Recombinant Macaca fascicularis FAD-dependent oxidoreductase domain-containing protein 1 (FOXRED1)

What is FOXRED1 and what is its primary function in mitochondrial biology?

FOXRED1 is a 486-amino acid FAD-dependent oxidoreductase that functions as a complex I-specific molecular chaperone in the mitochondria. It is crucial for the assembly and stability of mitochondrial respiratory chain complex I, the first and largest enzyme in the respiratory chain located in the inner mitochondrial membrane . This protein contains an FAD-dependent oxidoreductase domain and belongs to the family of D-amino acid oxidase . Through its chaperone function, FOXRED1 ensures the proper assembly of complex I, which is essential for mitochondrial energy production through oxidative phosphorylation.

Where is FOXRED1 localized within mitochondria?

FOXRED1 is primarily localized in the mitochondria, with western blot data confirming an expected band of approximately 53 kDa in the whole mitochondrial fraction . More specifically, FOXRED1 has been observed in both the intermembrane space (IMS) and the matrix of mitochondria. Evidence suggests that FOXRED1 precursor containing its mitochondrial import sequence is imported into mitochondria via a classical importation mechanism (such as the TOM/TIM machinery), after which the import sequence is cleaved within the matrix to release the mature protein . The mature protein then associates with the mitochondrial inner membrane, likely positioned near respiratory chain supercomplexes and adjacent to the complex I holoenzyme to perform its chaperone function .

How is FOXRED1 structure related to its function?

The structure of FOXRED1 provides important insights into its function:

• FOXRED1 contains an FAD-binding domain that is critical for its oxidoreductase activity

• Protein modeling using the Bacillus monomeric sarcosine oxidase structure as a template reveals that mutations affecting the FAD-binding site (such as R352W) can significantly impact protein function

• The protein sequence of Macaca fascicularis FOXRED1 (1-486aa) shares high homology with human FOXRED1, reflecting evolutionary conservation of this critical assembly factor

• Although FOXRED1 is not one of the known 45 subunits of complex I (as established by proteomics studies), it transiently interacts with complex I components during assembly

What expression systems are optimal for producing recombinant FOXRED1 protein?

E. coli has been successfully used as an expression system for recombinant Macaca fascicularis FOXRED1 protein . When expressing FOXRED1:

• Full-length protein (amino acids 1-486) can be expressed with an N-terminal His-tag for purification purposes

• The expressed protein typically yields greater than 90% purity as determined by SDS-PAGE

• The recombinant protein is often prepared as a lyophilized powder

• For reconstitution, it is recommended to use deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Adding 5-50% glycerol (final concentration) is advised for long-term storage at -20°C/-80°C

For working with the protein:

• Avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

• Tris/PBS-based buffer with 6% Trehalose, pH 8.0 is typically used as a storage buffer

How can researchers assess FOXRED1's role in complex I assembly experimentally?

Several complementary approaches can be employed to investigate FOXRED1's role in complex I assembly:

• Gene silencing/knockout studies:

  • RNA interference to silence FOXRED1 expression in human fibroblasts, which results in reduced complex I steady-state levels and activity

  • TALEN-mediated gene editing to disrupt the FOXRED1 gene in HEK293T cells, resulting in approximately 10% complex I levels, reduced complex I activity, and inability to grow on galactose media

• Rescue experiments:

  • Lentiviral-mediated FOXRED1 transgene expression to rescue complex I deficiency in patient fibroblasts

  • Overexpression of FOXRED1 containing patient mutations to evaluate their effect on complex I assembly

• Complex I assembly analysis:

  • Blue native PAGE (BN-PAGE) immunoblot analysis to detect native mitochondrial complex I and its assembly intermediates

  • Identification of subcomplexes (e.g., ~475 kDa subcomplex in FOXRED1 mutant cells compared to the mature complex)

  • Analysis of assembly intermediates (e.g., ~815 kDa intermediate that forms transiently in the absence of FOXRED1)

• Protein-protein interaction studies:

  • Co-immunoprecipitation to identify FOXRED1's interactions with complex I subunits

  • Western blot analysis of mitochondrial fractions to detect FOXRED1 localization

What functional assays can be used to evaluate the impact of FOXRED1 deficiency?

