Recombinant Human Methylsterol monooxygenase 1 (MSMO1)

What is MSMO1 and what is its primary function in human metabolism?

MSMO1 (methylsterol monooxygenase 1) is a protein-coding gene located on chromosome 4 that plays a critical role in human cholesterol biosynthesis. The protein is localized to the endoplasmic reticulum membrane and shares significant homology with the yeast ERG25 protein . MSMO1 contains metal binding motifs characteristic of membrane desaturases-hydroxylases and functions in cholesterol biosynthesis through the demethylation of specific sterol precursors .

At the molecular level, MSMO1 catalyzes a three-step monooxygenation reaction to remove methyl groups from 4,4-dimethyl and 4alpha-methylsterols, enabling their conversion into cholesterol . This enzymatic function places MSMO1 as a key player in the sterol biosynthesis pathway. The protein is also known by several alternative names in the literature, including DESP4, ERG25, MCCPD, and SC4MOL .

What are the key molecular characteristics of recombinant MSMO1 protein?

Recombinant Human MSMO1 is typically expressed as a transmembrane protein with specific structural and functional properties that reflect its native counterpart. The protein contains distinctive metal binding motifs that are essential for its catalytic activity . When working with recombinant MSMO1, researchers should note several key characteristics:

• Cellular localization: Primarily in the endoplasmic reticulum membrane and plasma membrane

• Molecular function: C-4 methylsterol oxidase activity and iron ion binding

• Enzymatic classification: EC 1.14.13.72 (oxidoreductase)

• Chromosomal location: Human ortholog is located at 4q32-q34

When designing experiments with recombinant MSMO1, consider that this is a multi-pass membrane protein that requires appropriate expression systems to maintain its native conformation and activity. Cell-free expression systems have been successfully employed to produce functional recombinant MSMO1 protein .

How does MSMO1 interact with other proteins in the cholesterol biosynthesis pathway?

MSMO1 functions within a complex network of proteins involved in cholesterol biosynthesis. Protein interaction analysis reveals that MSMO1 has significant functional relationships with several other enzymes in this pathway. Notably, IDI1 (isopentenyl-diphosphate delta isomerase 1) shows the highest correlation with MSMO1 expression (Spearman correlation: 0.58, p = 1.92e-26), suggesting coordinated regulation or functional dependency .

The protein interaction network of MSMO1 places it firmly within the cholesterol biosynthesis pathway, with particular connections to:

• Sterol biosynthesis enzymes

• Fatty acid metabolism proteins

• Endoplasmic reticulum membrane proteins

Researchers investigating MSMO1 should consider these interactions when designing studies, as perturbation of MSMO1 may have cascading effects on multiple aspects of lipid metabolism. Co-immunoprecipitation and proximity ligation assays are recommended methods for experimentally validating these protein interactions in your specific experimental system.

What is the significance of MSMO1 expression in cancer research?

MSMO1 has emerged as a significant factor in cancer research, particularly in cervical squamous cell carcinoma (CESC). Studies have demonstrated that MSMO1 is consistently upregulated in CESC compared to normal cervical tissue . This differential expression has important clinical implications.

Analysis of TCGA data showed that MSMO1 expression correlates with cancer progression and patient outcomes. Specifically, patients with high MSMO1 expression demonstrated:

These findings were particularly pronounced in the squamous cell carcinoma subgroup, where significant differences in OS (HR=2.47, 95% CI: 1.42–4.28, P=0.001), DSS (HR=2.90, 95% CI: 1.50–5.60, P=0.002), and PFS (HR=2.36, 95% CI: 1.35–4.12, P=0.003) were observed between high and low MSMO1 expression groups .

Researchers studying cancer should consider MSMO1 as a potential biomarker, especially in cervical cancer, where its expression level correlates with clinical stage and patient prognosis .

What methodologies are recommended for studying MSMO1 in liver disease models?

MSMO1 has been associated with various liver conditions including hepatitis, hepatocellular carcinoma, and fatty liver disease . When investigating MSMO1 in liver disease models, researchers should consider the following methodological approaches:

• Expression analysis: Quantitative PCR and Western blotting to measure MSMO1 transcript and protein levels in diseased versus healthy liver tissues.

