Recombinant Human ATP synthase lipid-binding protein, mitochondrial (ATP5G3)

What is ATP5G3 and what is its role in mitochondrial function?

ATP5G3 encodes one of three different precursors to subunit 9 (also known as subunit c) of the mitochondrial ATP synthase complex. This subunit is critical for proton transport across the inner mitochondrial membrane to the F1-ATPase, resulting in the generation of ATP . The mature protein encoded by ATP5G3 is identical to those produced by ATP5G1 and ATP5G2, though the leader peptide sequences differ while retaining the essential "RFS" motif critical for mitochondrial import and maturation . ATP5G3 is predominantly located in the inner mitochondrial membrane, where it forms part of the ATP synthase complex that acts as a molecular motor using the energy from proton gradients to drive ATP synthesis .

How does ATP5G3 differ from other ATP synthase subunit c isoforms?

While ATP5G3 produces a mature protein identical to those encoded by ATP5G1 and ATP5G2, its genomic location and expression patterns differ significantly. ATP5G3 maps to chromosome 2 in humans , whereas ATP5G1 and ATP5G2 are located on chromosomes 17 and 12, respectively . Sequence analysis reveals that ATP5G3 shares only about 80% identity with ATP5G1 and ATP5G2 in the DNA sequence encoding the mature protein . Furthermore, expression studies have shown that ATP5G3 is predominantly expressed among the three isoforms, accounting for approximately 75% of the transcripts in certain cell types like HAP1 cells . This differential expression suggests tissue-specific roles and regulatory mechanisms for each isoform.

What is the typical expression pattern of ATP5G3 across different tissues?

ATP5G3 is expressed in various tissues throughout the body , but expression levels vary significantly across different tissue types. Research has shown distinct expression patterns in tissues such as the hippocampus, cerebellum, and liver . In the context of pancreatic tissue, ATP5G3 showed upregulation in response to chronic alcohol consumption in rat models . The widespread but variable expression pattern of ATP5G3 reflects its fundamental role in energy metabolism across multiple tissue types, while suggesting potential tissue-specific functions or regulatory mechanisms.

What are the recommended approaches for studying ATP5G3 expression changes under stress conditions?

To effectively study ATP5G3 expression under stress conditions, researchers should consider a multi-method approach:

What gene silencing approaches are most effective for studying ATP5G3 function?

When designing gene silencing experiments for ATP5G3, researchers should consider:

• siRNA Transfection: Small interfering RNA (siRNA) targeting ATP5G3 has been successfully used to investigate its function in cell death mechanisms . When implementing this approach, researchers should design siRNAs specific to the unique regions of ATP5G3 to avoid off-target effects on ATP5G1 and ATP5G2.

• CRISPR-Cas9 Gene Editing: For complete gene disruption, CRISPR-Cas9 technology has proven effective. Guide RNAs (gRNAs) specific to exon III in ATP5G3 have been successfully used . To achieve complete elimination of all subunit c proteins, simultaneous targeting of all three genes (ATP5G1, ATP5G2, and ATP5G3) may be necessary, as demonstrated in HAP1 cell lines .

• Verification Methods: Following silencing, verification should include both RNA-level assessment via qRT-PCR and protein-level confirmation through Western blotting. Given that all three genes produce identical mature proteins, researchers must carefully validate the specificity of their silencing approach.

• Rescue Experiments: To confirm phenotype specificity, rescue experiments involving the reintroduction of the wild-type ATP5G3 gene are recommended to demonstrate reversibility of observed phenotypes.

How can researchers effectively distinguish between the three ATP synthase subunit c isoforms in experimental settings?

Distinguishing between ATP5G1, ATP5G2, and ATP5G3 presents challenges due to their identical mature protein sequences. Recommended approaches include:

How does ATP5G3 contribute to mitochondrial disease pathogenesis?

ATP5G3 mutations and dysfunction have been associated with several mitochondrial diseases through the following mechanisms:

• Energy Production Deficits: Disruption of ATP5G3 function compromises ATP synthesis, leading to energy deficiency in affected tissues. This is particularly impactful in high-energy-demanding tissues such as the nervous system and muscles, contributing to the neurological and myopathic symptoms seen in conditions like Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes (MELAS) .

• Oxidative Stress Regulation: ATP5G3 appears to have a protective role against oxidative stress-induced cell death. In cellular models, ATP5G3 knockdown increased susceptibility to sodium nitroprusside (SNP)-induced cytotoxicity . This protective function is mediated through the regulation of p38 MAPK signaling and Bcl-xL pathways , suggesting that ATP5G3 dysfunction may contribute to disease pathogenesis through increased cellular sensitivity to oxidative damage.

