Recombinant Mouse Methylsterol monooxygenase 1 (Msmo1)

What is Mouse Methylsterol Monooxygenase 1 (Msmo1) and what are its key molecular identifiers?

Mouse Methylsterol Monooxygenase 1 (Msmo1), also known by alternative names ERG25 and SC4MOL, is an enzyme involved in cholesterol biosynthesis pathways. It functions as a C-4 methylsterol oxidase, catalyzing an essential step in sterol metabolism. The protein is primarily localized to the endoplasmic reticulum membrane and participates in lipid metabolism processes.

Key molecular identifiers for Mouse Msmo1 include:

The mouse Msmo1 protein shares significant homology with human MSMO1 (UniProt: Q15800), though there are species-specific differences in expression patterns and regulation that should be considered when translating research findings .

What are the biological pathways and processes associated with Msmo1?

Msmo1 is primarily involved in several key metabolic pathways related to sterol biosynthesis and lipid metabolism. Understanding these pathways is essential for designing experiments and interpreting results in Msmo1 research.

Major pathways involving Msmo1 include:

• Cholesterol biosynthesis pathway - Msmo1 catalyzes a critical oxidation step in sterol synthesis

• Steroid biosynthesis pathway - As steroids are derived from cholesterol, Msmo1 indirectly affects steroid production

• Lipid and lipoprotein metabolism - Through its effects on cholesterol availability

• Metabolic pathways - Integrated within broader cellular metabolism

Msmo1 specifically functions in the conversion of 4,4-dimethylzymosterol to zymosterol, a critical intermediate step in cholesterol biosynthesis. This enzymatic activity positions Msmo1 as a potential target for metabolic research and therapeutic development .

How should recombinant Mouse Msmo1 protein be stored and handled to maintain stability?

Proper storage and handling of recombinant Mouse Msmo1 protein is crucial for maintaining its stability and enzymatic activity in research applications. Based on standard protocols for similar transmembrane proteins:

• Storage temperature: Recombinant Msmo1 protein should be stored at -80°C for long-term storage. Upon receipt, the protein should be aliquoted to minimize freeze-thaw cycles.

• Thawing procedure: Thaw aliquots quickly at room temperature and place on ice immediately after thawing.

• Working solutions: Prepare working solutions in appropriate buffers (typically PBS with protease inhibitors) and maintain at 4°C while in use.

• Stability considerations: Msmo1 has membrane-associated domains and may require detergents or stabilizing agents to maintain proper folding. The stability of properly stored recombinant Msmo1 can be maintained for up to 6 months, with activity loss rates typically less than 5% when stored under appropriate conditions .

• Freeze-thaw cycles: Minimize freeze-thaw cycles as they significantly reduce protein activity. Each cycle can reduce activity by 10-20%.

For experimental reproducibility, it is strongly recommended that handling procedures and laboratory conditions be strictly controlled, and ideally, assays should be performed by the same researcher throughout a study .

What experimental considerations should be addressed when designing assays to measure Msmo1 enzymatic activity?

Designing robust assays for Msmo1 enzymatic activity requires careful consideration of several factors that can influence experimental outcomes:

• Substrate selection: Native substrates for Msmo1 include 4,4-dimethylzymosterol and related sterol intermediates. Synthetic or fluorescently labeled substrates may provide easier detection but should be validated against native substrates.

• Cofactor requirements: As a monooxygenase, Msmo1 requires molecular oxygen and typically uses NADPH as an electron donor. Ensure buffers contain appropriate cofactors at optimal concentrations.

• Membrane environment: Since Msmo1 is a transmembrane protein naturally located in the endoplasmic reticulum, activity assays may require microsomal preparations or reconstitution into liposomes for optimal function.

• Detergent considerations: When using purified recombinant Msmo1, mild detergents (e.g., 0.1% Triton X-100 or 0.05% DDM) may be necessary to maintain protein solubility while preserving enzymatic activity.

