Recombinant Pongo abelii Fatty acyl-CoA reductase 1 (FAR1)

What is Fatty acyl-CoA reductase 1 (FAR1) from Pongo abelii and what is its primary function?

Fatty acyl-CoA reductase 1 from Pongo abelii (Sumatran orangutan) is an essential enzyme involved in the reduction of fatty acids to fatty alcohols. This enzymatic conversion represents a fundamental step in lipid metabolism across various species, including primates. The protein functions as a key component in the biosynthetic pathway of plasmalogens and other ether-containing lipids that play vital roles in cellular membrane structure and function. This NADPH-dependent process is essential for the proper formation of ether lipids, particularly plasmalogens, which are abundant in neural tissues, cardiac muscle, and immune cells.

What is the molecular structure of Pongo abelii FAR1?

The Pongo abelii FAR1 protein is cataloged in the UniProt database under accession number Q5R834. The full-length recombinant protein consists of 515 amino acids with the complete amino acid sequence beginning with "MVSIPEYYEGK" and ending with "FRASSTMRY". The protein exhibits the characteristic domains of the FAR family, including a large catalytic domain responsible for its enzymatic activity and a C-terminal region involved in membrane association. This primary structure is crucial for structure-function analyses and comparative studies with homologs from other species.

What are the recommended methods for studying FAR1 localization in cells?

To study FAR1 localization, immunofluorescence microscopy using specific antibodies against FAR1 is highly effective. Based on research findings, FAR1 is predominantly localized to peroxisomes . For experimental verification, researchers should consider:

• Co-localization studies with established peroxisomal markers

• Confocal microscopy to precisely determine subcellular localization

• FLAG-tagged FAR1 expression systems for detecting both recombinant and endogenous FAR1

As demonstrated in supplemental Figure 4A from research on FAR1, both recombinant FLAG-Far1 and endogenous Far1 can be detected in peroxisomes using these approaches .

How can researchers effectively measure FAR1 enzymatic activity?

For measuring FAR1 enzymatic activity, researchers should implement the following methodology:

• Prepare membrane fractions from cells expressing recombinant FAR1

• Conduct enzyme assays using radiolabeled or fluorescently-labeled fatty acyl-CoA substrates

• Measure fatty alcohol production via thin-layer chromatography, HPLC, or gas chromatography-mass spectrometry

• Include NADPH as an essential cofactor in reaction mixtures

• Optimize reaction conditions (pH, temperature, ionic strength) to ensure maximum enzyme activity

When establishing controls, include samples with heat-inactivated enzyme and reactions without NADPH to confirm the specificity of the enzymatic reaction.

How is FAR1 activity regulated at the post-translational level?

Research evidence indicates that FAR1 activity is primarily regulated at the post-translational level rather than through transcriptional control. Semiquantitative reverse transcription-PCR analysis has shown that restoration of normal plasmalogen levels in ADAPS-deficient fibroblasts and ZPEG251 cells did not alter cellular levels of FAR1 mRNA . This suggests that regulation occurs after protein synthesis.

The post-translational regulation mechanisms include:

• Protein stability control - FAR1 protein turnover is accelerated in the presence of plasmalogens

• Subcellular targeting - Newly synthesized FAR1 is efficiently transported to peroxisomes

• Protein degradation - FAR1 is subsequently degraded in cells replete with plasmalogens

Experimental evidence shows that FAR1 is stable when cells are cultured in the absence of plasmalogens but is largely degraded when cells are supplemented with hexadecylglycerol (HG), indicating that the rate of FAR1 protein turnover is accelerated in the presence of plasmalogens .

What experimental approaches can be used to study FAR1 protein turnover?

To effectively study FAR1 protein turnover, researchers should consider the following methodological approach:

• Establish cell lines with stable expression of tagged FAR1 (e.g., FLAG-FAR1)

• Culture cells under varying conditions (e.g., with/without plasmalogen precursors)

• Perform cycloheximide chase experiments to block new protein synthesis

• Collect samples at various time points (e.g., 0, 6, 9 hours)

• Analyze FAR1 protein levels via Western blotting with appropriate antibodies

• Quantify protein bands for degradation rate calculation

This approach, as demonstrated in previous research, can effectively reveal how cellular conditions affect FAR1 protein stability. For instance, researchers observed that "a large portion of Far1 was degraded in cells supplemented with 20 μm HG," indicating accelerated protein turnover in the presence of plasmalogens .

How does Pongo abelii FAR1 compare to FAR1 homologs in other primate species?

When conducting comparative analyses of FAR1 across primate species, researchers should focus on:

• Sequence homology analysis using multiple sequence alignment tools

• Phylogenetic tree construction to determine evolutionary relationships

• Comparison of key functional domains, particularly the catalytic region

• Identification of conserved residues that may be essential for enzymatic activity

• Analysis of species-specific variations that might reflect adaptive evolution

Understanding these similarities and differences provides insights into the evolutionary conservation of lipid metabolism pathways. The Sumatran orangutan (Pongo abelii) represents an important model for studying primate evolution, as it is one of three orangutan species and is critically endangered . Its classification within the Hominidae family makes it particularly valuable for comparative studies with human FAR1.

How should researchers address contradictory findings related to FAR1 function or regulation?