Several functional assays can be employed to assess the consequences of FOXRED1 deficiency:

• Mitochondrial respiration analysis:

  • Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

  • Determination of OCR/ECAR ratio, which shows a significant decrease in FOXRED1-deficient cells

• Complex I activity assays:

  • Assessment of OXPHOS activity

  • Specific measurement of complex I enzymatic activity

• Cell viability and growth assays:

  • Growth assessment in glucose vs. galactose media (FOXRED1-knockout cells are unable to grow on galactose media)

  • Cell survival under metabolic stress conditions

• Assembly intermediate characterization:

  • Tracking the formation and breakdown of complex I assembly intermediates

  • Temporal analysis of assembly progression in the presence/absence of FOXRED1

What genetic variants in FOXRED1 are associated with mitochondrial complex I deficiency?

Several pathogenic variants in FOXRED1 have been identified in patients with mitochondrial complex I deficiency:

• c.1054C>T (p.R352W):

  • Identified in a homozygous state in a child from a consanguineous Iranian-Jewish pedigree who presented with infantile-onset encephalomyopathy

  • Protein modeling suggested that this mutation could impinge on the FAD-binding site, affecting protein function

• c.733+1G>A:

  • A splicing mutation reported in compound heterozygous state with another variant

  • Leads to complex I deficiency with abnormal mitochondrial respiration

• Other variants:

  • Multiple pathogenic variants have been reported in patients with complex I deficiency

  • All identified pathogenic variants lead to loss of complex I activity and mitochondrial disease

  • Mutations in FOXRED1 are associated with Mitochondrial Complex I Deficiency, Nuclear Type 19 (OMIM #618241)

The genetic findings in all cases described are consistent with an autosomal recessive mode of inheritance .

What is the clinical spectrum associated with FOXRED1 mutations?

FOXRED1 mutations are associated with a range of clinical presentations:

• Infantile-onset encephalomyopathy:

  • Characterized by neurological and muscular symptoms beginning in infancy

  • Often severe clinical presentation

• Mitochondrial complex I deficiency:

  • The most commonly reported mitochondrial disorder presenting in childhood

  • Patients with complex I deficiency due to defects in nuclear genes usually have a severe clinical presentation and are associated with early age of death

• Variable phenotypic severity:

  • Some patients may have residual complex I activity, which may correlate with milder phenotypes

  • Overexpression of certain FOXRED1 mutations can partially rescue complex I assembly, explaining some phenotypic variability

How is FOXRED1 expression linked to cancer progression?

Research has revealed an intriguing relationship between FOXRED1 expression and cancer:

• Colorectal cancer prognosis:

  • FOXRED1 expression is significantly associated with histopathological grading, depth of invasion, lymph node metastasis, distant metastasis, and TNM stage in colorectal cancer (P<0.05 for each)

  • FOXRED1 mainly localizes in the cytoplasm in colorectal cancer tissues

• Survival correlation:

  • Colorectal cancer patients with higher expression of FOXRED1 had higher 3-year survival rates (P=0.003)

  • FOXRED1 has potential to be an independent prognostic factor for survival in colorectal cancer (P=0.04)

  • Low FOXRED1 expression correlates with poor prognosis of colorectal cancer

• Multivariate analysis of prognostic factors:

  Characteristics	Categories	B	SE	Wald	HR	95% CI	P-value
  FOXRED1 expression	High/Low	-1.021	0.497	4.218	0.360	0.136-0.954	0.040
  Differentiation	Well/moderately/poorly	0.659	0.237	7.726	1.933	1.214-3.076	0.005
  Lymph node metastasis	N0/N1/N2/N3	0.725	0.267	7.357	2.065	1.223-3.488	0.007
  Distant metastasis	M0/M1	1.545	0.402	14.808	4.689	2.134-10.302	0.000

  Table adapted from

• Mechanistic links:

  • As an assembly factor of respiratory chain complex I, FOXRED1 is important to its amount and activity

  • Respiratory chain complex I dysfunction is proposed to promote tumorigenesis through ROS alteration and AKT activation

  • Aberrations in mitochondrial complex I activity can enhance the aggressiveness of cancer cells

How can bioinformatic approaches enhance FOXRED1 functional studies?