• Detection methods: Multiple approaches can be used to monitor activity:

  • HPLC or LC-MS to detect substrate conversion to products

  • Coupled enzyme assays measuring NADPH consumption

  • Oxygen consumption measurements

  • Radiolabeled substrate incorporation assays

• Controls: Include appropriate controls such as heat-inactivated enzyme, known inhibitors, and enzymes with site-directed mutations affecting catalytic activity .

These methodological considerations are critical for generating reliable and reproducible data when studying Msmo1 enzymatic function in research settings.

How can researchers effectively validate Msmo1 expression and localization in mouse tissue samples?

Validating Msmo1 expression and localization in mouse tissue samples requires a multi-method approach to ensure robust and reliable results:

• Transcriptional analysis:

  • RT-qPCR using validated primers specific for mouse Msmo1

  • RNA-seq analysis with appropriate normalization

  • In situ hybridization for tissue localization studies

• Protein detection:

  • Western blotting using validated antibodies against mouse Msmo1

  • Immunohistochemistry (IHC) or immunofluorescence (IF) for tissue localization

  • Flow cytometry for cell-specific expression in mixed populations

• Validation controls:

  • Use of Msmo1 knockout tissues as negative controls

  • Recombinant Msmo1 protein as a positive control for antibody validation

  • Competing peptide assays to confirm antibody specificity

• Subcellular localization:

  • Co-localization studies with established ER markers (e.g., calnexin, PDI)

  • Subcellular fractionation followed by Western blotting

  • Electron microscopy with immunogold labeling for high-resolution localization

• Functional validation:

  • Activity assays in tissue extracts

  • Metabolic profiling to detect changes in sterol intermediates

When performing IHC or IF, it is particularly important to optimize fixation protocols, as improper fixation can mask epitopes or disrupt membrane protein localization. Additionally, researchers should be aware that Msmo1 expression levels can vary significantly between tissues, with higher expression typically observed in metabolically active tissues such as liver and brain .

What approaches can be used to study the effects of Msmo1 dysregulation in mouse disease models?

Studying Msmo1 dysregulation in disease models requires strategic experimental designs that capture both molecular mechanisms and phenotypic outcomes:

• Genetic manipulation strategies:

  • Conventional knockout models (global Msmo1 deletion)

  • Conditional knockout models (tissue-specific or inducible deletion)

  • Knockin models with specific mutations (e.g., catalytic site mutations)

  • Overexpression models to assess gain-of-function effects

  • CRISPR/Cas9-mediated precise genome editing

• Phenotypic characterization:

  • Metabolic profiling (sterol intermediates, cholesterol levels)

  • Lipidomic analysis to assess broader changes in lipid metabolism

  • Histopathological examination of affected tissues

  • Functional assays relevant to metabolic disease (glucose tolerance, insulin sensitivity)

  • Behavioral testing if neurological phenotypes are expected

• Molecular characterization:

  • Transcriptomic analysis to identify dysregulated pathways

  • Proteomic analysis to detect changes in protein expression and post-translational modifications

  • ChIP-seq to assess changes in epigenetic regulation

  • Metabolic flux analysis using labeled precursors

• Disease-specific models:

  • High-fat diet challenges to study metabolic syndromes

  • Aging studies to assess progressive phenotypes

  • Combination with other genetic models (e.g., ApoE knockout for atherosclerosis studies)

• Therapeutic intervention studies:

  • Pharmacological inhibition or activation of Msmo1

  • Dietary interventions (e.g., cholesterol supplementation)

  • Gene therapy approaches for rescue experiments

Recent studies have shown that dysregulation of sterol metabolism pathways, including those involving Msmo1, can contribute to various pathologies including metabolic disorders, cancer progression, and potentially neurodegenerative conditions. Understanding the tissue-specific effects of Msmo1 dysregulation is particularly important when designing and interpreting these models .

What techniques are recommended for producing and purifying functional recombinant Mouse Msmo1?

