Recombinant Mouse Protein SERAC1 (Serac1)

What is SERAC1 and what is its primary function in cellular biology?

SERAC1 (serine active site-containing protein 1) is a PGAP1-like protein that plays a critical role in phospholipid phosphatidylglycerol remodeling, specifically converting phosphatidylglycerol-34:1 (PG-34:1) to phosphatidylglycerol-36:1 (PG-36:1) . This function is essential for both mitochondrial function and intracellular cholesterol trafficking . The protein is strategically located at the interface between the endoplasmic reticulum and mitochondria, positioning it ideally for its role in lipid metabolism and mitochondrial membrane maintenance . Dysfunction of SERAC1 can lead to altered PG-34:1/PG-36:1 ratios, resulting in various mitochondrial disorders .

What is the molecular structure and domain organization of SERAC1?

The SERAC1 gene spans approximately 59 kb on chromosome 6q25.3 and contains 17 exons . The encoded protein is 654 amino acids in length and features a conserved serine-lipase domain with the consensus lipase motif GxSxG . SERAC1 belongs to the PGAP-like protein domain family (PFAM PF07819) . The functional domains of SERAC1 include:

• A serine-lipase domain that is critical for its enzymatic activity

• Multiple conserved regions across species, particularly around amino acid position 499

The domain organization is particularly important when assessing pathogenic variants, as mutations in different domains can result in varying phenotypic expressions.

How is SERAC1 expression regulated in different mouse tissues?

While the search results don't provide comprehensive information on tissue-specific expression patterns of mouse SERAC1, research indicates that SERAC1 is prominently expressed in the liver, similar to human expression patterns . Unlike other proteins such as Serum Amyloid A1 (which shows tissue-specific expression differences between humans and mice), SERAC1 appears to maintain consistent expression patterns across species, highlighting its evolutionarily conserved role . The conservation of the amino acid sequence at position 499 across ten different species further underscores the functional importance of this protein .

What are the known differences between mouse and human SERAC1?

The SERAC1 protein shows high conservation across species, particularly at functionally critical regions such as position 499, which is highly conserved among ten different species as demonstrated by amino acid sequence alignment . While the search results don't explicitly detail all differences between mouse and human SERAC1, the conservation of function suggests that mouse models provide relevant insights for human disease mechanisms. Both human and mouse SERAC1 function in phosphatidylglycerol remodeling and are associated with similar pathological outcomes when mutated, including neurological disorders characterized by movement abnormalities .

What are the best practices for working with recombinant mouse SERAC1 protein?

Based on general recombinant protein handling protocols, researchers working with recombinant mouse SERAC1 should consider the following best practices:

• Reconstitution: Lyophilized recombinant proteins should typically be reconstituted at appropriate concentrations (e.g., 100 μg/mL) in phosphate-buffered saline (PBS) .

• Storage: Use manual defrost freezers and avoid repeated freeze-thaw cycles to maintain protein integrity. Aliquoting the reconstituted protein is recommended to minimize freeze-thaw cycles .

• Formulation considerations: Recombinant proteins may be obtained in carrier-free (CF) formulations, which lack bovine serum albumin (BSA) or other carrier proteins. This is particularly important for applications where carrier proteins might interfere with experimental outcomes .

• Stability assessment: Regular verification of protein activity is essential, especially when investigating functional aspects of SERAC1.

How can researchers effectively detect and quantify SERAC1 in experimental models?

Effective detection and quantification of SERAC1 in experimental models may employ several complementary approaches:

• Western blotting: Using specific antibodies against mouse SERAC1 for protein detection in tissue lysates.

• Immunohistochemistry/Immunofluorescence: For localizing SERAC1 within cells and tissues, particularly to verify its positioning at the endoplasmic reticulum-mitochondria interface.

• qRT-PCR: For quantifying SERAC1 gene expression levels across different tissues or experimental conditions.

