Recombinant Pig FUN14 domain-containing protein 2 (FUNDC2)

What is the basic structure and localization of FUNDC2 in porcine cells?

FUNDC2, also known as HCBP6, is a highly conserved and ubiquitously expressed mitochondrial outer membrane protein . Research methodologies to determine its structure include protein crystallography and cryo-electron microscopy, which can reveal the spatial arrangement of the FUN14 domain. For localization studies, immunofluorescence microscopy with mitochondrial markers (such as MitoTracker) can confirm its presence on the outer mitochondrial membrane. For porcine-specific localization, researchers should isolate mitochondrial fractions from pig tissue samples and perform Western blot analysis using antibodies specific to porcine FUNDC2.

How does recombinant pig FUNDC2 compare to endogenous FUNDC2?

Methodologically, this comparison requires parallel expression and functional analyses. Researchers should express recombinant pig FUNDC2 with epitope tags (such as His or FLAG) in expression systems like E. coli or insect cells, followed by purification using affinity chromatography. The purified recombinant protein should then be compared to endogenous FUNDC2 isolated from pig tissues through activity assays, structural analysis, and interaction studies with known binding partners like SLC25A11. Mass spectrometry can identify post-translational modifications that might differ between recombinant and endogenous forms. When analyzing binding kinetics with SLC25A11, surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) should be employed to determine whether recombinant FUNDC2 maintains native binding properties.

What experimental models are best suited for studying pig FUNDC2 function?

To effectively study pig FUNDC2 function, researchers should employ a multi-model approach. Porcine cell lines (such as PK-15 or 3D4/31) can be used for initial studies, including CRISPR/Cas9-mediated knockout or overexpression systems. For more complex physiological analyses, primary cell cultures from pig cardiac or hepatic tissues provide a more relevant context for studying FUNDC2's role in ferroptosis. For in vivo studies, pig models with FUNDC2 modifications (knockout or tissue-specific overexpression) would be ideal but technically challenging; therefore, mouse models expressing pig FUNDC2 can serve as alternatives. When studying FUNDC2's interaction with SLC25A11 and its effect on mitochondrial glutathione transport, isolated mitochondria experiments with fluorescent glutathione analogs can provide direct functional evidence .

How does FUNDC2 mechanistically regulate ferroptosis in porcine cardiovascular tissues?

FUNDC2 regulates ferroptosis through multiple interconnected mechanisms. Experimental approaches to elucidate these mechanisms should include:

• Proximity labeling (BioID or APEX) to identify the complete FUNDC2 interactome in pig cardiac mitochondria

• Mitochondrial glutathione (mitoGSH) transport assays with radiolabeled glutathione in the presence of wild-type versus mutant FUNDC2

• Protein stability assays for SLC25A11 and GPX4 in the presence/absence of FUNDC2

Research has shown that FUNDC2 interacts with SLC25A11, the mitochondrial glutathione transporter, to regulate mitoGSH levels . Specifically, knockdown of SLC25A11 in FUNDC2-knockout cells reduced mitoGSH and increased sensitivity to erastin-induced ferroptosis . Furthermore, FUNDC2 affects the stability of both SLC25A11 and glutathione peroxidase 4 (GPX4), which are key regulators of ferroptosis . To fully characterize this mechanism in porcine tissues, researchers should perform targeted proteomic analyses of the mitochondrial ferroptosis pathway components in pig cardiac tissues under oxidative stress conditions.

What are the optimal expression systems and purification strategies for producing functional recombinant pig FUNDC2?

The optimal expression and purification strategy for recombinant pig FUNDC2 requires careful consideration of its membrane-associated nature. Methodologically, researchers should:

• Compare multiple expression systems (E. coli, yeast, insect cells, and mammalian cells) with varying solubilization tags (SUMO, MBP, or GST)

• Optimize membrane protein extraction using detergents like DDM, LDAO, or amphipols

• Implement a two-step purification strategy combining affinity chromatography and size exclusion chromatography

For quality control, circular dichroism spectroscopy should verify proper protein folding, while functional assays measuring interaction with SLC25A11 can confirm biological activity. When expressing recombinant pig FUNDC2, researchers should pay special attention to maintaining the native conformation of the FUN14 domain, as it is likely critical for mediating protein-protein interactions in the mitochondrial outer membrane.