Several bioinformatic approaches can provide valuable insights into FOXRED1 function:

• Co-expression analysis:

  • Bioinformatics analysis using the mouse GNF1M tissue atlas shows a strong positive correlation between expression of FOXRED1 and expression of known complex I subunits, implying a functional association

  • This correlation is stronger than for all previously identified assembly factors for complex I

• Phylogenetic profiling:

  • Phylogenetic profiling has been used to identify FOXRED1 as a candidate protein for complex I biogenesis

  • FOXRED1 was identified as one of 19 putative complex I assembly factors in a large-scale bioinformatic survey

• Protein modeling and structural analysis:

  • Protein modeling using the Bacillus monomeric sarcosine oxidase structure as a template can predict the impact of mutations on protein function

  • The FOXRED1 protein sequence can be used to build a protein model using tools like SWISS-MODEL

  • Template libraries can be searched with BLAST and HHBlits for evolutionary-related structures matching the target sequence

• Tissue expression analysis:

  • Steady-state mRNA transcripts for FOXRED1 have been detected in 12 human tissues, implying that the protein is widely expressed, in common with all previously identified complex I assembly factors

What experimental challenges are associated with studying FOXRED1 protein interactions?

Researchers face several challenges when investigating FOXRED1 protein interactions:

• Transient nature of interactions:

  • FOXRED1 interacts with complex I intermediates during assembly but may not be present in the mature complex

  • In the absence of FOXRED1, mtDNA-encoded complex I subunits are still translated and transiently assembled into a late-stage ~815 kDa intermediate before breaking down

  • Capturing these transient interactions requires specialized approaches

• Complex experimental systems:

  • Need for mitochondrial isolation to study FOXRED1 in its native environment

  • Requirement for membrane protein analysis techniques due to FOXRED1's association with the inner mitochondrial membrane

  • Specialized electrophoresis techniques like blue native PAGE are needed to preserve protein-protein interactions

• Methodological considerations:

  • Immunoprecipitation studies have shown that FOXRED1 co-immunoprecipitates with various complex I subunits

  • Careful selection of detergents and buffer conditions is required to maintain these interactions

  • Time-course studies may be necessary to capture the dynamic nature of FOXRED1's role in complex I assembly

How can researchers leverage FOXRED1 knowledge for therapeutic development?

Understanding FOXRED1 function opens several avenues for therapeutic development:

• Gene therapy approaches:

  • Lentiviral-mediated FOXRED1 transgene expression has been shown to rescue complex I deficiency in patient fibroblasts

  • This provides proof-of-concept for gene replacement therapy

• Drug discovery targets:

  • Understanding the assembly process of complex I and FOXRED1's role provides potential targets for small molecule intervention

  • Compounds that enhance residual FOXRED1 function might benefit patients with partial loss-of-function mutations

• Cancer therapeutic strategies:

  • Given the relationship between FOXRED1 expression and cancer prognosis, targeting this molecular pathway could be a potential treatment strategy for colorectal cancer

  • Modulation of mitochondrial complex I function through FOXRED1-related pathways might influence cancer progression

• Personalized medicine approaches:

  • Different FOXRED1 mutations may respond differently to therapeutic interventions

  • Overexpression of FOXRED1 containing patient mutations can rescue complex I assembly in some cases, suggesting mutation-specific approaches may be viable

Human Molecular Genetics | Volume 24 | Issue 10 | Pages 2952-2965 | 2015