Recombinant Bovine Protein SERAC1 (SERAC1)

What is the structure and function of SERAC1 protein?

SERAC1 (serine active site domain-containing protein 1) is a 654-amino acid protein containing a conserved serine-lipase domain with a consensus lipase motif GxSxG. It belongs to the PGAP-like protein domain family (PFAM PF07819) . The protein localizes to the outer mitochondrial membrane and functions at the interface between the endoplasmic reticulum and mitochondria .

Functionally, SERAC1:

• Participates in phospholipid remodeling, particularly converting phosphatidylglycerol-34:1 (PG-34:1) to phosphatidylglycerol-36:1 (PG-36:1)

• Facilitates intracellular cholesterol trafficking

• Acts as a component of the mitochondrial serine transporter system by interacting with SFXN1 to transport serine from cytosol to mitochondria

• Participates in the one-carbon metabolism cycle essential for nucleotide synthesis

What experimental models are available for studying SERAC1 function?

Several experimental models have been documented in the literature for SERAC1 research:

• Cellular Models:

  • HEK293T cell lines with SERAC1 knockdown or knockout

  • Patient-derived immortalized lymphocyte cells harboring SERAC1 mutations

• Animal Models:

  • Serac1^(-/-) knockout mice that recapitulate major clinical and biochemical phenotypes of MEGDEL syndrome

• In vitro Systems:

  • Recombinant protein expression systems for structural and functional studies

  • Mitochondrial assays to study SERAC1's role in phospholipid remodeling

When selecting an experimental model, researchers should consider the specific aspect of SERAC1 function they wish to investigate. For mitochondrial function studies, both cellular and animal models provide valuable platforms for investigating physiological impacts of SERAC1 dysfunction.

What methodological approaches can detect SERAC1 protein-protein interactions?

Several methodological approaches can be employed to study SERAC1 protein interactions:

• Co-immunoprecipitation (Co-IP): This approach has been utilized to demonstrate SERAC1's interaction with SFXN1, the mitochondrial serine transporter protein .

• Proximity Labeling Methods: BioID or APEX2 proximity labeling can identify proteins in close proximity to SERAC1 within the mitochondrial outer membrane environment.

• Fluorescence Resonance Energy Transfer (FRET): This technique can confirm direct protein-protein interactions in living cells.

• Yeast Two-Hybrid Screening: Although not specifically mentioned in the provided materials, this approach could identify novel interaction partners.

• Pull-down Assays with Recombinant Proteins: Using purified recombinant SERAC1 to identify direct binding partners from cellular lysates.

For bovine SERAC1 studies, these methods would need to be optimized using species-specific antibodies or recombinant proteins to ensure accurate detection of interaction partners.

How does SERAC1 contribute to the one-carbon metabolism cycle and how can this pathway be experimentally monitored?

SERAC1 contributes to the one-carbon metabolism cycle by facilitating serine transport into mitochondria through interaction with the SFXN1 transporter . This process is crucial for maintaining proper nucleotide pools within mitochondria.

Experimental monitoring approaches:

• Metabolite Analysis:

  • Liquid chromatography-mass spectrometry (LC-MS) to quantify one-carbon cycle intermediates

  • Monitor ratios of PG-34:1 to PG-36:1 as indicators of SERAC1 activity

  • Measure nucleotide levels in mitochondrial and cytosolic fractions

• Isotope Tracing:

  • Use ^13^C-labeled serine to track carbon flux through the one-carbon cycle

  • Analyze incorporation of labeled carbon into nucleotides and other downstream metabolites

• Functional Assays:

  • Serine transport assays using radioisotope-labeled serine to measure SERAC1-dependent mitochondrial serine import

  • Rescue experiments using nucleotide supplementation to verify the functional consequence of one-carbon cycle disruption

Research has demonstrated that loss of SERAC1 impairs the one-carbon cycle and disrupts the nucleotide pool balance, leading to mitochondrial DNA depletion. Importantly, both in vitro and in vivo supplementation of nucleosides/nucleotides can restore mitochondrial DNA content and function in SERAC1-deficient systems .

What methodological challenges exist in analyzing the effects of SERAC1 mutations, and how can researchers address them?

Methodological Challenges and Solutions:

• Phenotypic Variability:

  • Challenge: SERAC1 mutations can present with a spectrum of phenotypes ranging from severe MEGDEL syndrome to milder complicated hereditary spastic paraplegia (cHSP) .

  • Solution: Develop standardized phenotyping criteria and use multi-parameter assessment to capture the full range of effects. Implement matched controls and consider age-dependent effects in experimental design.