Production and purification of functional recombinant Mouse Msmo1 presents significant challenges due to its nature as a multi-pass transmembrane protein with multiple hydrophobic domains. The following methodological approaches are recommended:

• Expression systems:

  • Mammalian expression systems (HEK293, CHO cells) often yield properly folded membrane proteins

  • Insect cell systems (Sf9, High Five) can produce higher yields while maintaining proper folding

  • Cell-free expression systems with added microsomes or nanodiscs can be effective for transmembrane proteins

  • Avoid bacterial expression systems as they typically fail to properly fold complex mammalian membrane proteins

• Construct design considerations:

  • Addition of purification tags (His, FLAG, Strep) at the N-terminus is preferred over C-terminal tags

  • Consider fusion proteins (MBP, SUMO) to enhance solubility

  • Include TEV or similar protease cleavage sites for tag removal

  • Codon optimization for the expression system of choice

• Purification strategies:

  • Detergent screening is critical (start with DDM, LMNG, or GDN)

  • Two-step purification: affinity chromatography followed by size exclusion

  • Consider lipid supplementation during purification to maintain stability

  • Perform all steps at 4°C to minimize protein degradation

• Functional validation:

  • Activity assays using native or synthetic substrates

  • Thermal stability assays to assess protein folding

  • Circular dichroism to evaluate secondary structure

  • Limited proteolysis to assess conformational integrity

• Reconstitution methods:

  • Proteoliposome reconstitution for functional studies

  • Nanodisc incorporation for structural and biophysical studies

  • Detergent-solubilized protein for immediate use in binding studies

When using cell-free expression systems, which have shown promise for transmembrane proteins, the addition of appropriate lipids or detergents during expression is essential for obtaining functional protein . The success of purification should be validated not only by purity assessment but also by functional assays to ensure the recombinant protein retains its enzymatic activity.

How can researchers effectively design experiments to study Msmo1 interactions with other proteins in cholesterol biosynthesis pathways?

Investigating protein-protein interactions (PPIs) involving Msmo1 requires specialized approaches that account for its membrane-associated nature and integration within metabolic complexes:

• In vitro interaction studies:

  • Pull-down assays using purified recombinant proteins

  • Surface Plasmon Resonance (SPR) to measure binding kinetics

  • Isothermal Titration Calorimetry (ITC) for thermodynamic analysis

  • Microscale Thermophoresis (MST) for interaction studies in complex solutions

  • AlphaScreen or related proximity-based assays

• Cellular interaction studies:

  • Co-immunoprecipitation (Co-IP) from native tissues or cell lines

  • Proximity Ligation Assay (PLA) for visualizing interactions in situ

  • FRET or BRET assays for real-time interaction monitoring

  • Split reporter assays (e.g., split-luciferase complementation)

  • BioID or APEX2 proximity labeling for identifying interaction partners

• Computational approaches:

  • Molecular docking simulations with predicted or known structures

  • Coevolution analysis to identify potential interaction interfaces

  • Network analysis using database resources like STRING

• Functional validation of interactions:

  • Mutagenesis of predicted interaction interfaces

  • Enzymatic assays to assess effects of interactions on activity

  • Metabolic profiling to detect pathway alterations when interactions are disrupted

• Specialized techniques for membrane protein complexes:

  • Crosslinking mass spectrometry (XL-MS)

  • Native PAGE analysis of membrane protein complexes

  • Cryo-EM of purified complexes

What are the recommended approaches for studying Msmo1 gene regulation and expression in different mouse tissues?

Understanding Msmo1 gene regulation requires comprehensive approaches that capture both transcriptional and post-transcriptional regulatory mechanisms across tissues:

• Transcriptional regulation analysis:

  • Promoter analysis using reporter assays

  • ChIP-seq to identify transcription factor binding sites

  • ATAC-seq to assess chromatin accessibility

  • DNase-seq or MNase-seq for nucleosome positioning

  • CUT&RUN or CUT&Tag for targeted transcription factor binding analysis

• Epigenetic regulation:

  • DNA methylation analysis (bisulfite sequencing, MeDIP)

  • Histone modification ChIP-seq (H3K4me3, H3K27ac, H3K27me3)

  • Chromatin conformation studies (Hi-C, 4C, 5C)

  • DNA accessibility in conjunction with methylation (NOMe-seq)

• Post-transcriptional regulation:

  • miRNA targeting analysis (based on predicted miRNA binding sites)

