ATPIF1 Human

What is ATPIF1 and what is its primary function in human cells?

ATPIF1 is a nuclear-encoded, mitochondrial protein that serves as an endogenous inhibitor of ATP synthase. It binds to the catalytic interface between the α and β subunits of the F1 domain of ATP synthase, thereby regulating its activity . Since its identification in 1963, ATPIF1 has been recognized as highly conserved across species, suggesting its evolutionary importance . In humans, the ATPIF1 gene (ENSG00000130770) is located on chromosome 1 and undergoes alternative splicing to produce three transcript variants encoding distinct isoforms . The minimal inhibitory sequence of ATPIF1 comprises amino acids 14-47, with adjacent sequences (amino acids 10-13 and 48-56) helping to stabilize the ATP synthase-IF1 complex .

How is ATPIF1 gene expression regulated in human tissues?

ATPIF1 expression varies significantly among tissues, with highest levels found in organs with high energy demands such as heart, liver, kidney, and brain (particularly neurons) . Interestingly, tissues from the digestive tract show elevated relative expression of ATPIF1, while breast and lung tissues have nearly undetectable levels under normal conditions . At the transcriptional level, nuclear factor-κB (NF-κB) has been identified as a direct regulator of ATPIF1 expression . ATPIF1 is also overexpressed in various carcinomas including colon, lung, breast, ovaries, and liver tumors . Additionally, ATPIF1 expression in human mesenchymal stem cells but not in osteogenic differentiated cells suggests a potential role as a stemness marker involved in maintaining cellular quiescence .

What are the different isoforms of ATPIF1 and their functional differences?

The ATPIF1 gene undergoes alternative splicing, producing three distinct isoforms with different functional capabilities:

Based on mRNA expression levels, isoform 1 represents the major form expressed in human organs including liver, heart, kidney, and brain . Isoforms 2 and 3, which lack the complete minimal inhibitory sequence, are unlikely to inhibit ATP synthase activity, and their functional roles remain entirely unknown .

How does ATPIF1 regulate ATP synthase activity under normoxic versus hypoxic conditions?

ATPIF1 exhibits context-dependent regulation of ATP synthase:

During hypoxia or ischemia, when cellular pH decreases below 6.7, ATPIF1 forms active dimers that strongly inhibit the hydrolytic activity of ATP synthase, preventing wasteful ATP consumption when oxygen is limited . This function has traditionally been considered the primary role of ATPIF1.

Under normoxic conditions, recent evidence challenges the traditional view that ATPIF1 only inhibits ATP hydrolysis. Multiple studies now demonstrate that ATPIF1 can also inhibit the synthetic activity of ATP synthase during normal respiration . For instance, tissue-specific expression of constitutively active IF1-H49K has been shown to induce mitochondrial hyperpolarization and promote a shift from OXPHOS to aerobic glycolysis in neurons, hepatocytes, and hepatoma cells . Silencing of ATPIF1 under normoxic conditions leads to higher OXPHOS activity and cellular ATP levels compared to control cells .

What is the mechanism of ATPIF1 inhibition of ATP synthase?

ATPIF1 inhibits ATP synthase through a pH-dependent mechanism involving changes in its oligomeric state:

At low pH (<6.7), ATPIF1 exists predominantly as an active dimer formed through antiparallel coiled-coil interactions between the C-terminal regions of two monomers . This dimeric form binds to the catalytic interface between the α and β subunits of the F1 domain of ATP synthase, preventing catalytic activity .

At physiological or higher pH (>6.7), ATPIF1 forms tetramers and higher oligomers that are inactive and unable to inhibit ATP synthase . This pH-dependent regulation involves histidine residues, particularly histidine 49, which becomes protonated at low pH, destabilizing the tetramers and favoring active dimer formation .

The mutation of histidine 49 to lysine (IF1-H49K) creates a pH-independent constitutively active inhibitor, demonstrating the critical role of this residue in regulation . Recent data also suggest that tetramers of ATP synthase internally linked by IF1 dimers may represent an inhibited state, supporting IF1's ability to inhibit ATP synthesis in normally respiring mitochondria .