• Mass spectrometry: For detailed characterization of SERAC1 and its post-translational modifications.

• Functional assays: Measuring phosphatidylglycerol remodeling activity, specifically the conversion of PG-34:1 to PG-36:1, and evaluating the PG-34:1/PG-36:1 ratio as a functional readout of SERAC1 activity .

What are validated methodologies for studying SERAC1 mutations in mouse models?

Studying SERAC1 mutations in mouse models typically involves:

• Genetic confirmation: Sanger sequencing to verify SERAC1 variants, as demonstrated in studies of human patients where compound heterozygous variants were confirmed in a proband and her parents .

• Bioinformatic analysis: Using prediction software (PolyPhen-2, MutationTaster, PROVEAN, and SIFT) to assess the potential pathogenicity of novel variants .

• Biochemical assessments: Gas chromatography/mass spectrometry (GC/MS) to detect metabolic abnormalities, particularly increased 3-methylglutaconic acid in urine, which is a biochemical hallmark of SERAC1 dysfunction .

• Imaging studies: MRI to detect characteristic brain abnormalities, such as bilateral symmetric atrophy of the caudate head and anterior putamen with associated gliosis in affected individuals .

• Functional validation: Measuring phosphatidylglycerol ratios (PG-34:1/PG-36:1) to confirm the functional impact of mutations on SERAC1 activity .

What is the spectrum of phenotypes associated with SERAC1 mutations?

SERAC1 mutations are associated with a spectrum of phenotypes that vary in severity and age of onset:

• MEGDEL syndrome: A severe infantile-onset condition characterized by 3-methylglutaconic aciduria, dystonia, deafness, hepatopathy, encephalopathy, and Leigh-like lesions. This typically presents with feeding problems, liver failure, spasticity, dystonia, hearing loss, and truncal hypotonia, with a median lifespan of 9 years .

• Juvenile-onset complicated hereditary spastic paraplegia (cHSP): A milder phenotype with onset between 2-7 years of age, presenting with cognitive delay, spasticity, and slower progression than MEGDEL syndrome .

• Adult-onset movement disorders: A late-onset presentation with parkinsonism and progressive dystonia, as reported in two adult brothers with a homozygous splice site variant (c.[129-2A > C]). This represents a forme fruste of MEGDEL syndrome with significantly slower progression .

This phenotypic spectrum demonstrates that different SERAC1 variants can result in distinct clinical presentations, ranging from severe infantile-onset disease to milder adult-onset manifestations.

How do specific SERAC1 mutations correlate with disease severity?

The correlation between specific SERAC1 mutations and disease severity shows several patterns:

• Location of mutations: Variants located within the serine-lipase domain (such as c.1495A>G/p.Met499Val) may have different effects compared to those upstream of this domain (such as c.721_722del/p.Leu242fs) .

• Mutation type: Frameshift mutations leading to premature termination codons (like c.721_722delAG/p.Leu242fs) are often associated with more severe phenotypes due to potential nonsense-mediated decay or truncated protein products .

• Splice site variants: The homozygous variant c.[129-2A > C] affects the splice-acceptor site for exon 4, potentially resulting in exon skipping or activation of a cryptic splice site with consequent frameshift, leading to an adult-onset phenotype .

• Compound heterozygosity: The combined effect of two different variants (as observed in a case with c.1495A>G and c.721_722del) may result in intermediate phenotypes, with the specific manifestations depending on the residual function of the mutant proteins .

While the exact relationship between genotype and phenotype is not fully established, these patterns suggest that the nature and location of mutations influence disease severity and progression.

What are the biochemical markers of SERAC1 dysfunction?

Key biochemical markers of SERAC1 dysfunction include:

• 3-Methylglutaconic aciduria: Increased urinary excretion of 3-methylglutaconate is a primary biochemical marker, ranging from mild to significant elevation. In some cases, the abnormality may not be detected initially but becomes apparent with repeat testing .

