Recombinant Danio rerio Protein SERAC1 (serac1)

What is the SERAC1 protein and what are its primary functions?

SERAC1 (serine active site containing 1) is a protein whose complete function remains under investigation. Current evidence indicates that SERAC1 plays essential roles in phospholipid remodeling, particularly of phosphatidylglycerol, which serves as a precursor for cardiolipin synthesis. Additionally, SERAC1 facilitates intracellular cholesterol trafficking between cellular compartments . Recent studies have revealed that SERAC1 localizes to the outer mitochondrial membrane and functions as a component of the one-carbon metabolic cycle, interacting with the mitochondrial serine transporter protein SFXN1 to facilitate serine transport from the cytosol to mitochondria .

How is SERAC1 conserved between humans and Danio rerio (zebrafish)?

The SERAC1 protein demonstrates evolutionary conservation across vertebrate species, including between humans and zebrafish. Comparative sequence analysis reveals conserved functional domains, particularly in regions associated with phospholipid remodeling and the serine active site. This conservation makes zebrafish an excellent model organism for studying SERAC1 function, as findings may have translational relevance to human physiology and disease.

What cellular localization pattern does SERAC1 exhibit?

SERAC1 predominantly localizes at the interface between mitochondria and the endoplasmic reticulum, specifically in the mitochondria-associated membrane (MAM) fraction that is essential for phospholipid exchange between these organelles . More recent research has definitively identified SERAC1 as a protein component of the outer mitochondrial membrane, where it interacts with SFXN1 to facilitate serine transport from the cytosol to mitochondria .

How can recombinant Danio rerio SERAC1 be used to study mitochondrial phospholipid remodeling?

Recombinant Danio rerio SERAC1 provides a valuable tool for investigating phospholipid remodeling mechanisms in zebrafish models. Researchers can employ the recombinant protein in reconstitution experiments to assess its direct effects on phosphatidylglycerol species conversion, particularly the transformation between phosphatidylglycerol-34:1 and phosphatidylglycerol-36:1. Experimental approaches should include:

• In vitro phospholipid remodeling assays using purified recombinant SERAC1 with phosphatidylglycerol substrates

• Liposome-based systems to assess membrane integration and function

• Comparative analyses between wild-type and mutant SERAC1 proteins

• Identification of cofactors or binding partners that modulate SERAC1 activity

Analysis of phospholipid profiles before and after SERAC1 treatment can reveal specific substrate preferences and catalytic activities, providing insights into the molecular mechanisms underlying phospholipid remodeling in mitochondrial membranes .

What are the methodological considerations when using recombinant SERAC1 to investigate the one-carbon metabolic cycle?

Recent discoveries have identified SERAC1 as a component of the one-carbon metabolic cycle, specifically involved in mitochondrial serine transport through interaction with SFXN1 . When designing experiments to investigate this function:

• Construct in vitro transport assays using purified recombinant SERAC1 and SFXN1 in liposomal systems

• Measure serine transport rates with radiolabeled serine or fluorescent analogs

• Perform protein-protein interaction studies (co-immunoprecipitation, FRET, proximity ligation) to characterize the SERAC1-SFXN1 interface

• Compare transport efficiency in systems with wild-type versus mutant SERAC1

The experimental setup should include appropriate controls to distinguish direct effects of SERAC1 on serine transport from indirect effects on membrane composition or fluidity. Isotope tracing experiments can further reveal the metabolic fate of transported serine and its contribution to one-carbon metabolism .

How can zebrafish SERAC1 knockout models advance our understanding of MEGDEL syndrome pathophysiology?

MEGDEL syndrome (3-methylglutaconic aciduria with deafness, encephalopathy, and Leigh-like syndrome) is caused by SERAC1 mutations in humans . Zebrafish SERAC1 knockout models offer several advantages for investigating disease mechanisms:

• Optical transparency of zebrafish embryos allows real-time visualization of developmental processes and mitochondrial dynamics

• Relatively rapid development enables assessment of early phenotypes

• Amenability to high-throughput drug screening for potential therapeutic compounds

• Conservation of key metabolic and developmental pathways relevant to MEGDEL syndrome

Researchers should characterize these models at multiple levels:

These models can provide insights into the temporal progression of pathology and identify critical windows for therapeutic intervention .

What are the optimal conditions for expressing and purifying recombinant Danio rerio SERAC1?

Successful expression and purification of recombinant zebrafish SERAC1 requires careful consideration of several parameters:

• Expression system selection: For membrane-associated proteins like SERAC1, eukaryotic expression systems (insect cells, yeast) often yield better results than bacterial systems

• Fusion tags: Consider N-terminal tags (His6, GST, MBP) to enhance solubility while preserving C-terminal membrane interactions

• Buffer optimization: Include phospholipids or mild detergents in purification buffers to maintain native conformation

• Temperature and induction conditions: Lower temperatures (16-20°C) during induction may improve proper folding

A typical purification workflow would involve:

• Affinity chromatography using tag-specific resins

• Size exclusion chromatography to remove aggregates

• Functional validation assays to confirm activity

When troubleshooting expression issues, consider codon optimization for the expression system, using truncated constructs to improve solubility, or co-expression with potential binding partners like SFXN1 .

