Recombinant Human Protein SERAC1 (SERAC1)

What is the structure and cellular localization of SERAC1?

SERAC1 is a 654 amino acid protein with a conserved serine-lipase domain (containing the consensus lipase motif GxSxG) and belongs to the PGAP-like protein domain family (PFAM PF07819). The protein primarily localizes to the interface between the endoplasmic reticulum and mitochondria, which is crucial for its function in lipid metabolism and transport . Research has demonstrated that SERAC1 contains 17 exons spanning approximately 59 kb on chromosome 6q25.3 .

To study SERAC1 localization, immunofluorescence microscopy with organelle-specific markers is the preferred methodology. Co-localization experiments using antibodies against mitochondrial and endoplasmic reticulum markers together with anti-SERAC1 antibodies can provide precise subcellular localization information. For protein structure studies, recombinant expression followed by structural analysis techniques including X-ray crystallography or cryo-electron microscopy would be appropriate methodological approaches.

What are the primary molecular functions of SERAC1?

SERAC1 serves multiple critical functions in cellular metabolism:

To investigate these functions, researchers typically employ gene knockout/knockdown approaches followed by metabolite profiling, lipidomics analysis focusing on phosphatidylglycerol species ratios, and interaction studies using co-immunoprecipitation or proximity ligation assays.

Disease Models and Clinical Relevance

Several models have been developed to study SERAC1 deficiency:

• Knockout mouse models: Serac1−/− mice demonstrate phenotypes that mimic the major diagnostic clinical and biochemical features of MEGD(H)EL syndrome .

• Cell culture models:

  • HEK293T cells with SERAC1 depletion

  • Patient-derived immortalized lymphocyte cells

  • Fibroblast models from affected individuals

Methodology for developing these models typically involves CRISPR/Cas9-mediated gene editing for cellular models and traditional knockout approaches for animal models. When establishing new models, researchers should consider:

• Confirmation of SERAC1 depletion at mRNA and protein levels

• Assessment of mitochondrial DNA content

• Evaluation of phosphatidylglycerol profiles, particularly PG34:1/PG36:1 ratios

• Functional mitochondrial assays (respiration, membrane potential)

• One-carbon metabolism analysis

How does SERAC1 deficiency lead to mitochondrial dysfunction?

The mechanism linking SERAC1 deficiency to mitochondrial dysfunction involves multiple interconnected pathways:

To study these mechanisms, researchers can employ metabolic flux analysis using isotope-labeled serine, comprehensive mitochondrial functional assays (oxygen consumption measurement, ATP production), mtDNA quantification through qPCR, and phospholipid profiling using mass spectrometry.

What therapeutic approaches show promise for SERAC1-related disorders?

Current research has identified several potential therapeutic strategies:

• Nucleoside/nucleotide supplementation: Both in vitro and in vivo studies have demonstrated that supplementation with nucleosides or nucleotides can restore mtDNA content and mitochondrial function in SERAC1-deficient models . This approach addresses the downstream effects of one-carbon cycle disruption.

• Symptomatic treatments: For clinical manifestations, treatments focused on managing spasticity and drooling have shown effectiveness in affected individuals .

• Hearing interventions: Hearing aids and cochlear implants have been utilized, though they generally have not significantly improved communication skills in severely affected patients .

Experimental approaches for studying therapeutic efficacy include:

• Measurement of mtDNA content before and after treatment

• Assessment of mitochondrial respiratory function

• Evaluation of clinical parameters in animal models

• Monitoring of biochemical markers (3-methylglutaconic acid levels)

• Analysis of phosphatidylglycerol profiles

How can researchers effectively measure and analyze phosphatidylglycerol remodeling in SERAC1 studies?

Phosphatidylglycerol remodeling analysis is central to SERAC1 research since the protein specifically catalyzes the conversion of PG-34:1 to PG-36:1. The most effective methodological approach includes:

• Lipidomic analysis: Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) provides the most accurate quantification of different phosphatidylglycerol species.

• PG34:1/PG36:1 ratio calculation: This ratio serves as a biomarker for SERAC1 function, with increased ratios indicating SERAC1 dysfunction .

• Tissue-specific analysis: Different tissues may show varying degrees of phosphatidylglycerol abnormalities, so comparative analysis across tissues can provide insights into differential effects.

• Time-course studies: Monitoring changes in phosphatidylglycerol species over time, particularly in response to interventions, can help establish causality in pathogenic mechanisms.

• Correlation with functional outcomes: Linking phosphatidylglycerol profiles to mitochondrial function, mtDNA content, and clinical parameters helps establish the relevance of these lipid changes.

When interpreting results, researchers should consider that the degree of phosphatidylglycerol abnormalities appears to correlate with phenotypic severity, with milder changes observed in less severe juvenile-onset cases compared to infantile-onset MEGD(H)EL cases .