• 3-Methylglutaric acid: Mild increases may be observed alongside 3-methylglutaconate .

• Altered phosphatidylglycerol ratio: Increased PG-34:1/PG-36:1 ratio reflects impaired SERAC1 function in remodeling phosphatidylglycerols .

• Normal liver function tests: Despite the association with MEGDEL syndrome, which can include hepatopathy, liver function tests may remain normal in milder phenotypes or adult-onset forms .

These biochemical abnormalities serve as important diagnostic markers and can help monitor disease progression or response to potential therapeutic interventions.

What neuroimaging findings are associated with SERAC1 mutations?

Neuroimaging findings in individuals with SERAC1 mutations vary according to phenotype:

• MEGDEL syndrome: Leigh-like lesions on brain MRI, typically showing bilateral basal ganglia abnormalities .

• Adult-onset forms: Bilateral symmetric atrophy of the caudate head and anterior putamen with associated gliosis, as documented in two brothers with adult-onset parkinsonism and dystonia .

• Spinal abnormalities: Patients with complicated hereditary spastic paraplegia may show abnormalities on spinal MRI, though specific findings are not detailed in the search results .

These imaging findings provide important diagnostic clues and help in distinguishing SERAC1-related disorders from other neurological conditions with similar clinical presentations.

What is the mechanistic relationship between SERAC1 dysfunction and neurological disorders?

The mechanistic link between SERAC1 dysfunction and neurological disorders involves several interconnected pathways:

• Impaired phosphatidylglycerol remodeling: SERAC1 is essential for converting PG-34:1 to PG-36:1, a process critical for maintaining proper mitochondrial membrane composition. Disruption of this process leads to altered phospholipid ratios .

• Mitochondrial dysfunction: The altered phospholipid composition compromises mitochondrial function, particularly affecting tissues with high energy demands such as the brain .

• Disturbed cholesterol trafficking: SERAC1 plays a role in intracellular cholesterol trafficking, and its dysfunction may alter cholesterol distribution within neurons, affecting membrane properties and signaling pathways .

• Selective vulnerability: The basal ganglia appear particularly vulnerable to SERAC1 dysfunction, explaining the predominance of movement disorders (dystonia, parkinsonism) in the clinical presentation .

• Progressive nature: The accumulation of dysfunctional mitochondria and altered lipid metabolism likely contributes to the progressive neurodegeneration observed in affected individuals .

Understanding these mechanisms is crucial for developing targeted therapeutic approaches for SERAC1-related disorders.

What experimental approaches can be used to study SERAC1 interactions with other proteins?

Several experimental approaches can be employed to study SERAC1 interactions with other proteins:

• Co-immunoprecipitation (Co-IP): To identify proteins that physically interact with SERAC1 in cellular contexts.

• Proximity labeling techniques: Such as BioID or APEX, which can identify proteins in close proximity to SERAC1, particularly at the endoplasmic reticulum-mitochondria interface.

• Yeast two-hybrid screening: To identify potential binding partners of SERAC1.

• Protein microarrays: To screen for interactions between SERAC1 and a large panel of proteins.

• Cross-linking mass spectrometry: To capture transient interactions and identify binding sites.

• Fluorescence resonance energy transfer (FRET): To study protein-protein interactions in living cells.

These techniques can help elucidate the broader interactome of SERAC1 and identify potential regulatory mechanisms or additional functions beyond phosphatidylglycerol remodeling.

How can researchers develop and validate therapeutic approaches for SERAC1-related disorders?

Developing therapeutic approaches for SERAC1-related disorders requires:

• Target validation: Confirming that modulating SERAC1 function or its downstream pathways can ameliorate disease phenotypes in cellular and animal models.

• Gene therapy strategies: Developing AAV-based or other vectors for delivering functional SERAC1 genes to affected tissues, particularly the central nervous system.

• Small molecule screening: Identifying compounds that can enhance residual SERAC1 function or bypass the need for SERAC1 by activating alternative phospholipid remodeling pathways.