What experimental approaches are best for studying ATPIF1's role in cellular bioenergetics?

To effectively study ATPIF1's role in bioenergetics, researchers should employ multiple complementary approaches:

• Kinetic Mode Assay: This method, developed by Barrientos and detailed in Bio-Protocol, measures ATP synthase activity in the presence or absence of oligomycin, providing clear evidence of ATPIF1 inhibition under basal cellular conditions .

• Genetic Manipulation: Using isolated mitochondria and permeabilized cells with ATPIF1 content manipulated through silencing, knockout, or overexpression provides robust experimental systems . Both loss-of-function (silencing, knockout) and gain-of-function (overexpression of wild-type or constitutively active variants) should be employed .

• Integrated Measurements: Assessment of related parameters including cellular respiration, mitochondrial membrane potential (ΔΨm), and mitochondrial ROS production provides a more complete understanding of ATPIF1's effects .

• Multiple Model Systems: Findings from cell culture should be validated with in vivo models, as the researchers note that "the findings from cells in culture do not necessarily have to reproduce the results obtained in vitro with recombinant proteins" .

• Appropriate Controls: Experiments should include rigorous controls and methodologies that minimize unnecessary cell manipulation to prevent artifacts .

How does ATPIF1 contribute to metabolic reprogramming and the Warburg effect?

ATPIF1 plays a significant role in promoting the Warburg effect - the metabolic shift toward enhanced aerobic glycolysis observed in cancer cells:

By inhibiting ATP synthase activity, ATPIF1 compromises oxidative phosphorylation (OXPHOS) efficiency, forcing cells to increase glycolytic metabolism to meet energy demands . Multiple studies demonstrate this effect: overexpression of ATPIF1 or the constitutively active IF1-H49K variant induces mitochondrial hyperpolarization (higher ΔΨm) and promotes a metabolic shift from OXPHOS to aerobic glycolysis in neurons, hepatocytes, hepatoma cells, mouse intestinal epithelium, human skeletal muscle cells, and rat insulinoma cells .

Conversely, silencing of ATPIF1 in rat liver epithelial F258 cells prevents the glycolytic shift induced by the carcinogen benzo[a]pyrene (B[a]P) . This metabolic reprogramming driven by ATPIF1 is likely to promote metabolic dysfunction, chronic inflammation, and cancer development .

What methodological considerations are important when measuring ATPIF1 effects on ATP synthase?

When measuring ATPIF1's effects on ATP synthase, researchers should consider several methodological issues:

How do changes in ATPIF1 expression affect oxidative phosphorylation versus glycolysis?

Changes in ATPIF1 expression directly impact the balance between OXPHOS and glycolysis:

ATPIF1 Overexpression Effects:

• Inhibition of ATP synthase activity

• Increased mitochondrial membrane potential (hyperpolarization)

• Reduced OXPHOS capacity and ATP production via mitochondria

• Enhanced glycolytic flux to compensate for ATP deficiency

• Metabolic shift toward aerobic glycolysis (Warburg effect)

ATPIF1 Silencing/Knockout Effects:

• Enhanced ATP synthase activity

• Increased OXPHOS efficiency and capacity

• Higher cellular ATP levels from mitochondrial sources

• Reduced reliance on glycolysis

• Prevention of glycolytic shift in response to carcinogens

What is the evidence for ATPIF1's role in cancer development and progression?

Multiple lines of evidence support ATPIF1's involvement in cancer:

ATPIF1 is significantly overexpressed in carcinomas from various organs, including colon, lung, breast, ovaries, and liver . The relative mitochondrial content of ATPIF1 increases significantly in these carcinomas compared to normal tissues, suggesting participation in oncogenesis . ATPIF1 promotes the Warburg effect through inhibition of ATP synthase, forcing cancer cells to rely more heavily on aerobic glycolysis .