When facing contradictory findings in FAR1 research, implement the following methodological framework:

• Identify the type of contradiction present in the literature or experimental data:

  • Self-contradictory findings within a single study

  • Contradicting results between different studies

  • Conditional contradictions involving multiple interconnected factors

• Evaluate the quality and reliability of each data source using:

  • Methodological rigor assessment

  • Sample size and statistical power analysis

  • Reproducibility across independent laboratories

  • Technical variations in experimental conditions

• Design experiments to directly test contradictory hypotheses:

  • Use multiple complementary techniques to measure the same parameter

  • Include appropriate positive and negative controls

  • Vary experimental conditions systematically to identify context-dependent effects

  • Consider conditional factors that might reconcile apparently contradictory findings

Research on contradiction detection indicates that sophisticated analytical approaches, particularly when enhanced with Chain of Thought prompting, can effectively identify contradictions regardless of document proximity . This suggests that systematic review methodologies can help reconcile apparently contradictory findings in the scientific literature.

What are the established and potential applications of recombinant Pongo abelii FAR1 in research?

Recombinant Pongo abelii FAR1 has various applications in immunological and biochemical research. The protein can be utilized in:

• Enzyme-linked immunosorbent assays (ELISA) for studying antibody responses against conserved enzymes in lipid metabolism

• Structure-function analyses to understand the catalytic mechanism of fatty acid reduction

• Comparative biochemistry studies examining evolutionary adaptations in lipid metabolism among primates

• Investigation of plasmalogen biosynthesis pathways and their role in membrane biology

• Development of in vitro systems to study ether lipid production

Additionally, understanding FAR1 function provides valuable insights into lipid metabolism disorders and potential therapeutic targets for diseases involving aberrant lipid processing.

What methodological approaches are recommended for studying the relationship between FAR1 activity and plasmalogen biosynthesis?

To investigate the relationship between FAR1 activity and plasmalogen biosynthesis, researchers should implement the following methodological framework:

• Cell Model Systems:

  • Use cell lines with deficiencies in plasmalogen synthesis (e.g., ZPEG251 cells)

  • Establish stable transfection systems expressing wild-type or mutant FAR1

  • Create knockdown/knockout models using siRNA or CRISPR-Cas9 technology

• Analytical Techniques:

  • Lipidomic analysis using mass spectrometry to quantify plasmalogens and precursors

  • Enzymatic assays measuring FAR1 activity under various conditions

  • Metabolic labeling with isotope-tagged precursors to track flux through the pathway

• Experimental Manipulations:

  • Supplement cultures with plasmalogen precursors (e.g., hexadecylglycerol)

  • Modulate FAR1 expression levels through inducible expression systems

  • Apply inhibitors of specific steps in the plasmalogen biosynthetic pathway

• Data Analysis:

  • Correlate FAR1 protein levels with plasmalogen content

  • Analyze enzyme kinetics in relation to substrate availability

  • Model the relationship between FAR1 activity and end-product accumulation

Research has demonstrated that FAR1 protein turnover is accelerated in cells replete with plasmalogens, suggesting a feedback regulatory mechanism . This relationship can be further explored using the above methodological approaches.

What are the key considerations when designing experiments involving recombinant Pongo abelii FAR1?

When designing experiments with recombinant Pongo abelii FAR1, researchers should consider:

• Expression System Selection:

  • Prokaryotic systems (E. coli) for high yield but potential issues with folding

  • Eukaryotic systems (yeast, insect, mammalian cells) for proper post-translational modifications

  • Cell-free systems for rapid production avoiding cellular regulatory mechanisms

• Purification Strategy:

  • Affinity tags selection (His, FLAG, GST) based on downstream applications

  • Purification conditions that maintain enzyme stability and activity

  • Quality control assessments (SDS-PAGE, Western blot, mass spectrometry)

• Experimental Controls:

  • Enzymatically inactive mutants as negative controls

  • Known FAR family enzymes for comparative analyses

  • Substrate specificity controls to validate functional activity

• Methodological Approach Selection:

  • Qualitative vs. quantitative methods based on research question

  • Primary vs. secondary research approaches

  • Descriptive vs. experimental designs

• Data Analysis Strategies:

  • Statistical methods appropriate for quantitative data

  • Thematic analysis for qualitative observations

  • Mixed methods approach when appropriate

Proper experimental design is integral to generating reliable and reproducible data on FAR1 function and regulation.

What are the current gaps in our understanding of Pongo abelii FAR1 and future research directions?

Despite advances in understanding Pongo abelii FAR1, several knowledge gaps remain that warrant further investigation:

• Structural Biology:

  • High-resolution crystal structure determination

  • Structure-function relationships between protein domains

  • Substrate binding mechanisms and catalytic site architecture

• Regulatory Networks:

  • Comprehensive characterization of post-translational modifications

  • Identification of protein interaction partners

  • Elucidation of cellular signaling pathways affecting FAR1 activity

• Comparative Biology:

  • Detailed comparison with FAR1 from other primate species

  • Evolutionary analysis of substrate specificity changes

  • Functional adaptation of FAR1 in different tissues and species

• Physiological Significance:

  • Tissue-specific roles in lipid metabolism

  • Response to metabolic stress conditions

  • Potential involvement in disease processes