• Mitochondrial Permeability Transition Pore (PTP) Regulation: Studies investigating the components of the PTP have explored the role of ATP synthase subunits, including those encoded by ATP5G3 . Alterations in PTP function due to ATP5G3 dysfunction could contribute to mitochondrial swelling, cytochrome c release, and cell death pathways implicated in mitochondrial diseases.

• Tissue-Specific Impacts: Given the differential expression of ATP5G3 across tissues, dysfunction may have tissue-specific consequences, potentially explaining the variable clinical presentations of mitochondrial diseases associated with ATP5G3 mutations .

What is the evidence linking ATP5G3 to neurodegenerative disorders?

While direct evidence specifically linking ATP5G3 to neurodegenerative disorders is still emerging, several lines of evidence suggest potential connections:

• Mitochondrial Dysfunction in Neurodegeneration: Mitochondrial dysfunction is a well-established component of neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases. As a critical component of ATP synthase, ATP5G3 dysfunction could contribute to the bioenergetic deficits observed in these conditions.

• Oxidative Stress Protection: Research has demonstrated that ATP5G3 plays a protective role against oxidative stress-induced cell death . Given that oxidative stress is a key pathogenic mechanism in neurodegenerative disorders, ATP5G3 dysfunction could exacerbate neuronal vulnerability to oxidative damage.

• Cell Death Regulation: ATP5G3 has been implicated in the regulation of autophagic cell death and lysosome-associated cell death pathways , which are increasingly recognized as important in neurodegenerative disease progression.

• Association with Mitochondrial Diseases: ATP5G3 mutations have been linked to mitochondrial diseases with neurological manifestations, including MELAS , suggesting potential roles in neurological pathology.

• Expression in Neural Tissues: ATP5G3 is expressed in brain regions such as the hippocampus and cerebellum , areas frequently affected in neurodegenerative conditions.

How does ATP5G3 function relate to metabolic disorders such as obesity and alcoholism?

Research has suggested several connections between ATP5G3 and metabolic disorders:

• Alcohol Metabolism: Studies have identified ATP5G3 as a gene upregulated in the pancreas of ethanol-fed rats , suggesting a role in the cellular response to chronic alcohol consumption. This upregulation may represent an adaptive response to altered metabolic demands or mitochondrial stress induced by ethanol exposure.

• Potential Links to Obesity: Gene expression correlation studies have suggested potential associations between ATP5G3 expression levels and obesity-related traits . This connection may reflect the central role of mitochondrial function in energy expenditure and metabolic regulation.

• Tissue-Specific Regulation: The expression patterns of ATP5G3 vary significantly across tissues involved in metabolism, including the liver . This tissue-specific regulation may contribute to the differential impacts of metabolic disorders on various organ systems.

• Bioenergetic Adaptation: As a component of ATP synthase, ATP5G3 plays a crucial role in cellular energy production. Alterations in ATP5G3 expression or function may reflect or contribute to the bioenergetic adaptations observed in metabolic disorders.

What are the key regulatory mechanisms controlling ATP5G3 expression under normal and pathological conditions?

The regulation of ATP5G3 expression involves complex mechanisms that remain partially understood:

• Transcriptional Regulation: Differential expression of ATP5G3 across tissues suggests tissue-specific transcriptional regulatory mechanisms. Transcriptome QTL mapping indicates that ATP5G3 is differentially regulated in hippocampus, cerebellum, and liver , pointing to tissue-specific transcription factors and regulatory elements.

• Stress-Responsive Regulation: ATP5G3 expression changes under oxidative stress conditions , suggesting the presence of stress-responsive elements in its promoter region. The mechanisms by which oxidative stress signals are transduced to alter ATP5G3 expression warrant further investigation.

• Post-transcriptional Regulation: The stability and translation of ATP5G3 mRNA may be subject to regulation by microRNAs and RNA-binding proteins, particularly under stress conditions. These mechanisms could contribute to rapid adjustments in ATP5G3 levels in response to cellular demands.

• Epigenetic Mechanisms: Epigenetic modifications such as DNA methylation and histone modifications likely contribute to the tissue-specific and condition-dependent expression patterns of ATP5G3. Studies exploring the epigenetic landscape of the ATP5G3 gene under various conditions would provide valuable insights into its regulation.