How does ATPIF1 activity influence immune cell function in the tumor microenvironment?

ATPIF1 plays a critical role in T cell function within tumor microenvironments:

Studies using genetic approaches have demonstrated that ATPIF1 deficiency impairs immune activities of CD8+ T cells, leading to quicker tumor growth in ATPIF1-knockout mice with B16 melanoma and Lewis lung cancer . ATPIF1-knockout T cells show reduced proliferation and IFN-γ secretion accompanied by metabolic reprogramming toward increased glycolysis and decreased OXPHOS after activation .

Single-cell RNA sequencing and flow cytometry confirmed increased T cell exhaustion in tumor-infiltrating leukocytes from ATPIF1-knockout mice . Conversely, ATPIF1 overexpression in T cells enhances expression of IFN-γ and Granzyme B, increases the subset of central memory T cells in CAR-T cells, and improves survival rates in tumor-bearing mice . These findings suggest ATPIF1 is a potential molecular target for enhancing antitumor immunity, particularly in CAR-T cell therapy .

What is the relationship between ATPIF1 phosphorylation and disease states?

ATPIF1 phosphorylation represents a critical regulatory mechanism with implications for disease:

ATPIF1 is phosphorylated at serine 39 (S39) by protein kinase A (PKA) or a mitochondrial PKA-like activity . This phosphorylation prevents ATPIF1 from interacting with ATP synthase, thereby blocking its inhibitory function and allowing increased ATP synthase activity .

Cancer cells and human carcinomas contain variable amounts of phosphorylated and dephosphorylated ATPIF1 , suggesting altered phosphorylation states may contribute to metabolic dysregulation in disease. β-adrenergic stimulation promotes ATPIF1 phosphorylation and inactivation, increasing ATP synthase activity in mouse heart , indicating hormonal regulation of this mechanism with potential implications for cardiac disease.

The phosphorylation status of ATPIF1 can be experimentally manipulated - it is prevented by PKA inhibitors and stimulated by db-cAMP in cell culture models . This regulatory mechanism provides a potential therapeutic target for diseases characterized by bioenergetic dysfunction.

How does ATPIF1 deficiency affect T cell function and antitumor immunity?

ATPIF1 deficiency significantly compromises T cell function and antitumor immunity:

Genetic inactivation of ATPIF1 impairs CD8+ T cell immune activities, resulting in accelerated tumor growth in mouse models of B16 melanoma and Lewis lung cancer . At the cellular level, ATPIF1-knockout T cells exhibit multiple functional deficits:

• Reduced proliferative capacity

• Decreased IFN-γ secretion

• Metabolic reprogramming toward increased glycolysis and decreased oxidative phosphorylation

• Increased T cell exhaustion in tumor infiltrating leukocytes as revealed by single-cell RNA sequencing and confirmed by flow cytometry

These findings demonstrate that ATPIF1 is essential for maintaining optimal T cell antitumor activity, potentially through its role in regulating cellular bioenergetics. The metabolic shift observed in ATPIF1-deficient T cells likely impairs their ability to sustain effector functions within the challenging metabolic environment of tumors .

What are the effects of ATPIF1 overexpression on CAR-T cell therapy?

ATPIF1 overexpression enhances CAR-T cell efficacy through multiple mechanisms:

Studies have demonstrated that increasing ATPIF1 expression in T cells leads to:

• Enhanced expression of effector molecules including IFN-γ and Granzyme B, critical for antitumor activity

• Increased proportion of central memory T cell subsets in CAR-T cells, which are associated with improved persistence and efficacy

• Improved survival rates in NALM-6 tumor-bearing mice treated with ATPIF1-overexpressing CAR-T cells

These findings suggest ATPIF1 as a potential molecular target for optimizing CAR-T cell therapy. The researchers conclude that "ATPIF1 is a potential molecular target in the modulation of antitumor immunity of CD8+ T cells in cancer immunotherapy. Induction of ATPIF1 activity may promote CAR-T activity in cancer therapy" .