What are the unresolved questions regarding SERAC1's role in the one-carbon cycle?

While SERAC1 has been identified as a component of the one-carbon cycle, several aspects remain incompletely understood:

• The precise biochemical step(s) at which SERAC1 acts within the one-carbon cycle pathway requires further elucidation.

• The relationship between SERAC1's lipid remodeling function and its role in one-carbon metabolism presents a conceptual challenge. Researchers should design experiments that can distinguish between:

  • Direct involvement in one-carbon metabolism reactions

  • Indirect effects through altered membrane composition affecting enzyme localization or function

  • Regulatory roles affecting expression or activity of other one-carbon cycle components

• The cell type-specific requirements for SERAC1 in one-carbon metabolism have not been fully mapped.

Methodological approaches to address these questions include:

• In vitro reconstitution of one-carbon cycle reactions with purified components

• Metabolic flux analysis using isotope-labeled precursors

• Comparative analysis across different cell types

• Structural studies to identify potential catalytic or regulatory domains beyond the known lipase motif

How do we reconcile the varying clinical phenotypes associated with different SERAC1 mutations?

The clinical spectrum of SERAC1 mutations ranges from severe infantile-onset MEGD(H)EL syndrome to milder juvenile-onset cHSP. This phenotypic variability raises important research questions:

• Genotype-phenotype correlation: No clear relationship between specific SERAC1 variants and clinical phenotypes has been established . Research should focus on systematically cataloging variants and associated phenotypes.

• Modifier genes: The influence of genetic background and modifier genes on phenotypic expression requires investigation through whole-genome sequencing approaches and comparative studies across families.

• Biochemical thresholds: The hypothesis that milder biochemical abnormalities (e.g., less severely altered PG34:1/PG36:1 ratios) lead to milder phenotypes requires validation across larger cohorts .

• Tissue-specific effects: The differential vulnerability of various tissues to SERAC1 dysfunction remains poorly understood. Single-cell approaches and tissue-specific conditional knockout models may help address this question.

Methodological approaches should include:

• Comprehensive phenotyping of large patient cohorts

• Detailed biochemical profiling correlated with clinical severity

• Functional characterization of variant SERAC1 proteins

• Development of tissue-specific knockout models

• Rescue experiments with different SERAC1 variants

What are the key considerations for developing a SERAC1 overexpression system for structure-function studies?

When designing systems for SERAC1 overexpression:

• Expression vector selection:

  • Mammalian vectors are preferred for maintaining native post-translational modifications

  • Inclusion of epitope tags (e.g., FLAG, His) should be carefully positioned to avoid interfering with the serine-lipase domain or membrane localization signals

• Cell line considerations:

  • HEK293T cells have been successfully used in previous SERAC1 studies

  • Consider SERAC1-null backgrounds to avoid confounding effects of endogenous protein

• Functional domain analysis:

  • Generate truncation constructs to map domains responsible for:

    • SFXN1 interaction

    • Phosphatidylglycerol remodeling

    • Mitochondrial localization

  • Site-directed mutagenesis of the lipase motif (GxSxG) to confirm catalytic function

• Interaction studies:

  • Co-immunoprecipitation with SFXN1 and other potential partners

  • Proximity labeling techniques to identify the SERAC1 interactome

  • Membrane fraction enrichment to study protein complexes

• Functional readouts:

  • PG34:1/PG36:1 ratio measurement

  • Serine transport assays

  • One-carbon cycle flux analysis

  • mtDNA content assessment

What approaches can address the technical challenges in studying SERAC1 in primary patient samples?

Working with patient samples presents specific challenges that require methodological considerations:

• Limited material availability:

  • Develop micro-scale assays requiring minimal tissue

  • Establish immortalized cell lines from patient samples

  • Consider reprogramming to iPSCs followed by directed differentiation to relevant cell types

• Tissue heterogeneity:

  • Single-cell approaches to account for cellular heterogeneity

  • Cell sorting to enrich for specific populations

  • Laser capture microdissection for tissue-specific analysis

• Biomarker validation:

  • 3-methylglutaconic acid detection in urine is highly sensitive (present in 98% of cases)

  • PG34:1/PG36:1 ratio measurement requires specialized lipidomic facilities

  • Development of simplified assays suitable for clinical laboratories

• Functional assays:

  • Mitochondrial respiration in permeabilized cells

  • Seahorse analysis for bioenergetic profiling

  • Serine transport measurement using radioisotope-labeled serine

• Data integration:

  • Correlation of biochemical, genetic, and clinical data

  • Longitudinal sampling to capture disease progression

  • Comparison with other mitochondrial disorders to identify common and distinct features