• Immunohistochemistry: To visualize the distribution and expression pattern of MSMO1 in liver tissue sections, particularly focusing on zones of inflammation or necrosis.

• Animal models: Consider using both genetic models (MSMO1 knockout or overexpression) and disease induction models (high-fat diet, chemical induction of hepatitis).

• Pathway analysis: Given MSMO1's role in cholesterol metabolism, incorporate measurements of lipid profiles and related metabolic enzymes.

• Co-expression studies: Analyze MSMO1 in relation to inflammatory markers and mediators of liver injury, as MSMO1 has been associated with inflammation and necrosis .

When designing liver disease studies involving MSMO1, it's important to consider the protein's role in both cholesterol synthesis and inflammatory processes, as these pathways may be differentially affected depending on the specific liver pathology under investigation.

How can researchers effectively measure MSMO1 activity in experimental disease models?

Measuring MSMO1 enzymatic activity in experimental disease models requires specialized techniques that account for its membrane-bound nature and specific catalytic function. The following methodological approaches are recommended:

• In vitro enzyme activity assay: Using recombinant MSMO1 or microsomal fractions containing the native protein, measure the conversion of 4,4-dimethyl and 4α-methylsterols to their demethylated products using HPLC-MS/MS.

• Substrate tracking: Employ isotope-labeled sterol precursors to track MSMO1-mediated metabolism in cell culture or animal models.

• Inhibitor studies: Use known inhibitors of MSMO1 to validate the specificity of measured activity and establish dose-response relationships.

• Genetic manipulation: Compare MSMO1 activity in wild-type versus knockout/knockdown models to establish baseline and altered enzymatic function.

• Metabolomic profiling: Analyze the sterol profile in tissues and biological fluids to indirectly assess MSMO1 activity through substrate accumulation or product depletion.

When designing such experiments, consider that MSMO1 requires iron as a cofactor , so ensure appropriate metal availability in in vitro assays. Additionally, since MSMO1 is an integral membrane protein, detergent solubilization conditions must be carefully optimized to maintain enzymatic activity while isolating the protein from membranes.

What are the most effective experimental designs for studying MSMO1's role in cancer progression?

Based on current research findings, effective experimental designs for studying MSMO1's role in cancer progression should integrate multiple approaches:

• Clinical correlation studies: Analyze MSMO1 expression in tumor samples with matched normal tissues, correlating expression with clinical parameters such as tumor stage, grade, and patient survival. Consider using a methodology similar to that employed in CESC studies, where receiver operating characteristic (ROC) curve analysis yielded an AUC of 0.751 for MSMO1 as an independent prognostic factor .

• Mechanistic cell-based studies:

  • MSMO1 knockdown/overexpression in cancer cell lines

  • Assessment of proliferation, migration, invasion, and apoptosis

  • Analysis of cholesterol metabolism and steroid hormone production

  • Investigation of downstream signaling pathways

• In vivo models:

  • Xenograft models with MSMO1-modulated cancer cells

  • Genetically engineered mouse models

  • Correlation of tumor growth with MSMO1 expression

  • Therapeutic targeting of MSMO1 or its pathway

• Multi-omics approach: Integrate transcriptomics, proteomics, and metabolomics to comprehensively understand how MSMO1 alterations affect multiple cellular processes.

• Translational research: Evaluate MSMO1 as a potential therapeutic target or biomarker using patient-derived samples and preclinical models.

When designing these experiments, researchers should account for the stage-dependent expression of MSMO1, as studies have shown that higher cancer stages correlate with increased MSMO1 expression (F value = 4.1) .

How can researchers interpret conflicting data regarding MSMO1 expression across different tumor types?