How does ATPIF1 regulate metabolic reprogramming in activated T cells?

ATPIF1 plays a crucial role in T cell metabolic reprogramming during activation:

T cell activation normally involves a shift toward both increased glycolysis and enhanced oxidative phosphorylation to meet elevated energy demands. ATPIF1 appears essential for maintaining proper balance between these metabolic pathways .

In ATPIF1-knockout T cells, activation leads to excessive glycolysis with insufficient OXPHOS activity . This imbalanced metabolic response likely impairs T cell function since both pathways are critical for optimal immune responses. Energy supply is identified as "a primary factor in the control of T cell activities, especially in the glucose-limiting tumor microenvironment, in which T cells suffer energy deficiency from low supplies of glucose and other nutrients" .

ATPIF1's role in T cell metabolism appears somewhat paradoxical compared to its function in cancer cells. While ATPIF1 promotes glycolysis in cancer cells by inhibiting OXPHOS, its activity in T cells seems necessary for maintaining adequate OXPHOS during activation . This context-dependent function highlights the complex regulatory role of ATPIF1 in cellular metabolism.

How does phosphorylation regulate ATPIF1 activity?

Phosphorylation represents a critical regulatory mechanism for ATPIF1 activity:

ATPIF1 is phosphorylated at serine 39 (S39) by protein kinase A (PKA) or a mitochondrial PKA-like activity . This post-translational modification prevents ATPIF1 from interacting with ATP synthase, thereby blocking its inhibitory function . In effect, phosphorylation inactivates ATPIF1, allowing increased ATP synthase activity.

The phosphorylation state of ATPIF1 is dynamically regulated in response to cellular signaling:

• PKA inhibitors prevent ATPIF1 phosphorylation

• Dibutyryl-cAMP (db-cAMP) stimulates ATPIF1 phosphorylation

• β-adrenergic stimulation promotes phosphorylation and inactivation of ATPIF1 in mouse heart

Cancer cells, human carcinomas, and mouse tissues that express ATPIF1 contain variable amounts of phosphorylated and dephosphorylated ATPIF1, indicating this regulatory mechanism operates under physiological and pathological conditions .

How does pH regulate ATPIF1 structure and function?

pH exerts profound effects on ATPIF1 structure and function through modulation of its oligomeric state:

At low pH (<6.7), ATPIF1 exists predominantly as a dimer formed through antiparallel coiled-coil interactions between the C-terminal regions of two monomers . This dimeric form represents the active state that can bind and inhibit ATP synthase .

At physiological or higher pH (>6.7), ATPIF1 forms tetramers and higher oligomers, which are inactive forms unable to inhibit ATP synthase . This pH-dependent structural reorganization involves histidine residues, particularly histidine 49, which becomes protonated at low pH, destabilizing tetramers and favoring active dimer formation .

The mutation of histidine 49 to lysine (IF1-H49K) results in a pH-independent constitutively active IF1 dimer, demonstrating the critical role of this residue in pH-dependent regulation . This pH-sensitivity allows ATPIF1 to respond to cellular conditions, with maximal inhibitory activity during hypoxia or ischemia when cellular pH decreases .

What interactions exist between ATPIF1 phosphorylation and pH-dependent regulation?

While the search results don't explicitly address interactions between phosphorylation and pH-dependent regulation of ATPIF1, we can infer potential relationships based on our understanding of both mechanisms:

Both regulatory mechanisms affect ATPIF1's ability to interact with ATP synthase - phosphorylation directly prevents binding, while pH controls the formation of active dimers versus inactive tetramers . These mechanisms likely operate independently but may interact under certain physiological or pathological conditions.

For example, during cellular stress conditions that lower pH (such as hypoxia), ATPIF1 would tend to form active dimers, but concurrent PKA-mediated phosphorylation could override this activation by preventing binding to ATP synthase regardless of oligomeric state. Conversely, in conditions where PKA activity is reduced, pH would become the dominant regulatory factor determining ATPIF1 activity.