• Genotype-Phenotype Correlation:

  • Challenge: No clear relationship between specific SERAC1 variants and phenotypes has been established .

  • Solution: Employ comprehensive mutation analysis coupled with functional assays to classify variants. Use CRISPR-Cas9 to introduce specific mutations in cellular or animal models for direct comparison.

• Functional Assessment:

  • Challenge: Determining the functional impact of missense variants in the serine-lipase domain versus frameshift mutations.

  • Solution: Develop in vitro enzyme activity assays to measure lipase function. For novel variants, use predictive software in combination with conservation analysis:

  Analysis Tool	Result for Missense Variant	Interpretation
  PolyPhen-2	Probably Damaging	Likely pathogenic
  SIFT	Deleterious	Likely pathogenic
  MutationTaster	Disease Causing	Likely pathogenic
  Conservation	Highly conserved across species	Functionally important

• Biochemical Markers:

  • Challenge: Validating biochemical indicators of SERAC1 dysfunction.

  • Solution: Monitor 3-methylglutaconic acid levels, lactate levels, phosphatidylglycerol ratios, and mitochondrial DNA content as quantitative biomarkers .

How can researchers design experiments to investigate SERAC1's dual role in phospholipid remodeling and cholesterol trafficking?

Experimental Design Strategies:

• Subcellular Fractionation and Imaging:

  • Separate mitochondria-associated membranes (MAMs) where SERAC1 functions at the ER-mitochondria interface

  • Use super-resolution microscopy with fluorescently tagged SERAC1 to visualize its dynamic localization

• Lipid Profiling:

  • Employ lipidomics to quantitatively assess changes in phospholipid composition, particularly phosphatidylglycerol species

  • Compare PG-34:1/PG-36:1 ratios in wild-type versus SERAC1-deficient systems

  • Monitor cardiolipin synthesis and remodeling, as cardiolipin is derived from phosphatidylglycerol

• Cholesterol Transport Assays:

  • Use fluorescent cholesterol analogs to track intracellular movement

  • Measure cholesterol distribution in cellular compartments using filipin staining

  • Assess effects of SERAC1 mutations on cholesterol-dependent cellular processes

• Domain-Specific Mutagenesis:

  • Create targeted mutations in different functional domains of SERAC1:

    • Serine-lipase domain (amino acids ~300-500) for phospholipid remodeling

    • Other domains potentially involved in protein-protein interactions or cholesterol binding

  • Express these mutants in SERAC1-deficient cells to assess rescue of specific functions

• Biochemical Reconstitution:

  • Purify recombinant SERAC1 protein (wild-type and mutant versions)

  • Reconstitute with artificial membranes to directly measure phospholipid remodeling activity

  • Test cholesterol transfer between membrane vesicles in vitro

What approaches can be used to study the tissue-specific effects of SERAC1 deficiency?

SERAC1 deficiency manifests with tissue-specific effects, particularly affecting the nervous system, hearing, liver, and mitochondria-rich tissues . To investigate these tissue-specific manifestations:

• Tissue-Specific Conditional Knockout Models:

  • Generate tissue-specific Cre-loxP conditional knockout mice to eliminate SERAC1 in specific tissues (brain, liver, cochlea)

  • Compare phenotypes across different tissue-specific knockouts to isolate primary versus secondary effects

• Cell-Type Specific Analyses:

  • Derive different cell types (neurons, hepatocytes, cochlear hair cells) from patient iPSCs or through directed differentiation of SERAC1-knockout stem cells

  • Compare mitochondrial function, phospholipid composition, and cellular responses across cell types

• Multi-Omics Integration:

  • Apply transcriptomics, proteomics, and metabolomics to tissues from SERAC1-deficient models

  • Identify tissue-specific pathways affected by SERAC1 dysfunction

  • Data integration table example:

  Tissue	Transcriptomic Changes	Proteomic Changes	Metabolomic Changes	Functional Impact
  Brain	↓ mitochondrial genes	↓ respiratory chain complexes	↑ lactate, ↓ ATP	Energy deficit, neurodegeneration
  Liver	↑ stress response genes	↓ metabolic enzymes	Altered lipid profiles	Hepatic dysfunction
  Cochlea	↓ ion transport genes	↓ membrane proteins	Altered phospholipids	Hearing loss

• Ex Vivo Tissue Explants:

  • Culture tissue explants from SERAC1-deficient models

  • Test tissue-specific responses to metabolic stressors

  • Evaluate tissue-specific rescue with nucleotide supplementation or gene therapy approaches

What PCR and sequencing protocols are optimal for detecting SERAC1 mutations?