• Feedback Regulation: ATP levels and mitochondrial function status may feed back to regulate ATP5G3 expression as part of a homeostatic mechanism to maintain appropriate ATP synthase levels.

What are the most promising therapeutic strategies targeting ATP5G3 for mitochondrial diseases?

Developing therapeutic approaches targeting ATP5G3 presents both opportunities and challenges:

What are the primary technical difficulties in producing recombinant ATP5G3 protein, and how can they be addressed?

Producing functional recombinant ATP5G3 presents several technical challenges:

• Hydrophobicity Issues: As a subunit of a membrane-embedded complex, ATP5G3-encoded protein is highly hydrophobic, creating difficulties in expression and purification. Solutions include:

  • Using specialized expression systems designed for membrane proteins

  • Incorporating solubility tags such as MBP (maltose-binding protein) or GST (glutathione S-transferase)

  • Employing detergent screening to identify optimal solubilization conditions

• Proper Folding: Ensuring correct folding of the recombinant protein is crucial. Strategies include:

  • Expression at lower temperatures to slow protein synthesis and allow proper folding

  • Co-expression with chaperones to assist folding

  • Cell-free expression systems that can accommodate membrane proteins

• Post-translational Processing: The mature ATP5G3 protein requires removal of the leader peptide. Approaches to address this include:

  • Engineering constructs with appropriate cleavage sites

  • Co-expression with processing enzymes

  • In vitro processing following initial purification

• Functional Assessment: Verifying the functionality of recombinant ATP5G3 is challenging outside its native complex. Potential solutions include:

  • Reconstitution into liposomes or nanodiscs to assess proton translocation

  • Co-expression with other ATP synthase components

  • Development of functional assays specific to subunit c properties

How can researchers effectively analyze ATP5G3 interactions with other proteins in the mitochondrial membrane?

Investigating ATP5G3 protein interactions requires specialized approaches for membrane proteins:

• Crosslinking Strategies: Chemical crosslinking combined with mass spectrometry can capture transient or stable interactions. Zero-length crosslinkers or spacer-arm crosslinkers can be selected based on the interaction interface being studied.

• Co-immunoprecipitation Adaptations: Standard co-IP protocols must be modified for membrane proteins:

  • Use of gentle, non-ionic detergents that preserve protein-protein interactions

  • Adjustment of buffer conditions to maintain native-like membrane environment

  • Two-step purification approaches to reduce non-specific binding

• Proximity Labeling Technologies: BioID or APEX2 proximity labeling can identify proteins in close proximity to ATP5G3 in living cells, offering advantages for studying membrane protein complexes.

• Stable Isotope Labeling Approaches: SILAC (Stable Isotope Labeling with Amino acids in Cell culture) has been successfully applied to ATP synthase studies and can be used to quantitatively assess changes in protein interactions under different conditions.

• Cryo-electron Microscopy: High-resolution structural analysis using cryo-EM has provided valuable insights into ATP synthase complex organization and can reveal detailed interaction interfaces between subunit c and other components.

What experimental controls are essential when studying ATP5G3 in relation to oxidative stress response?

When investigating ATP5G3's role in oxidative stress responses, several critical controls should be implemented:

• Gene Silencing Controls:

  • Include scrambled siRNA controls to account for non-specific effects of transfection

  • Validate knockdown efficiency at both mRNA (using qRT-PCR) and protein levels

  • Consider silencing other ATP5G isoforms to distinguish isoform-specific effects

• Oxidative Stress Induction Controls:

  • Include both fresh and photodegraded sodium nitroprusside (SNP) treatments to distinguish between effects of the compound itself versus its degradation products

  • Use iron chelators like deferoxamine (DFO) to confirm the involvement of iron-dependent mechanisms

  • Employ alternative oxidative stress inducers to confirm that observed effects are not specific to a particular stressor

• Pathway Validation Controls:

  • Use specific inhibitors and activators of suspected signaling pathways (e.g., p38 inhibitors and activators) to confirm mechanistic connections

  • Combine ATP5G3 silencing with pathway component silencing (e.g., co-transfection with sip38 or siBcl-xL) to validate proposed mechanisms

• Cell Death Assessment Controls:

  • Employ multiple assays to distinguish between different forms of cell death (apoptosis, autophagic cell death, necrosis)

  • Include lysosomal inhibitors when investigating autophagic or lysosome-associated cell death mechanisms

  • Monitor p62 protein levels as an indicator of autophagic flux