How do post-translational modifications affect pig FUNDC2 function in ferroptosis regulation?

To investigate the impact of post-translational modifications (PTMs) on pig FUNDC2 function, researchers should implement a comprehensive PTM mapping strategy:

• Perform phosphoproteomic analysis of FUNDC2 isolated from pig tissues under normal and ferroptotic conditions

• Generate site-specific mutants (phosphomimetic and non-phosphorylatable) at identified modification sites

• Assess the impact of these mutations on FUNDC2's interaction with SLC25A11 and GPX4

Research suggests that FUNDC2 modulates ferroptotic stress by regulating mitoGSH levels through its interaction with SLC25A11 . PTMs likely play a critical role in this regulation, potentially serving as molecular switches that determine FUNDC2's activity under different cellular conditions. For example, phosphorylation events might alter FUNDC2's binding affinity for SLC25A11, thereby affecting mitochondrial glutathione transport and cellular resistance to ferroptosis.

What techniques are most effective for studying FUNDC2-SLC25A11 interactions in porcine mitochondria?

For studying FUNDC2-SLC25A11 interactions in porcine mitochondria, researchers should employ a multi-technique approach:

• Co-immunoprecipitation (Co-IP) with antibodies against endogenous proteins

• Förster Resonance Energy Transfer (FRET) or Bioluminescence Resonance Energy Transfer (BRET) to analyze interactions in intact mitochondria

• Crosslinking mass spectrometry to identify the specific interaction domains

Studies have demonstrated that FUNDC2 interacts with SLC25A11 to regulate mitochondrial glutathione levels . This interaction appears to be functionally significant, as knockdown of SLC25A11 in FUNDC2-knockout cells reduced mitoGSH and increased sensitivity to erastin-induced ferroptosis . To characterize the interaction domains, researchers should perform domain mapping experiments using truncated versions of both proteins. Additionally, hydrogen-deuterium exchange mass spectrometry can identify conformational changes that occur upon binding, providing insights into the molecular mechanism of the interaction.

How can researchers effectively measure FUNDC2-mediated changes in mitochondrial glutathione levels?

To accurately measure FUNDC2-mediated changes in mitochondrial glutathione levels, researchers should:

• Isolate intact mitochondria from pig tissues or cells with varying FUNDC2 expression levels

• Employ both direct and indirect measurement techniques:

  • Direct: LC-MS/MS quantification of glutathione species (GSH/GSSG) in purified mitochondrial fractions

  • Indirect: Mitochondria-targeted glutathione-sensitive fluorescent probes (mito-Grx1-roGFP2)

• Validate findings through complementary approaches such as isotope tracing of glutathione precursors

Research has shown that FUNDC2 regulates mitoGSH levels through its interaction with SLC25A11 . When designing experiments to measure these changes, researchers must carefully control for potential artifacts during mitochondrial isolation and ensure that measurements reflect the in vivo state. Time-course studies following induction of ferroptosis can provide valuable insights into the dynamics of FUNDC2-mediated glutathione regulation.

What are the optimal in vivo models for studying the role of FUNDC2 in ferroptosis-related pathologies?

For studying FUNDC2's role in ferroptosis-related pathologies, researchers should consider the following in vivo approaches:

• Generate conditional FUNDC2 knockout pigs using CRISPR/Cas9 technology with tissue-specific promoters

• Develop pig models of doxorubicin-induced cardiomyopathy to study FUNDC2's role in cardiac ferroptosis

• Implement non-invasive imaging techniques (MRI with ferroptosis-specific contrast agents) to monitor disease progression

Research has shown that knockout of FUNDC2 protected mice from doxorubicin-induced cardiac injury by preventing ferroptosis . This suggests that similar protective effects might be observed in porcine models. When developing these models, researchers should carefully consider the timing and dosing of ferroptosis inducers, as well as the methods for assessing tissue damage and ferroptotic markers.

How should researchers analyze and interpret contradictory data regarding FUNDC2 function?