The relative contribution of each regulatory mechanism likely varies across tissue types, disease states, and metabolic conditions. Future research specifically investigating how these regulatory mechanisms interact would provide valuable insights into ATPIF1 regulation under complex physiological and pathological conditions.

What are the most effective genetic models for studying ATPIF1 function?

Several genetic models have proven effective for investigating ATPIF1 function:

In Vitro and Cellular Models:

• CRISPR/Cas9-mediated ATPIF1 knockout cell lines (HCT116, Jurkat)

• RNAi-mediated silencing of ATPIF1 in various cell types

• Transient and stable transfection with ATPIF1 overexpression constructs

• Expression of mutant variants, particularly the constitutively active IF1-H49K

• Primary cells from genetic models, including T cells, skeletal muscle cells, and hepatocytes

Animal Models:

• ATPIF1 knockout mice, which show accelerated tumor growth in cancer models

• Tissue-specific expression of constitutively active IF1-H49K variant

• Genetic loss- and gain-of-function models of ATPIF1 in different mouse tissues (brain, kidney, heart, and colon)

These diverse genetic models provide complementary approaches for understanding ATPIF1 function across different biological contexts. The combination of in vitro, cellular, and animal models allows for comprehensive investigation of ATPIF1's roles in metabolism, cancer biology, and immune function.

How can researchers accurately assess ATPIF1 expression levels in different tissues?

Accurate assessment of ATPIF1 expression requires consideration of several methodological approaches:

• Protein Quantification:

  • Western blotting with validated antibodies, normalizing ATPIF1 levels to appropriate loading controls

  • Analysis of ATPIF1 relative to other mitochondrial proteins (e.g., IF1/β-F1-ATPase ratio) to account for differences in mitochondrial content

  • Immunohistochemistry for tissue localization and relative expression

• mRNA Analysis:

  • Quantitative RT-PCR for ATPIF1 transcript variants

  • RNA sequencing for comprehensive transcriptome analysis

  • Single-cell RNA sequencing to assess cell-type specific expression

• Subcellular Localization:

  • Immunofluorescence microscopy to confirm mitochondrial localization

  • Subcellular fractionation to assess ATPIF1 distribution between mitochondria and other cellular compartments

• Post-translational Modification Analysis:

  • Detection of phosphorylated versus non-phosphorylated forms using phospho-specific antibodies

  • Phosphatase treatments to distinguish between phosphorylation states

• Functional Assessments:

  • Correlation of expression levels with ATP synthase inhibition

  • Metabolic analysis to connect expression levels with functional outcomes

For comprehensive analysis, multiple complementary approaches should be employed to account for potential limitations of individual methods.

What are the limitations of current methodologies for studying ATPIF1?

Current methodologies for studying ATPIF1 have several important limitations:

• Translational Gaps:

  • Findings from cells in culture may not reproduce in vivo results

  • In vitro studies with recombinant proteins provide mechanistic insights but lack cellular complexity

  • Limited studies in entire organisms and different organs restrict understanding of tissue-specific functions

• Technical Challenges:

  • Potential artifacts from excessive cell manipulation

  • Difficulty distinguishing direct ATPIF1 effects from secondary metabolic adaptations

  • Variability in expression levels across cell lines complicates interpretation

• Isoform Analysis:

  • Limited understanding of the roles of different ATPIF1 isoforms

  • Most studies focus on isoform 1, with isoforms 2 and 3 remaining poorly characterized

• Post-translational Modification Detection:

  • Challenges in preserving and accurately quantifying phosphorylation status

  • Need for better tools to distinguish between differently modified ATPIF1 forms

• Context Dependency:

  • ATPIF1 effects may depend on cellular energy demand, metabolic state, and cell type

  • Difficult to recapitulate the complexity of tissue microenvironments in experimental models

Addressing these limitations requires multidisciplinary approaches, development of new methodologies, and integration of findings across different experimental systems to build a comprehensive understanding of ATPIF1 biology.