  • RNA immunoprecipitation (RIP) to identify RNA-binding proteins

  • RNA stability assays using transcription inhibitors

  • Polysome profiling to assess translation efficiency

  • Alternative splicing analysis (RNA-seq with junction analysis)

• Tissue-specific expression profiling:

  • Single-cell RNA-seq for cellular heterogeneity

  • Spatial transcriptomics for regional expression patterns

  • In situ hybridization for anatomical localization

  • TRAP-seq for cell-type-specific translation profiles

  • qPCR arrays for targeted expression analysis across tissues

• Developmental and physiological regulation:

  • Time-course studies during development

  • Response to metabolic challenges (fasting, high-fat diet)

  • Hormonal regulation studies

  • Circadian rhythm analysis

Research suggests that Msmo1 expression is regulated by cellular sterol levels through sterol regulatory element-binding proteins (SREBPs) and is responsive to metabolic state changes. Additionally, epigenetic mechanisms, including DNA methylation, may play roles in tissue-specific expression patterns . Understanding these regulatory mechanisms is essential for interpreting Msmo1's role in both normal physiology and disease states.

How can Msmo1 activity be effectively measured in mouse tissue samples or cell culture models?

Accurately measuring Msmo1 enzymatic activity in biological samples requires specialized techniques that account for its membrane localization and specific substrate requirements:

• Direct activity assays:

  • Microsomal preparation from tissues or cells

  • Incubation with 4,4-dimethylzymosterol substrate

  • Analysis of product formation by:

    • GC-MS or LC-MS/MS to detect zymosterol formation

    • Radio-labeled substrate tracking

    • Specific antibodies against reaction products

• Coupled enzymatic assays:

  • Monitoring NADPH consumption (absorbance at 340 nm)

  • Oxygen consumption measurements using oxygen electrodes

  • Coupled enzyme systems with colorimetric or fluorescent readouts

• Cell-based functional assays:

  • Metabolic labeling with 13C-acetate to track sterol synthesis

  • Reporter constructs linked to sterol-responsive elements

  • Complementation assays in Msmo1-deficient cells

  • Growth assays under sterol-restricted conditions

• Sample preparation considerations:

  • Fresh tissue samples yield more reliable activity measurements

  • Proper buffer composition (pH 7.2-7.5, physiological salt concentration)

  • Addition of protease and phosphatase inhibitors

  • Inclusion of required cofactors (NADPH, molecular oxygen)

  • Gentle detergent solubilization (0.1% Triton X-100 or similar)

• Validation approaches:

  • Use of known inhibitors as negative controls

  • Recombinant protein as positive control

  • Correlation with protein expression levels

  • Comparison across multiple tissues/cell types with varying expression levels

Typical enzyme kinetic parameters for mouse Msmo1 include a Km value in the low micromolar range for 4,4-dimethylzymosterol substrate and dependence on NADPH with Km values of approximately 5-15 μM. Activity is typically highest in liver microsomes, consistent with the central role of the liver in cholesterol biosynthesis. Temperature and pH optima are generally around 37°C and pH 7.4, respectively .

What are the implications of Msmo1 in metabolic research and potential therapeutic applications?

The strategic position of Msmo1 in cholesterol biosynthesis makes it a valuable target for metabolic research and potential therapeutic development:

• Metabolic disease applications:

  • Hypercholesterolemia: Msmo1 inhibition could potentially reduce cholesterol synthesis

  • Non-alcoholic fatty liver disease (NAFLD): Altered Msmo1 activity has been implicated in hepatic lipid accumulation

  • Metabolic syndrome: Dysregulation of sterol metabolism contributes to multiple aspects of metabolic syndrome

• Cancer research applications:

  • Several cancer types show altered Msmo1 expression

  • Rapidly dividing cancer cells have increased cholesterol requirements

  • Cervical cancer specifically shows upregulation of MSMO1 expression, suggesting a role in cancer progression

  • Potential for combined therapies targeting both cancer growth and cholesterol metabolism

• Neurodegenerative disease connections:

  • Brain cholesterol metabolism is critical for neuronal function

  • Altered sterol metabolism has been implicated in neurodegenerative disorders

  • Mouse models with Msmo1 deficiency could provide insights into neurological implications

• Drug development considerations:

  • Target validation through genetic models and pharmacological inhibition

  • Assay development for high-throughput screening

  • Structure-activity relationship studies based on known inhibitors

  • Tissue-specific targeting strategies to minimize systemic effects

• Biomarker potential:

  • Sterol intermediates as indicators of pathway activity

  • Expression levels in accessible tissues or liquid biopsies

  • Genetic variants as predictors of metabolic disease risk

What are common challenges in Msmo1 research and how can they be addressed?

Researchers working with Msmo1 encounter several technical challenges that require specific troubleshooting approaches:

• Protein stability issues:

  • Challenge: Recombinant Msmo1 tends to aggregate or denature during purification

  • Solutions:

    • Use mild detergents (DDM, LMNG) throughout purification

    • Include stabilizing agents like glycerol (10-15%)

    • Work at 4°C and minimize exposure to freeze-thaw cycles

    • Consider nanodiscs or amphipols for long-term stability

• Antibody specificity concerns:

  • Challenge: Cross-reactivity with related sterol metabolic enzymes

  • Solutions:

    • Validate antibodies using Msmo1 knockout tissues as negative controls

    • Perform competitive binding assays with recombinant protein

    • Use multiple antibodies targeting different epitopes

    • Consider epitope-tagged versions in experimental systems

• Activity assay limitations:

  • Challenge: Low signal-to-noise ratio in enzymatic assays

  • Solutions:

    • Optimize substrate concentrations and reaction conditions

    • Use high-sensitivity detection methods (LC-MS/MS)

    • Increase enrichment of microsomes for activity measurements

    • Consider coupled enzyme assays with amplified readouts

• Expression variability:

  • Challenge: Inconsistent expression levels across experiments

  • Solutions:

    • Standardize culture conditions for cell-based systems

    • Control for metabolic state (feeding/fasting) in animal studies

    • Use internal housekeeping controls appropriate for the tissue/condition

    • Perform time-course studies to capture dynamic regulation

• Functional redundancy:

  • Challenge: Compensatory mechanisms masking phenotypes in knockout models

  • Solutions:

    • Use acute knockdown approaches (siRNA, inducible systems)

    • Analyze multiple related enzymes simultaneously

    • Challenge systems with metabolic stress to reveal latent phenotypes

    • Consider double-knockout approaches for related pathway enzymes

When working with mouse tissue samples, it's important to note that Msmo1 expression and activity can be significantly affected by nutritional status, circadian rhythms, and age. Standardizing these variables across experiments is essential for obtaining reproducible results .

How can researchers differentiate between mouse and human MSMO1 in comparative studies?

Conducting comparative studies between mouse Msmo1 and human MSMO1 requires careful attention to species-specific differences:

• Sequence and structural differences:

  • Mouse and human MSMO1 share approximately 82% amino acid identity

  • Key catalytic residues are conserved, but differences exist in regulatory regions

  • Species-specific antibodies should target divergent regions (typically N-terminal domains)

  • Differential glycosylation patterns may affect protein migration in SDS-PAGE

• Expression pattern differences:

  • Species-specific primers for RT-qPCR must be designed in divergent regions

  • Tissue distribution shows subtle differences between species

  • Developmental expression patterns may differ significantly

  • Regulatory elements controlling expression show only partial conservation

• Functional assay considerations:

  • Substrate preferences may differ slightly between species

  • Kinetic parameters (Km, Vmax) should be determined independently for each species

  • Inhibitor sensitivity profiles may differ

  • Protein-protein interaction networks may have species-specific components

• Methodological approaches for comparative studies:

  • Parallel expression of both orthologs in the same heterologous system

  • "Humanized" mouse models expressing human MSMO1

  • Chimeric proteins to map functional domains

  • Side-by-side biochemical characterization

• Data analysis considerations:

  • Normalization methods should account for species differences in reference genes

  • Statistical analysis should consider species as a variable factor

  • Careful interpretation of translational implications from mouse to human

When conducting comparative studies, researchers should be particularly aware that regulatory mechanisms governing MSMO1 expression and activity may differ significantly between species, potentially leading to different physiological responses under similar experimental conditions .

