Recombinant Macaca mulatta FUN14 domain-containing protein 2 (FUNDC2)

What is FUNDC2 and how does it relate to other FUN14 domain-containing proteins?

FUNDC2 belongs to the FUN14 domain-containing protein family, which includes the better-characterized FUNDC1. While specific information about FUNDC2 functions is limited, FUNDC1 serves as an integral mitochondrial outer-membrane protein that mediates the formation of mitochondria-associated endoplasmic reticulum membranes (MAMs) . FUNDC1 has been implicated in angiogenesis, hypoxia-induced mitophagy, and hepatic ferroptosis, suggesting potential related functions for FUNDC2 . Research approaches for FUNDC2 would likely parallel those used for FUNDC1, including protein localization studies, interaction partner identification, and functional assays under various cellular stresses.

Why use Macaca mulatta as a model for studying FUNDC2?

Macaca mulatta (rhesus macaque) represents an invaluable non-human primate model for biomedical research due to its close evolutionary relationship with humans . Approximately 86% of human genes are represented in macaque transcriptomes, making them excellent models for studying human-relevant protein functions . The species-specificity of the immune system necessitates studying proteins like FUNDC2 in appropriate models when investigating immune-related disorders or when developing therapeutic approaches . The high genetic similarity between macaques and humans allows for more reliable extrapolation of experimental results to human applications compared to rodent models.

What are the primary methods for isolating RNA for recombinant protein expression studies in Macaca mulatta?

RNA isolation from Macaca mulatta tissues for recombinant protein expression studies should follow established protocols using reagents like Trizol (Invitrogen) . The process involves:

• Tissue homogenization in Trizol reagent

• RNA extraction following manufacturer's protocol

• Validation of RNA quality by electrophoresis in formaldehyde-containing agarose gels to confirm intact 28S and 18S ribosomal RNA bands

• mRNA subtraction using poly(A) tract isolation systems (e.g., PolyA Tract mRNA isolation system by Promega)

• First-strand cDNA synthesis using oligo(dT) primers and reverse transcriptase

• Second-strand synthesis using DNA pol I and RNase H

This approach ensures high-quality RNA for downstream applications like PCR amplification of the target gene.

How do I design primers for amplifying the FUNDC2 gene from Macaca mulatta samples?

When designing primers for Macaca mulatta FUNDC2 amplification, consider the following methodology:

• Obtain reference sequences from databases like NCBI for both human and available macaque FUNDC2 sequences

• Identify conserved regions between human and macaque sequences to design primers

• Design primers with appropriate restriction sites for subsequent cloning

• Ensure primers have compatible melting temperatures (within 2-3°C of each other)

• Validate primer specificity using in silico PCR tools

• Include appropriate overhangs for the selected expression system

For example, when amplifying genes from M. mulatta, researchers have successfully used polymerase chain reaction (PCR) with specifically designed primers containing restriction sites compatible with the intended expression vector .

What alternative splicing events might affect FUNDC2 expression and function in Macaca mulatta?

Alternative splicing (AS) events significantly impact protein diversity and function in primates. Based on transcriptome studies in Macaca fascicularis, approximately 4,314 AS events have been identified across various tissues . Intriguingly, 10.4% of AS events are associated with transposable element (TE) insertions, which could potentially affect FUNDC2 expression and function . When investigating FUNDC2 variants:

• Perform tissue-specific transcriptome analysis to identify potential FUNDC2 splice variants

• Examine intron-exon boundaries for potential alternative splice sites

• Validate predicted variants using RT-PCR and sequencing

• Compare expression patterns of different splice variants across tissues

• Assess functional differences between splice variants using in vitro assays

Understanding these variants is crucial as they may confer tissue-specific functions or differential responses to cellular stress conditions.

How do post-translational modifications affect FUNDC2 function in mitochondrial dynamics?

While specific data on FUNDC2 post-translational modifications (PTMs) is limited, studies on related proteins like FUNDC1 suggest PTMs play crucial roles in regulating function. For FUNDC1, phosphorylation states regulate mitophagy activity and interaction with binding partners like LC3 . To investigate PTMs in FUNDC2:

• Perform mass spectrometry analysis of purified recombinant FUNDC2 to identify potential PTM sites

• Create phosphomimetic and phosphodeficient mutants through site-directed mutagenesis

• Compare wild-type and mutant FUNDC2 function in cellular assays

• Investigate kinases and phosphatases that may regulate FUNDC2 activity

• Assess how cellular stress conditions (hypoxia, oxidative stress) affect FUNDC2 PTM patterns

Understanding these modifications would provide insights into how FUNDC2 activity is regulated in different cellular contexts.