When faced with conflicting data regarding MSMO1 expression across different tumor types, researchers should employ a systematic approach to interpretation:

• Methodological assessment: Evaluate whether differences stem from:

  • Analytical techniques (microarray vs. RNA-seq vs. protein-based methods)

  • Sample preparation protocols

  • Data normalization approaches

  • Statistical analysis methods

• Biological context consideration:

  • Assess tumor microenvironment differences

  • Consider cancer-specific metabolic adaptations

  • Evaluate tissue-specific baseline expression of MSMO1

  • Analyze correlations with cholesterol requirements in different tumor types

• Resolution strategies:

  • Perform meta-analysis of existing datasets

  • Design verification studies using standardized protocols across multiple tumor types

  • Use multiple detection methods on the same samples

  • Integrate findings with functional studies to determine biological relevance

• Data integration:

  • Cross-reference MSMO1 expression with mutation profiles and copy number variations

  • Correlate expression with pathway activation signatures

  • Assess co-expression patterns with known interacting proteins, particularly IDI1 which shows the highest correlation with MSMO1

The available data shows MSMO1 is highly expressed in cervical cancer , but researchers should be cautious when extrapolating these findings to other cancer types without direct experimental evidence.

What techniques are recommended for studying the interaction between MSMO1 and its highly correlated gene partner IDI1?

Given the strong correlation between MSMO1 and IDI1 (Spearman correlation: 0.58, p = 1.92e-26) , investigating their interaction requires specialized techniques:

• Co-expression analysis:

  • Perform qRT-PCR and Western blotting to quantify expression levels in various tissues

  • Use single-cell RNA sequencing to identify cell populations where both genes are co-expressed

  • Employ dual-color fluorescence in situ hybridization to visualize co-expression patterns

• Protein-protein interaction studies:

  • Co-immunoprecipitation with antibodies against MSMO1 and IDI1

  • Proximity ligation assay to detect in situ protein interactions

  • FRET/BRET analysis using fluorescently tagged proteins to detect direct interactions

  • Cross-linking mass spectrometry to map interaction interfaces

• Functional dependency assessment:

  • Genetic manipulation: knockdown/knockout of one gene followed by expression analysis of the other

  • Rescue experiments to restore function

  • Double knockdown/knockout to assess synergistic effects

• Pathway analysis:

  • Metabolomic profiling following manipulation of either gene

  • Flux analysis using isotope-labeled precursors

  • Analysis of cholesterol pathway intermediates

• Promoter analysis:

  • Identify common transcription factors regulating both genes

  • Chromatin immunoprecipitation to confirm binding of transcription factors

  • Reporter assays to assess promoter activity under various conditions

Given their involvement in the cholesterol biosynthesis pathway, researchers should also consider contextual factors such as cellular sterol levels and feedback regulation mechanisms when designing experiments to study MSMO1-IDI1 interactions.

What are the critical factors to consider when designing experiments with recombinant MSMO1?

When working with recombinant MSMO1, researchers should consider several critical factors to ensure experimental success:

• Expression system selection:

  • MSMO1 is a transmembrane protein localized to the endoplasmic reticulum

  • Cell-free expression systems have been successfully used

  • Mammalian expression systems may provide better post-translational modifications

  • Consider using human cell lines for most physiologically relevant studies

• Protein solubilization and purification:

  • As a multi-pass membrane protein, MSMO1 requires appropriate detergents

  • Optimize detergent type and concentration to maintain enzymatic activity

  • Consider using nanodiscs or liposomes for functional studies

• Cofactor requirements:

  • MSMO1 contains metal binding motifs and requires iron ion for activity

  • Ensure buffers contain appropriate metal ions

  • Consider the redox state of the metal cofactors

• Substrate preparation:

  • MSMO1 acts on 4,4-dimethyl and 4α-methylsterols

  • These substrates have limited solubility in aqueous solutions

  • Develop appropriate formulation strategies (e.g., cyclodextrin complexation)

• Activity assay design:

  • MSMO1 catalyzes a three-step monooxygenation reaction

  • Develop sensitive detection methods for reaction intermediates and products

  • Consider coupling reactions to other enzymes for spectrophotometric detection

Researchers should validate their recombinant MSMO1 preparations by comparing the enzymatic parameters to those of the native protein whenever possible.

How can researchers overcome challenges in measuring MSMO1 expression in tissue samples?

