropriate bioinformatic pipelines to identify novel variants. Functional validation of newly identified variants requires fibroblast studies, enzyme activity assays, and potentially model organism complementation assays.

How is ATP5D expression measured in experimental and clinical samples?

Multiple methodologies can be employed to measure ATP5D at transcript and protein levels:

• ELISA: Effectively used to detect ATP5D protein in seminal plasma of fertile versus infertile males

• qRT-PCR: For quantifying ATP5D mRNA expression in tissue samples

• Western blotting: For protein detection and semi-quantitative analysis

• Immunohistochemistry: For visualizing ATP5D distribution in tissue sections

• Mass spectrometry: For precise quantification and detection of post-translational modifications

When analyzing clinical samples, researchers should consider using multiple complementary techniques and include appropriate controls to account for sample variability.

How does ATP5D expression differ across human tissues and pathological states?

ATP5D is expressed in multiple human tissues, reflecting the universal requirement for ATP synthesis. Notably, research has demonstrated significantly reduced ATP5D protein levels in the seminal plasma of infertile males compared to fertile controls . While ATP5D expression patterns in other pathological conditions are still being characterized, its altered expression in male infertility suggests it may serve as a potential biomarker. Researchers investigating tissue-specific expression should employ RNA-seq, proteomics, and tissue microarrays to establish comprehensive expression profiles in normal and disease states.

What is the molecular pathophysiology of ATP5D mutations in human disease?

Biallelic mutations in ATP5F1D cause a distinct metabolic disorder characterized by episodic lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia . The pathophysiology involves:

• Impaired ATP synthase assembly: Mutations disrupt the proper assembly of the F1FO complex

• Reduced Complex V activity: Leading to compromised ATP production capacity

• Mitochondrial cristae abnormalities: Fibroblasts from affected individuals show significant decreases in mitochondrial cristae

• Metabolic derangements: Including organic aciduria and hyperammonemia

Research approaches should include comprehensive metabolomics, mitochondrial functional assays, and electron microscopy to fully characterize the cascade of events resulting from ATP5D dysfunction.

What experimental models are most effective for studying ATP5D function and pathology?

Several experimental models have proven valuable for studying ATP5D:

• Patient-derived fibroblasts: Demonstrate impaired ATP synthase assembly and cristae abnormalities

• Drosophila models: Knockdown of ATPsynδ (the ATP5F1D homolog) causes developmental defects, with human wild-type ATP5F1D capable of rescuing the phenotype while mutant versions show partial rescue

• Mouse models: ATP5D knockout and knockin models generated using CRISPR/Cas9 technology demonstrate fertility phenotypes

• iPSC-derived models: Allow study of tissue-specific effects in relevant cell types

Each model offers distinct advantages: cell models allow biochemical analysis, Drosophila provides rapid generation time for genetic studies, while mouse models better recapitulate mammalian physiology.

How does ATP5D dysfunction contribute to male infertility?

Research has established a potential role for ATP5D in male fertility through multiple experimental approaches:

• Expression correlation: ATP5D protein levels are significantly reduced in seminal plasma of infertile males

• Knockout effects: ATP5D knockout male mice showed significantly prolonged fertility time and impaired testicular histomorphology

• Functional implications: The involvement likely relates to the high energy requirements of spermatogenesis and sperm motility

Experimental approaches to investigate this association include ELISA measurement of ATP5D in seminal plasma, histological examination of testicular tissue, sperm functional assays, and metabolomic analysis of seminal fluid. Researchers should note that while expression levels are altered in infertility, sequencing studies did not detect mutations in the coding regions of ATP5D in infertile males .

What therapeutic strategies are being investigated for ATP5D-associated disorders?

While specific therapies for ATP5D-associated disorders are still emerging, several approaches warrant investigation:

• Metabolic interventions: Management of metabolic acidosis and hyperammonemia

• Mitochondrial biogenesis enhancers: Compounds that stimulate mitochondrial function

• Gene therapy approaches: Viral vector-mediated delivery of functional ATP5F1D

• Small molecule screening: Identification of compounds that might stabilize ATP synthase assembly

• Precision medicine: Development of mutation-specific therapies for particular variants

Research methodologies should include high-throughput drug screening, patient-derived cell models for therapeutic testing, and appropriate animal models for preclinical evaluation.

How do ATP5D mutations specifically affect ATP synthase assembly and structure?