What are the tissue-specific expression patterns of FUNDC2 in Macaca mulatta and how do they compare to human expression profiles?

Tissue-specific expression analysis is critical for understanding protein function in context. In transcriptome studies of Macaca fascicularis, 175 tissue-specific transcripts were identified across 16 different tissues . While specific FUNDC2 expression data is not provided in the search results, a methodological approach to determine this would include:

• Quantitative RT-PCR analysis of FUNDC2 expression across multiple tissues

• RNA-Seq analysis of different tissues to obtain expression profiles

• Western blot analysis using FUNDC2-specific antibodies to confirm protein expression

• Immunohistochemistry to localize FUNDC2 in tissue sections

• Comparative analysis with human tissue expression data

Such analysis would reveal whether FUNDC2 shows preferential expression in specific tissues, providing clues to its physiological functions.

What expression systems are optimal for producing recombinant Macaca mulatta FUNDC2?

When selecting an expression system for recombinant Macaca mulatta FUNDC2, consider the following options and their methodological implications:

For Macaca mulatta proteins, P. pastoris has been successfully used to express recombinant proteins with yields of approximately 3-4 mg from 1 L of inductive media . The choice should be guided by the intended application and required protein characteristics.

How can I assess the functional activity of recombinant FUNDC2 in vitro?

Functional assessment of recombinant FUNDC2 requires multiple complementary approaches:

• Binding assays to identify interaction partners:

  • Co-immunoprecipitation with potential binding partners

  • Surface plasmon resonance to determine binding kinetics

  • Yeast two-hybrid screening to discover novel interactions

• Subcellular localization studies:

  • Fluorescent tagging and microscopy

  • Subcellular fractionation and Western blotting

  • Protease protection assays for topology determination

• Cell-based functional assays:

  • Overexpression and knockdown studies

  • Mitochondrial morphology and function analysis

  • Cell survival under stress conditions (hypoxia, nutrient deprivation)

If FUNDC2 functions similarly to FUNDC1, assess its potential role in mitophagy through co-localization with LC3 under various stress conditions and its impact on mitochondrial dynamics .

What experimental design considerations are essential when using FUNDC2 in Macaca mulatta studies?

The Experimental Design Assistant (EDA) provides valuable guidance for designing experiments with non-human primates . Key considerations include:

• Sample size calculation:

  • Use power analysis to determine the minimum number of animals required

  • Consider variability in FUNDC2 expression levels between individuals

• Randomization and blinding:

  • Implement randomization in treatment group assignments

  • Ensure analysis is performed blind to treatment conditions

• Controls and validation:

  • Include appropriate positive and negative controls

  • Validate antibody specificity for macaque FUNDC2

  • Use multiple methodologies to confirm findings

• Ethical considerations:

  • Follow the 3Rs (Replacement, Reduction, Refinement) principles

  • Ensure proper approval from institutional review boards

Additionally, construct a visual representation of your experimental plan using tools like the EDA to facilitate transparency and collaboration with colleagues .

How can I optimize Western blot protocols for detecting FUNDC2 in Macaca mulatta samples?

Optimizing Western blot protocols for macaque FUNDC2 requires careful consideration of several factors:

• Sample preparation:

  • Use appropriate tissue/cell lysis buffers with protease inhibitors

  • Optimize protein extraction protocols for subcellular fractions (especially for mitochondrial proteins)

• Antibody selection:

  • Test antibodies raised against human FUNDC2 for cross-reactivity with macaque protein

  • Consider using antibodies generated against recombinant fragment proteins for improved specificity

• Protocol optimization:

  • Determine optimal primary antibody dilution (typically start with 1:1000)

  • Optimize blocking conditions to reduce background

  • Consider using PVDF membranes for better protein retention and sensitivity

• Controls:

  • Include recombinant FUNDC2 as a positive control

  • Use tissue samples known to express high levels of FUNDC2

  • Include loading controls appropriate for the subcellular fraction being analyzed

Given the high sequence similarity between human and macaque proteins, antibodies designed for human FUNDC1/FUNDC2 detection may work for macaque samples, but validation is essential .

How does FUNDC2 sequence and function differ between Macaca mulatta and humans?