Measuring MSMO1 expression in tissue samples presents several challenges due to its membrane localization and relatively moderate expression levels. Here are methodological approaches to overcome these challenges:

• RNA-based detection:

  • Design primers spanning exon-exon junctions to avoid genomic DNA amplification

  • Consider alternative splicing of MSMO1 when designing primers

  • Use digital droplet PCR for enhanced sensitivity in low-expression samples

  • Include appropriate reference genes for normalization

• Protein-based detection:

  • Optimize tissue fixation protocols to preserve membrane protein epitopes

  • Consider antigen retrieval methods specific for membrane proteins

  • Validate antibodies using positive and negative controls, including MSMO1 knockout tissues

  • Compare multiple antibodies targeting different epitopes

• Sample preparation optimization:

  • For fresh tissue, rapid processing is critical to prevent RNA degradation

  • For fixed tissue, standardize fixation time to ensure consistent protein detection

  • Consider laser capture microdissection for cell-type specific analysis

• Quantification strategies:

  • Use appropriate scoring systems for immunohistochemistry (e.g., H-score, Allred score)

  • Employ digital image analysis for objective quantification

  • Consider multiplex immunofluorescence to simultaneously detect MSMO1 and cell-type markers

• Validation approaches:

  • Correlate mRNA and protein expression when possible

  • Compare expression across multiple detection platforms

  • Include appropriate biological controls (e.g., tissues known to express high/low levels of MSMO1)

When studying MSMO1 in cancer tissues, researchers should consider the heterogeneity of expression within tumors and include adequate sampling to capture this variability.

What are the most common experimental pitfalls when studying MSMO1 and how can they be avoided?

Researchers studying MSMO1 may encounter several experimental pitfalls. Here are the most common issues and strategies to avoid them:

• Antibody cross-reactivity:

  • MSMO1 shares sequence similarity with other sterol metabolizing enzymes

  • Solution: Validate antibody specificity using knockout controls and peptide competition assays

  • Solution: Use multiple antibodies targeting different epitopes

• Enzyme instability:

  • As a membrane protein, MSMO1 may lose activity during purification

  • Solution: Optimize buffer conditions (pH, ionic strength, glycerol content)

  • Solution: Consider using microsomal preparations instead of purified protein

• Substrate solubility issues:

  • Sterol substrates have limited aqueous solubility

  • Solution: Use appropriate solubilization methods (cyclodextrins, mild detergents)

  • Solution: Optimize substrate concentration to avoid precipitation

• Cofactor depletion:

  • MSMO1 requires iron for activity

  • Solution: Supplement reaction buffers with appropriate metal ions

  • Solution: Monitor metal content by ICP-MS

• Expression level variability:

  • MSMO1 expression varies across tissues and disease states

  • Solution: Include appropriate biological controls

  • Solution: Use larger sample sizes to account for biological variation

• Alternative splicing confusion:

  • MSMO1 has multiple splice variants

  • Solution: Design isoform-specific primers for PCR

  • Solution: Use antibodies that can distinguish between isoforms

• Cell culture artifacts:

  • Cell culture conditions can affect cholesterol metabolism

  • Solution: Standardize serum concentrations and cell density

  • Solution: Consider serum-free conditions with defined lipid supplements

By anticipating these challenges and implementing the suggested solutions, researchers can significantly improve the reliability and reproducibility of their MSMO1 studies.

What are promising therapeutic targeting strategies for MSMO1 in cancer treatment?

Based on the current understanding of MSMO1's role in cancer progression, particularly its association with poor prognosis in cervical cancer , several promising therapeutic targeting strategies warrant investigation:

• Direct enzyme inhibition:

  • Design small molecule inhibitors targeting the catalytic site

  • Develop allosteric modulators affecting enzyme conformation

  • Create irreversible inhibitors targeting critical residues in the active site

• Gene expression modulation:

  • Employ siRNA or antisense oligonucleotides for transient knockdown

  • Develop CRISPR-Cas9 approaches for complete gene inactivation

  • Explore epigenetic modifiers to downregulate MSMO1 expression

• Metabolic pathway intervention:

  • Target downstream effectors of MSMO1-mediated cholesterol synthesis

  • Develop combination therapies with other cholesterol pathway inhibitors

  • Explore synthetic lethality approaches with interacting pathways

• Immunotherapeutic approaches:

  • Generate antibody-drug conjugates targeting MSMO1-expressing cells

  • Develop CAR-T cells recognizing MSMO1-overexpressing cancer cells

  • Investigate immune checkpoint inhibitors in combination with MSMO1 targeting

• Biomarker-guided therapy:

  • Stratify patients based on MSMO1 expression levels (high vs. low)

  • Develop companion diagnostics for MSMO1-targeted therapies

  • Monitor MSMO1 expression during treatment to assess response

Researchers pursuing these strategies should consider the potential off-target effects, particularly in tissues where MSMO1 plays crucial physiological roles in cholesterol homeostasis.

How might emerging single-cell technologies enhance our understanding of MSMO1 biology?

Emerging single-cell technologies offer unprecedented opportunities to advance our understanding of MSMO1 biology:

• Single-cell RNA sequencing (scRNA-seq):

  • Map MSMO1 expression across cell types within heterogeneous tissues

  • Identify cell populations where MSMO1 is co-expressed with interaction partners like IDI1

  • Discover novel cellular contexts for MSMO1 function

  • Track expression changes during cellular differentiation or disease progression

• Single-cell proteomics:

  • Quantify MSMO1 protein levels in individual cells

  • Correlate protein expression with activity states

  • Investigate post-translational modifications affecting MSMO1 function

• Spatial transcriptomics/proteomics:

  • Visualize MSMO1 expression patterns within tissue architecture

  • Correlate expression with microenvironmental features

  • Identify spatial relationships with interacting proteins

• CRISPR screens at single-cell resolution:

  • Identify genetic dependencies related to MSMO1 function

  • Discover synthetic lethal interactions

  • Map genetic networks controlling MSMO1 expression

• Single-cell metabolomics:

  • Profile sterol intermediates in individual cells

  • Correlate metabolite levels with MSMO1 expression

  • Track metabolic flux through MSMO1-dependent pathways

These technologies will likely reveal cell type-specific functions of MSMO1 that are currently obscured in bulk analysis methods. They may also identify rare cell populations with unique MSMO1 expression patterns or regulatory mechanisms that could serve as targets for therapeutic intervention.

What research questions remain unanswered regarding MSMO1's role in normal physiology and disease?

Despite significant advances in understanding MSMO1, several important research questions remain unanswered:

• Tissue-specific functions:

  • How does MSMO1 function differ across tissues with varying cholesterol requirements?

  • Are there tissue-specific interaction partners that modulate MSMO1 activity?

  • What compensatory mechanisms exist in tissues with low MSMO1 expression?

• Regulatory mechanisms:

  • How is MSMO1 expression regulated during development and in response to metabolic changes?

  • What transcription factors and epigenetic mechanisms control MSMO1 expression?

  • How does post-translational modification affect MSMO1 activity?

• Cancer biology:

  • Through what mechanisms does MSMO1 overexpression contribute to cancer progression?

  • Is MSMO1's role in cancer primarily related to cholesterol metabolism or does it have non-canonical functions?

  • Why does MSMO1 expression correlate with poor prognosis in cervical cancer ?

• Metabolic disease connections:

  • What is MSMO1's contribution to metabolic disorders such as fatty liver disease ?

  • How does MSMO1 function change in insulin resistance and diabetes?

  • Could MSMO1 be a therapeutic target for metabolic disorders?

• Drug metabolism role:

  • What is the full scope of MSMO1's role in drug metabolism beyond eldecalcitol ?

  • Could polymorphisms in MSMO1 affect drug efficacy or toxicity?

  • Is MSMO1 inducible by xenobiotics through mechanisms similar to cytochrome P450 enzymes?

Addressing these questions will require integrative approaches combining genetics, biochemistry, cell biology, and clinical research. The answers will not only advance our fundamental understanding of cholesterol metabolism but may also reveal new therapeutic opportunities.