• Metabolic bypass strategies: Developing approaches to normalize phosphatidylglycerol ratios or supplement key metabolites affected by SERAC1 dysfunction.

• Mitochondrial-targeted therapies: Testing agents that improve mitochondrial function independently of SERAC1, such as antioxidants or compounds that enhance mitochondrial biogenesis.

• Biomarker development: Establishing reliable biomarkers (such as PG-34:1/PG-36:1 ratios or urinary 3-methylglutaconate levels) to monitor disease progression and treatment response.

Validation would require testing in appropriate cellular and animal models before advancing to clinical trials.

What is the significance of SERAC1 conservation across species for translational research?

The high conservation of SERAC1 across species has several important implications for translational research:

• Evolutionary importance: The conservation of amino acid sequences, particularly at functionally critical positions like Met499, underscores the fundamental importance of SERAC1 in cellular biology across diverse organisms .

• Model validity: The conservation supports the use of mouse models to study SERAC1 function and dysfunction, as findings are likely to be relevant to human disease mechanisms.

• Functional domains: Highly conserved regions likely represent crucial functional domains that could be targeted for therapeutic intervention or used as biomarkers.

• Cross-species comparisons: Studying species-specific differences in SERAC1 function or regulation may provide insights into mechanisms of resilience or vulnerability to SERAC1 dysfunction.

• Predictive value: Conservation data helps predict the pathogenicity of novel variants, as mutations affecting highly conserved residues are more likely to be deleterious .

This conservation facilitates the translation of findings from model organisms to human applications, accelerating the development of diagnostic and therapeutic approaches for SERAC1-related disorders.

What are the most pressing knowledge gaps in SERAC1 research?

Several critical knowledge gaps exist in SERAC1 research that require further investigation:

• Complete phenotypic spectrum: While three distinct phenotypes associated with SERAC1 mutations have been described (MEGDEL syndrome, juvenile-onset complicated hereditary spastic paraplegia, and adult-onset dystonia-parkinsonism), additional intermediate or divergent phenotypes may exist .

• Genotype-phenotype correlations: The relationship between specific SERAC1 variants and clinical manifestations remains incompletely understood .

• Tissue-specific effects: The mechanisms underlying tissue-specific vulnerability to SERAC1 dysfunction, particularly the predilection for neurological manifestations, require further exploration.

• Interacting partners: The complete interactome of SERAC1 and its integration into broader cellular pathways remains to be fully elucidated.

• Compensation mechanisms: Understanding why some individuals with SERAC1 mutations develop symptoms later in life could reveal important compensatory mechanisms.

Addressing these knowledge gaps will advance our understanding of SERAC1 biology and pathology, potentially leading to new therapeutic strategies.

How might advanced technologies enhance our understanding of SERAC1 function?

Advanced technologies that could significantly enhance our understanding of SERAC1 function include:

• CRISPR-Cas9 gene editing: For creating precise models of SERAC1 mutations in cellular and animal systems, enabling detailed study of variant-specific effects.

• Single-cell omics: To investigate cell type-specific effects of SERAC1 dysfunction and identify particularly vulnerable cell populations.

• Advanced imaging techniques: Such as super-resolution microscopy to visualize SERAC1 localization and dynamics at the ER-mitochondria interface with unprecedented detail.

• Lipidomics: To comprehensively characterize alterations in the lipid landscape resulting from SERAC1 dysfunction, beyond the known changes in phosphatidylglycerol species.

• Induced pluripotent stem cells (iPSCs): Derived from patients with SERAC1 mutations and differentiated into relevant cell types (neurons, hepatocytes) to study disease mechanisms in human cells.

• Organoid models: To recapitulate the complexity of affected tissues and study SERAC1 function in more physiologically relevant systems.

These technologies will provide deeper insights into the molecular mechanisms of SERAC1 function and dysfunction, potentially revealing new therapeutic targets.