Understanding evolutionary differences between macaque and human FUNDC2 requires comparative analysis:

• Sequence comparison:

  • Perform alignment analysis of FUNDC2 sequences from both species

  • Identify conserved domains and species-specific variations

  • Calculate sequence identity percentages for different functional domains

• Structural analysis:

  • Predict protein structures using homology modeling

  • Compare functional motifs and potential binding sites

  • Identify differences that might affect protein-protein interactions

• Functional comparison:

  • Express both macaque and human FUNDC2 in cellular models

  • Compare subcellular localization patterns

  • Assess functional parameters under identical experimental conditions

While specific FUNDC2 comparison data is not available, transcriptome sequencing of Macaca fascicularis shows approximately 86% of human genes are represented among the genes sequenced, suggesting high but not complete conservation .

What models are best suited for studying FUNDC2 interactions with the mitochondrial quality control machinery?

Selecting appropriate models for studying FUNDC2 interactions with mitochondrial quality control machinery should consider:

• Cell line models:

  • Macaque-derived cell lines for species-specific interactions

  • Human cell lines for translational relevance

  • Knockout/knockdown systems to study loss-of-function effects

• Primary cell models:

  • Primary fibroblasts or myoblasts from macaque tissues

  • Primary neurons for studying mitochondrial dynamics in neuronal contexts

• In vitro reconstitution:

  • Purified recombinant proteins for direct interaction studies

  • Isolated mitochondria for functional assays

• Stress conditions to examine:

  • Hypoxia (to mimic conditions known to affect FUN14 domain proteins)

  • Oxidative stress using specific compounds

  • Nutrient deprivation to induce mitophagy

If FUNDC2 functions similarly to FUNDC1, it may interact with mitochondrial quality control machinery under stress conditions, making stress-response models particularly valuable .

How can transcriptome sequencing improve our understanding of FUNDC2 regulation in Macaca mulatta?

Transcriptome sequencing provides powerful insights into gene regulation. Based on studies with Macaca fascicularis, the following methodological approaches would be valuable:

• Tissue-specific expression profiling:

  • Sequence transcriptomes from multiple tissues (as done with M. fascicularis across 16 tissues)

  • Identify tissue-specific expression patterns of FUNDC2

  • Discover potential tissue-specific isoforms

• Alternative splicing analysis:

  • Identify potential alternative splicing events in FUNDC2

  • Analyze the role of transposable elements in FUNDC2 exonization events

  • Compare splicing patterns between macaque and human

• Regulatory network identification:

  • Identify co-expressed genes to infer functional relationships

  • Discover potential transcription factors regulating FUNDC2

  • Analyze promoter regions for regulatory elements

Such analysis could reveal regulatory mechanisms controlling FUNDC2 expression under different physiological conditions and in response to cellular stresses.

What are the most promising research directions for understanding FUNDC2 function in mitochondrial biology?

Based on knowledge of related proteins and general mitochondrial biology, promising research directions include:

• Defining the role of FUNDC2 in mitophagy and mitochondrial quality control

• Investigating FUNDC2 involvement in cellular responses to metabolic stress

• Exploring potential roles in disease models where mitochondrial dysfunction is implicated

• Comparing functions between macaque and human FUNDC2 to evaluate translational potential

• Identifying small molecules that modulate FUNDC2 activity for experimental tools

These directions would build upon known functions of FUN14 domain-containing proteins while exploring the specific roles of FUNDC2.

How can cross-species comparative studies of FUNDC2 inform human disease models?

Cross-species comparative studies offer valuable insights for translational research:

• Identification of conserved functional domains that are likely essential for FUNDC2 function

• Discovery of species-specific adaptations that might affect drug responses

• Development of more predictive preclinical models for testing therapeutics targeting mitochondrial pathways

• Understanding evolutionary adaptations in mitochondrial quality control mechanisms

• Identification of compensatory mechanisms that might exist in one species but not another

Given that approximately 86% of human genes are represented in macaque transcriptomes, macaques provide a strong model for studying human-relevant functions while acknowledging potential species differences .

What novel technologies are emerging for studying mitochondrial proteins like FUNDC2?

Emerging technologies that could advance FUNDC2 research include:

• CRISPR/Cas9 gene editing:

  • Generation of precise knockouts or tagged endogenous proteins in macaque cells

  • Introduction of human variants to study functional differences

• Proximity labeling techniques:

  • BioID or APEX2 fusion proteins to identify proximal interactors in living cells

  • Mapping the spatial organization of FUNDC2 in mitochondrial membranes

• Advanced imaging:

  • Super-resolution microscopy to visualize mitochondrial dynamics

  • Live-cell imaging with fluorescent sensors to monitor mitochondrial function

• Single-cell transcriptomics:

  • Analysis of cell-to-cell variation in FUNDC2 expression

  • Identification of rare cell populations with unique FUNDC2 functions