Recombinant Mouse ATP synthase subunit f, mitochondrial (Atp5j2)

Advanced Research Questions

• What is the relationship between ATP synthase subunit f and mitochondrial pathologies like Alzheimer's disease?

  The connection between ATP synthase and Alzheimer's disease (AD) is increasingly recognized, though the specific role of subunit f (Atp5j2) requires further investigation. Mitochondrial dysfunction is a key feature of AD, which has led to the development of the mitochondrial cascade hypothesis .

  Several studies have implicated ATP synthase in AD pathology:

  1. Decreased expression: Several ATP synthase subunits show decreased expression in affected brain regions of AD patients, including the posterior cingulate cortex, hippocampal field CA1, middle temporal gyrus, and entorhinal cortex .

  2. Oxidative modifications: The α-subunit of ATP synthase shows 4-HNE modification (lipoxidation) in AD patients, correlating with disease progression as measured by neurofibrillary tangle (NFT) presence. This modification results in reduced ATP synthase activity .

  3. Interaction with amyloid β: Studies with transgenic mice expressing mutant APP (corresponding to familial AD) showed increased expression of the α-subunit, potentially as a cellular stress response to maintain energy production .

  Although these studies do not specifically focus on subunit f, they highlight how ATP synthase dysfunction contributes to AD pathogenesis. The F0 domain, which includes subunit f, is critical for proton translocation and ATP production. Dysfunction of this domain could contribute to the bioenergetic deficits observed in AD.

  Future research should investigate whether Atp5j2 undergoes specific modifications in AD and how these might affect ATP synthase function and mitochondrial bioenergetics in the context of neurodegeneration.

• How can site-directed mutagenesis of Atp5j2 inform our understanding of F0 domain function?

  Site-directed mutagenesis of Atp5j2 can reveal critical functional regions and mechanism of action within the F0 domain of ATP synthase. This approach has been highly informative for other ATP synthase subunits, particularly regarding the directionality and efficiency of the ATP synthase motor.

  Key strategies and findings from mutagenesis studies of ATP synthase that could be applied to Atp5j2 include:

  1. Conserved residue mutation: Studies of the a-subunit revealed that the strictly conserved arginine (R176) is crucial for motor directionality, protonation state control, and separation of proton half-channels. Mutation to alanine (R176A) drastically reduced the rotation barrier in the hydrolysis direction, while mutation to lysine (R176K) increased the barrier in the synthesis direction . Similar mutations of conserved residues in Atp5j2 could reveal its specific contributions to motor function.

  2. Cysteine-scanning mutagenesis: This approach has been used to map aqueous accessible regions in the F0 domain. For example, the cG58C substitution in one study was highly sensitive to modification, and a single modified c subunit was sufficient to completely inhibit the complex . Similar scanning of Atp5j2 could map its topology and identify critical functional regions.

  3. Phosphomimetic mutations: Studies of the β subunit showed that phosphomimetic mutations (e.g., T262E) abolished ATP synthase activity while nonphosphorylatable mutations (T262A) maintained normal function . Identifying potential phosphorylation sites in Atp5j2 and creating similar mutations could reveal regulatory mechanisms.

  When designing mutagenesis experiments for Atp5j2, researchers should focus on:

  • Conserved residues identified through multiple sequence alignments

  • Regions predicted to interact with other subunits

  • Potential post-translational modification sites

  • Residues at the membrane interface or within predicted transmembrane segments

• What are the challenges and approaches for studying the integration of recombinant Atp5j2 into functional ATP synthase complexes?

  Studying the integration of recombinant Atp5j2 into functional ATP synthase complexes presents several challenges that require sophisticated experimental approaches:

  Challenges:

  1. Membrane protein integration: As part of the F0 domain, Atp5j2 is a membrane protein that must properly integrate into the lipid bilayer, which is challenging in recombinant systems.

  2. Complex assembly: ATP synthase assembly involves multiple subunits assembled in a specific order. The assembly sequence of subunits into different modules and between modules is not entirely clear .

  3. Functional assessment: Determining whether recombinant Atp5j2 has properly integrated requires functional assays of the entire complex.

  4. Native environment: The mitochondrial inner membrane provides a specific environment that is difficult to replicate in experimental systems.

  Approaches:

  1. Reconstitution systems: Purified recombinant Atp5j2 can be reconstituted with other ATP synthase subunits in liposomes to study complex assembly and function. Clear native PAGE (CN-PAGE) with mild detergents can then be used to analyze complex formation .

  2. Genetic complementation: In systems where endogenous Atp5j2 can be depleted or knocked out, complementation with recombinant variants can determine functional integration.

  3. Submitochondrial particles (SMPs): These inside-out vesicles derived from mitochondria provide a native-like environment for studying ATP synthase function. Cyclophilin D binding to ATP synthase has been studied in SMPs, showing decreased ATP synthesis and hydrolysis rates .

  4. Stable isotope labeling: Combining recombinant protein expression with stable isotope labeling and mass spectrometry can track the incorporation of recombinant Atp5j2 into ATP synthase complexes.

  5. Cryo-EM structural analysis: Recent advances in cryo-EM allow visualization of membrane protein complexes, providing insights into whether recombinant Atp5j2 integrates correctly into the structure.

• How does the expression and regulation of Atp5j2 differ across tissues and under pathological conditions?

  The expression and regulation of ATP synthase subunits, including Atp5j2, can vary significantly across tissues and under pathological conditions. This variation reflects tissue-specific energy demands and responses to stress:

  Tissue-specific expression:

  While the search results don't provide comprehensive data on tissue-specific expression of Atp5j2, we can infer patterns based on known ATP synthase regulation:

  1. High-energy demand tissues: Tissues with high energy demands like brain, heart, and skeletal muscle typically show higher expression of ATP synthase components.

  2. Tissue-specific isoforms: Some ATP synthase subunits have tissue-specific isoforms. For Atp5j2, alternatively spliced transcript variants encoding different isoforms have been identified , suggesting potential tissue-specific regulation.

  Pathological conditions:

  Several pathological conditions affect ATP synthase expression and function:

  1. Alzheimer's disease: Studies have shown decreased expression of several ATP synthase subunits in affected brain regions of AD patients . The α-subunit shows oxidative modifications correlating with disease progression.

  2. Transgenic disease models: In transgenic mouse models expressing mutant APP (corresponding to familial AD), there was a 12.2-fold increase in α-subunit expression compared to non-transgenic controls , potentially as a stress response to maintain energy production.

  3. Neurogenesis defects: Adult neurogenesis defects in AD may arise from impaired function of hippocampal neuronal stem cells (NSCs). A study using iPSC-derived NSCs with familial AD-associated PS1 mutation M146L observed decreased expression of the ATP synthase complex .

  Regulatory mechanisms:

  ATP synthase subunits, potentially including Atp5j2, are regulated through various mechanisms:

  1. Post-translational modifications: Phosphorylation of ATP synthase subunits can significantly alter activity. For example, phosphomimetic mutations in the β subunit (T262E) abolished activity while nonphosphorylatable mutations (T262A) maintained normal function .

  2. Protein-protein interactions: Regulatory proteins like cyclophilin D can bind to ATP synthase and decrease both ATP synthesis and hydrolysis rates . In the absence of cyclophilin D or when its binding is inhibited by cyclosporin A, matrix adenine nucleotide levels are affected.

  Future research should focus on characterizing tissue-specific expression patterns of Atp5j2 and identifying specific regulatory mechanisms that control its expression and function under different physiological and pathological conditions.