What methods are most effective for assessing SERAC1 function in phospholipid remodeling experiments?

To investigate SERAC1's role in phospholipid remodeling, researchers should employ multiple complementary approaches:

• Lipidomic analysis using mass spectrometry:

  • Liquid chromatography-mass spectrometry (LC-MS) to quantify specific phospholipid species

  • Monitor ratios of phosphatidylglycerol-34:1 to phosphatidylglycerol-36:1, as these are specifically altered in SERAC1 deficiency

• In vitro enzymatic assays:

  • Design assays with defined phospholipid substrates

  • Include appropriate cofactors (potential metal ions, ATP)

  • Monitor product formation over time using HPLC or TLC

• Cellular models:

  • Compare SERAC1 knockout vs. wild-type cells

  • Complement with wild-type or mutant SERAC1 expression

  • Assess mitochondrial membrane composition and function

• Visualization techniques:

  • Use fluorescently-labeled lipids to track metabolism

  • Employ filipin staining to assess cholesterol distribution

When interpreting results, researchers should consider that SERAC1 may function as part of a larger complex, and activities observed in purified systems may differ from those in cellular contexts.

How can zebrafish SERAC1 models inform therapeutic strategies for MEGDEL syndrome?

Recent research with Serac1-/- mice has demonstrated that nucleoside/nucleotide supplementation can restore mtDNA content and mitochondrial function . This finding suggests potential therapeutic avenues for MEGDEL syndrome that can be further investigated using zebrafish models:

• Nucleoside/nucleotide supplementation trials:

  • Determine optimal formulations and dosing regimens

  • Assess developmental stage-specific responses

  • Monitor biochemical and phenotypic outcomes

• One-carbon metabolism modulators:

  • Test serine supplementation strategies

  • Evaluate folate pathway interventions

  • Investigate methyl donor supplementation

• Phospholipid-based therapies:

  • Explore cardiolipin or precursor supplementation

  • Test compounds that stabilize existing cardiolipin

• Gene therapy approaches:

  • Develop zebrafish models with specific SERAC1 mutations

  • Test gene replacement strategies using various delivery vectors

The transparency and rapid development of zebrafish make them particularly suitable for evaluating treatment efficacy on organogenesis and nervous system development in real-time .

What biochemical markers should be monitored when studying SERAC1 function in zebrafish models?

When investigating SERAC1 function or screening potential therapeutic interventions, researchers should monitor multiple biochemical parameters:

These markers provide a comprehensive assessment of SERAC1's multiple functions and can serve as outcome measures in therapeutic studies.

What are the unexplored aspects of SERAC1 function in zebrafish development?

Despite recent advances in understanding SERAC1 function, several important questions remain unexplored:

• Developmental expression patterns of SERAC1 in zebrafish:

  • Temporal and spatial expression during embryogenesis

  • Tissue-specific functions during organogenesis

  • Regulation of expression under different metabolic conditions

• Tissue-specific effects of SERAC1 deficiency:

  • Why certain tissues (brain, inner ear, liver) are particularly affected

  • Compensatory mechanisms in less affected tissues

  • Metabolic adaptations to SERAC1 deficiency

• Interaction partners beyond SFXN1:

  • Comprehensive interactome analysis in different cellular compartments

  • Dynamic changes in protein interactions during development

  • Tissue-specific protein complexes

• Evolutionary adaptations of SERAC1 function:

  • Species-specific differences in SERAC1 activity

  • Compensatory pathways in different vertebrate lineages

These investigations could reveal additional functions of SERAC1 beyond its established roles in phospholipid remodeling and serine transport .

How might CRISPR-Cas9 gene editing advance zebrafish SERAC1 research?

CRISPR-Cas9 technology offers powerful approaches to investigate SERAC1 function in zebrafish:

• Generation of precise disease-mimicking mutations:

  • Engineer specific mutations identified in MEGDEL patients

  • Create allelic series with varying degrees of functional impairment

  • Introduce tagged versions of SERAC1 at endogenous loci

• Domain-specific functional analysis:

  • Target specific functional domains to dissect their roles

  • Create conditional alleles for temporal control of gene disruption

  • Perform structure-function analyses through targeted mutations

• High-throughput screening:

  • Create reporter lines for monitoring SERAC1-dependent processes

  • Develop zebrafish models for drug screening

  • Perform genetic modifier screens

• Cell-type specific studies:

  • Use tissue-specific promoters to drive Cas9 expression

  • Investigate cell-autonomous vs. non-cell-autonomous effects

  • Study tissue-specific rescue

These approaches can provide unprecedented insights into SERAC1 biology and potentially identify new therapeutic targets for MEGDEL syndrome .