Based on published protocols, the following methodological approach is recommended for SERAC1 mutation detection:

DNA Isolation:

• Extract DNA from whole blood using QIAamp DNA Blood Mini kit or equivalent

• Alternatively, use saliva or skin fibroblasts as DNA sources

PCR Amplification of SERAC1 Gene:

• Design primers spanning all 17 exons and flanking intronic regions

• For exon 10, which contains several reported pathogenic variants, use:

  • Forward primer: 5'-TCCAACCAAGAGCTAAGCAG-3'

  • Reverse primer: 5'-TGAACATATCATGAGGGGTAGAG-3'

• PCR conditions:

  • Initial denaturation: 94°C for 3 minutes

  • 30 cycles of: 94°C for 30 seconds, 55°C for 45 seconds, 72°C for 2 minutes

  • Final extension: 72°C for 10 minutes

• Use high-fidelity DNA polymerase for accurate amplification

Sequencing Methods:

• Sanger Sequencing:

  • Purify PCR products and perform cycle sequencing using BigDye Terminator v3.1

  • Analyze on automated DNA sequencer (e.g., ABI 3130)

• Next-Generation Sequencing:

  • Whole Exome Sequencing (WES) for comprehensive variant detection

  • Target enrichment of SERAC1 and related genes

  • Minimum coverage of 30× recommended for reliable variant calling

Variant Analysis:

• Use bioinformatics software (e.g., Alamut Visual) to predict pathogenicity

• Cross-reference variants with databases: ClinVar, ExAC, ESP

• Apply ACMG guidelines for variant classification

How can researchers effectively design and validate CRISPR-Cas9 knockout models for SERAC1 functional studies?

CRISPR-Cas9 Strategy for SERAC1 Knockout:

• Guide RNA Design:

  • Target early exons (exons 1-5) or the serine-lipase domain (located within exons 10-14)

  • Design multiple gRNAs using tools like CHOPCHOP, CRISPOR, or Benchling

  • Select gRNAs with high on-target and low off-target scores

  • Example targets: conserved catalytic residues in the serine-lipase domain

• Delivery Methods:

  • For cell lines: lipofection, electroporation, or lentiviral vectors

  • For animal models: embryo microinjection or electroporation

• Validation of Knockout Efficiency:

  Validation Method	Technique	Expected Outcome
  Genomic Validation	PCR + Sanger sequencing	Confirm indel mutations at target site
  mRNA Expression	RT-qPCR	Reduced/absent SERAC1 transcript
  Protein Detection	Western blot	Absence of SERAC1 protein
  Functional Validation	Phospholipid ratio analysis	Increased PG-34:1/PG-36:1 ratio
  Phenotypic Confirmation	mtDNA quantification	Reduced mtDNA content

• Controls:

  • Include wild-type controls

  • Generate heterozygous models as intermediate phenotype controls

  • Create rescue models by reintroducing wild-type SERAC1

• Phenotypic Characterization:

  • Assess mitochondrial function (oxygen consumption, membrane potential)

  • Measure one-carbon cycle metabolites

  • Quantify nucleotide pools

  • Evaluate phospholipid remodeling

  • Test rescue with nucleoside/nucleotide supplementation

What are the recommended protocols for investigating mitochondrial function in SERAC1-deficient models?

Research has established that SERAC1 deficiency impairs mitochondrial function through disruption of phospholipid remodeling and nucleotide supply . The following protocols are recommended:

Mitochondrial Respiration Analysis:

• Oxygen consumption measurement using Seahorse XF Analyzer

• Parameters to assess:

  • Basal respiration

  • ATP-linked respiration

  • Maximal respiratory capacity

  • Spare respiratory capacity

  • Proton leak

Mitochondrial DNA Content Analysis:

• Quantitative PCR comparing mitochondrial to nuclear DNA ratio

• Primers targeting conserved mtDNA regions and single-copy nuclear genes

• Calculate relative mtDNA content using the 2^-ΔΔCt^ method

Mitochondrial Membrane Potential:

• JC-1 or TMRM staining followed by flow cytometry or confocal microscopy

• Analyze distribution patterns in different cellular compartments

Mitochondrial Morphology:

• Live-cell imaging using MitoTracker dyes

• Transmission electron microscopy for ultrastructural analysis

• Quantify parameters like mitochondrial number, size, and cristae density

Cardiolipin Analysis:

• Lipidomics approach using LC-MS/MS

• Analyze cardiolipin species profile and content

• Compare phosphatidylglycerol species ratios (PG-34:1/PG-36:1)

One-Carbon Metabolism Assessment:

• Measure serine transport into isolated mitochondria

• Analyze flux through one-carbon cycle using isotope-labeled precursors

• Quantify nucleotide pools in mitochondrial and cytosolic fractions

Rescue Experiments:

• Nucleoside/nucleotide supplementation:

  • dNTP mix (dATP, dGTP, dCTP, dTTP) at 50-200 μM

  • Assess restoration of mtDNA content and mitochondrial function

• Gene complementation with wild-type SERAC1

What therapeutic approaches are being investigated for SERAC1-related disorders, and how can their efficacy be assessed?

Recent research has identified potential therapeutic strategies for SERAC1-related disorders, with nucleotide supplementation showing promising results:

Current Therapeutic Approaches:

• Nucleoside/Nucleotide Supplementation:

  • Both in vitro and in vivo studies have demonstrated that nucleoside/nucleotide supplementation can restore mtDNA content and mitochondrial function in SERAC1-deficient systems

  • This approach addresses the underlying mtDNA depletion caused by impaired one-carbon metabolism

• Gene Therapy Approaches:

  • Viral vector-mediated gene delivery (AAV vectors)

  • Targeted to affected tissues (brain, liver, cochlea)

• Mitochondrial-Targeted Therapies:

  • Compounds that enhance mitochondrial function (CoQ10, riboflavin)

  • Antioxidants to reduce oxidative stress resulting from mitochondrial dysfunction

Efficacy Assessment Methods:

• Biochemical Markers:

  • 3-methylglutaconic acid levels in urine

  • Lactate levels in blood and CSF

  • PG-34:1/PG-36:1 ratio in tissues and cultured cells

  • mtDNA content in tissues and blood

• Functional Assays:

  • Mitochondrial respiratory chain activity

  • ATP production capacity

  • Mitochondrial membrane potential

  • Serine transport efficiency

• Clinical Parameters:

  • Neurological function assessment

  • Hearing tests

  • Liver function tests

  • Growth and developmental parameters

• Experimental Design for Therapeutic Testing:

  Study Phase	Model System	Outcome Measures	Duration
  Preclinical	Cell models	mtDNA content, mitochondrial function	1-4 weeks
  Preclinical	Mouse models	Tissue-specific biomarkers, behavioral tests	4-12 weeks
  Clinical	Patient trials	Clinical parameters, biochemical markers	6-24 months

Research has shown that MEGD(H)EL syndrome shares molecular features with mtDNA depletion syndrome, suggesting that therapeutic approaches developed for mtDNA depletion disorders may be applicable to SERAC1-related conditions .

How do researchers differentiate between primary and secondary effects of SERAC1 dysfunction in experimental systems?

Distinguishing primary from secondary effects of SERAC1 dysfunction is crucial for understanding disease mechanisms and developing targeted therapies:

Methodological Approaches:

• Temporal Analysis:

  • Conduct time-course experiments to establish the sequence of cellular and biochemical changes

  • Primary effects occur earlier, while secondary effects develop progressively

  • Monitor parameters at different time points following SERAC1 knockout or knockdown

• Direct Target Identification:

  • Identify molecules that directly interact with SERAC1 (e.g., SFXN1)

  • Examine immediate biochemical consequences of SERAC1 loss (phospholipid remodeling)

  • Use proximity labeling methods to identify direct protein partners

• Rescue Experiments:

  • Systematic rescue of specific pathways:

    • Restore serine transport with alternative transporters

    • Supplement nucleotides to bypass one-carbon metabolism defects

    • Provide phospholipid precursors to compensate for remodeling defects

  • Analyze which interventions rescue which phenotypes

• Multi-omics Integration:

  • Compare transcriptomic, proteomic, and metabolomic changes

  • Map altered pathways to known SERAC1 functions

  • Use network analysis to distinguish hub effects from peripheral consequences

• Single-Cell Analysis:

  • Examine cell-to-cell variability in response to SERAC1 deficiency

  • Identify cell populations most sensitive to primary SERAC1 function

Established Primary Effects of SERAC1 Dysfunction:

• Impaired phosphatidylglycerol remodeling

• Defective serine transport into mitochondria

• Disruption of one-carbon metabolism

• mtDNA depletion

Secondary Effects:

• Mitochondrial respiratory chain dysfunction

• Elevated lactate and 3-methylglutaconic acid

• Neurological symptoms

• Hearing loss