When confronting contradictory data regarding FUNDC2 function, researchers should follow a systematic analytical approach:

• Evaluate methodological differences between studies (cell types, species differences, experimental conditions)

• Perform comprehensive literature meta-analysis to identify patterns in contradictory findings

• Design decisive experiments that directly address the conflicting results, including positive and negative controls

For example, if contradictory results emerge regarding FUNDC2's role in ferroptosis across different porcine tissues, researchers should compare glutathione metabolism, iron handling, and lipid peroxidation pathways in these tissues. Tissue-specific factors might explain divergent functions of FUNDC2. Additionally, researchers should consider the presence of FUNDC2 isoforms or post-translational modifications that might confer tissue-specific functions.

What is the current status of research on FUNDC2 and viral interactions in porcine models?

Current research on FUNDC2 and viral interactions in porcine models is limited, but related studies on viral recombination in pigs provide methodological guidance:

• Researchers should explore potential interactions between FUNDC2 and porcine viruses using:

  • Viral infection studies in pig cells with FUNDC2 knockdown/overexpression

  • Proteomic analyses to identify virus-FUNDC2 interactions

  • Transcriptomic profiling to assess FUNDC2 expression changes during viral infection

Recent studies have identified recombinant porcine enteroviruses in pig farms , and recombination events in Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) . While direct links to FUNDC2 have not been established, these findings highlight the importance of studying host-pathogen interactions in porcine models. Researchers investigating FUNDC2's potential role in viral infections should employ metagenomics approaches similar to those used to detect recombinant viruses in pig farms .

How can structural data on FUNDC2 inform the development of targeted research tools?

Structural data on FUNDC2 can guide the development of research tools through:

• Structure-based design of:

  • Domain-specific antibodies for immunoprecipitation and imaging

  • High-affinity peptide inhibitors for specific FUNDC2 interactions

  • PROTAC or degrader molecules for targeted FUNDC2 degradation

• Identification of critical residues for:

  • Site-directed mutagenesis to create function-specific FUNDC2 variants

  • Development of conformation-specific probes to track FUNDC2 activation states

When developing these tools, researchers should focus on the FUN14 domain and regions mediating interaction with SLC25A11, as these are likely critical for FUNDC2's role in regulating ferroptosis through mitochondrial glutathione transport .

Experimental Design Table for FUNDC2 Research

This comprehensive table provides researchers with a structured approach to investigating various aspects of recombinant pig FUNDC2 function, highlighting appropriate controls and anticipated challenges for each experimental direction.

What are the critical knowledge gaps in our understanding of pig FUNDC2 function?

• Tissue-specific expression patterns and functions of FUNDC2 in porcine models

• The complete interactome of FUNDC2 beyond SLC25A11 and GPX4

• Evolutionary conservation of FUNDC2 function across species and potential pig-specific adaptations

• Regulatory mechanisms controlling FUNDC2 expression and activity under normal and stressed conditions

Research has shown that knockout of FUNDC2 protected mice from doxorubicin-induced cardiac injury by preventing ferroptosis , but the broader implications of this protection in other tissue types and disease models remain unexplored. Addressing these gaps requires comprehensive tissue expression profiling, interactome studies, and comparative analyses across species.

How might recombinant pig FUNDC2 be utilized as a research tool for studying ferroptosis mechanisms?

Recombinant pig FUNDC2 can serve as a valuable research tool for studying ferroptosis mechanisms through several methodological approaches:

• As a competitive inhibitor of endogenous FUNDC2-SLC25A11 interactions

• For in vitro reconstitution of mitochondrial glutathione transport systems

• As bait for identifying novel ferroptosis regulators in pull-down experiments

By producing functional domains of recombinant pig FUNDC2, researchers can develop tools to manipulate mitochondrial glutathione transport and ferroptosis pathways in experimental systems. For example, a recombinant protein containing only the SLC25A11-binding domain could be used to competitively inhibit the FUNDC2-SLC25A11 interaction, providing temporal control over this pathway without genetic manipulation.

What novel methodologies should be developed to advance pig FUNDC2 research?

To advance pig FUNDC2 research, several novel methodologies should be developed:

• Pig-specific FUNDC2 nanobodies for super-resolution imaging and acute inhibition

• Mitochondria-targeted biosensors for real-time monitoring of FUNDC2-dependent processes

• Organ-on-chip models incorporating primary pig cells for studying tissue-specific FUNDC2 functions

• CRISPR-based screening platforms to identify genetic modifiers of FUNDC2 activity