The p.Pro82Leu and p.Val106Gly mutations in ATP5F1D affect conserved residues that are critical for proper protein folding or interactions with other subunits . Functional studies have shown:

• Cultured skin fibroblasts from affected individuals exhibit impaired assembly of F1FO ATP synthase

• Subsequent reduction in complex V activity

• Drosophila complementation studies demonstrate that mutant proteins retain partial function but cannot fully rescue developmental defects

Researchers investigating these effects should employ blue native PAGE, co-immunoprecipitation studies, cryo-electron microscopy, and molecular modeling to characterize structural perturbations caused by specific mutations.

What are the optimal approaches for investigating ATP5D interactions with other ATP synthase components?

To comprehensively characterize ATP5D interactions within the ATP synthase complex:

• Blue native PAGE: Allows analysis of intact ATP synthase complexes

• Co-immunoprecipitation: Identifies direct binding partners

• Proximity labeling: BioID or APEX2 approaches can identify proteins in close proximity

• Cryo-electron microscopy: Provides structural information about the assembled complex

• Cross-linking mass spectrometry: Maps specific interaction interfaces

Researchers should combine multiple complementary approaches to build a comprehensive interaction map of ATP5D within the ATP synthase complex.

How can researchers effectively analyze ATP5D evolutionary conservation for functional insights?

ATP5D homologs are present across species, providing opportunities for evolutionary analysis:

• The Drosophila homolog (ATPsynδ) functionally complements human ATP5F1D

• Cross-species rescue experiments identify functionally conserved domains

• Comparative genomics can highlight critical residues maintained throughout evolution

• Phylogenetic analysis can reveal evolutionary relationships and functional constraints

Research approaches should combine computational phylogenetic analysis with experimental cross-species complementation to identify functionally critical domains and residues.

What considerations are important when establishing ATP5D as a biomarker for male fertility?

Research suggests ATP5D may serve as a potential biomarker for male fertility , requiring:

• Standardized measurement protocols: Consistent ELISA or other quantitative methods

• Large-scale validation: Studies across diverse populations to establish reference ranges

• Correlation with clinical parameters: Sperm count, morphology, and other fertility metrics

• Mechanism elucidation: Understanding how ATP5D affects spermatogenesis and sperm function

• Comparative biomarker analysis: Determining ATP5D's value relative to existing fertility biomarkers

Researchers pursuing biomarker development should conduct prospective studies with appropriate statistical power and diverse patient populations to validate clinical utility.

What are the critical unanswered questions regarding ATP5D function in human disease?

Despite progress in understanding ATP5D, several important questions remain:

• Tissue-specific effects: Why do ATP5D mutations predominantly affect specific tissues despite ubiquitous expression?

• Phenotypic variability: What factors explain the clinical heterogeneity in patients with ATP5D mutations?

• Regulatory mechanisms: How is ATP5D expression regulated across tissues and developmental stages?

• Interaction partners: Are there tissue-specific interaction partners that modify ATP5D function?

• Potential roles beyond ATP synthesis: Does ATP5D have functions independent of its role in ATP synthase?

These questions represent critical areas for future investigation that will advance our understanding of ATP5D biology and pathology.

How can systems biology approaches advance our understanding of ATP5D in cellular energetics?

Integrative approaches to study ATP5D include:

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data

• Network analysis: Identifying ATP5D within broader mitochondrial and cellular networks

• Mathematical modeling: Creating predictive models of ATP synthase function incorporating ATP5D dynamics

• Single-cell approaches: Characterizing cell-to-cell variability in ATP5D expression and function

• In silico drug screening: Computational approaches to identify potential therapeutic compounds

These systems-level approaches can provide comprehensive insights into ATP5D's role within broader cellular energetic networks.

What are the recommended protocols for ATP5D functional analysis in patient samples?

For researchers working with patient samples to analyze ATP5D function:

• ATP5D protein quantification: Validated ELISA assays for seminal plasma and other biofluids

• Mitochondrial functional assessment: Oxygen consumption rate measurements in patient fibroblasts

• ATP synthesis capacity: Luciferase-based ATP determination assays

• Mitochondrial morphology: Electron microscopy protocols for cristae analysis

• ATP synthase assembly: Blue native PAGE followed by immunoblotting for complex V subunits

Standardized protocols with appropriate controls are essential for meaningful cross-study comparisons and clinical correlations.