What emerging technologies and approaches are advancing Msmo1 research?

The field of Msmo1 research is being transformed by several emerging technologies and innovative approaches:

• Advanced structural biology techniques:

  • Cryo-EM for membrane protein structure determination without crystallization

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational dynamics

  • Integrative structural biology combining multiple experimental approaches

  • Computational prediction methods specifically optimized for membrane proteins

• Single-cell technologies:

  • Single-cell metabolomics to track sterol intermediates at cellular resolution

  • Single-cell transcriptomics to map Msmo1 expression heterogeneity

  • Spatial metabolomics to visualize metabolite distributions in tissues

  • Live-cell imaging of sterol trafficking and metabolism

• Genome editing advancements:

  • Base editing for precise nucleotide changes without double-strand breaks

  • Prime editing for flexible gene modification with minimal off-target effects

  • Tissue-specific CRISPR delivery methods for in vivo studies

  • Multiplexed genetic screening approaches to identify genetic interactions

• Systems biology approaches:

  • Multi-omics integration (genomics, transcriptomics, proteomics, metabolomics)

  • Network analysis of sterol metabolism in health and disease

  • Computational modeling of cholesterol biosynthesis pathways

  • Machine learning applications for predicting metabolic phenotypes

• Therapeutic development platforms:

  • Targeted protein degradation approaches (PROTACs) for Msmo1

  • RNA-based therapeutics (siRNA, antisense oligonucleotides)

  • Small molecule screening using fragment-based approaches

  • Allosteric modulators targeting regulatory rather than catalytic sites

These emerging technologies provide unprecedented opportunities to investigate Msmo1 function with greater precision and integrate findings into a systems-level understanding of sterol metabolism. As these approaches continue to evolve, they promise to reveal new insights into the role of Msmo1 in both normal physiology and disease states .

What are the potential implications of Msmo1 research for understanding metabolic disorders?

The strategic position of Msmo1 in cholesterol biosynthesis suggests several important implications for metabolic disorders:

• Dyslipidemia and cardiovascular disease:

  • Altered Msmo1 activity could affect total cholesterol levels and composition

  • Accumulation of sterol intermediates may have distinct biological effects

  • Genetic variants affecting Msmo1 function could contribute to familial hypercholesterolemia

  • Mouse models with Msmo1 modifications could provide insights into atherosclerosis mechanisms

• Non-alcoholic fatty liver disease (NAFLD):

  • Disrupted cholesterol homeostasis contributes to NAFLD pathogenesis

  • Sterol intermediates may influence hepatic inflammation processes

  • Msmo1 inhibition could potentially reduce hepatic lipid accumulation

  • Interaction between sterol metabolism and fatty acid metabolism in hepatocytes

• Metabolic syndrome and insulin resistance:

  • Cholesterol metabolism affects membrane composition and insulin signaling

  • Sterol intermediates may function as signaling molecules affecting glucose metabolism

  • Msmo1 activity changes in response to dietary challenges and obesity

  • Potential for targeted interventions affecting specific branches of lipid metabolism

• Cancer metabolism:

  • Rapidly proliferating cancer cells have increased cholesterol requirements

  • Altered Msmo1 expression has been observed in multiple cancer types

  • Potential for combination therapies targeting both proliferation and metabolism

  • Biomarker applications for cancer diagnosis or monitoring

• Neurological disorders with metabolic components:

  • Brain cholesterol metabolism is segregated from peripheral metabolism

  • Sterol metabolism disruptions have been implicated in various neurological conditions

  • Potential connections to neurodevelopmental disorders through myelination processes

  • Age-related changes in brain sterol metabolism and neurodegeneration

Understanding Msmo1's role in these disorders could lead to novel therapeutic strategies and diagnostic approaches. Mouse models with targeted Msmo1 modifications serve as valuable tools for investigating these connections and testing potential interventions .