Recombinant Rat Methylsterol monooxygenase 1 (Msmo1)

What is Methylsterol monooxygenase 1 (Msmo1) and what is its biochemical function?

Methylsterol monooxygenase 1 (Msmo1), also known as C-4 methylsterol oxidase, is an enzyme involved in cholesterol biosynthesis. It functions as a methyl sterol oxidase in the Bloch pathway, catalyzing the demethylation of C4-methyl sterols. The enzyme has the Enzyme Commission number EC=1.14.13.72, indicating its role as an oxidoreductase that incorporates molecular oxygen during catalysis . Msmo1 plays a parallel role to FAXDC2, which appears to function predominantly in the Kandutsch-Russell pathway of cholesterol biosynthesis .

What are the alternative names and identifiers for rat Msmo1?

Rat Methylsterol monooxygenase 1 has been known by several names in scientific literature:

• C-4 methylsterol oxidase

• Neuropep 1

• RANP-1

• Previously known as Sc4mol (sterol-C4-methyl oxidase-like)

The nomenclature was updated in 2011 to reflect human and mouse nomenclature standards . Key database identifiers include:

• UniProt accession: O35532

• RGD ID: 13700067

• Ensembl Gene: ENSRNOG00000032297

• Ensembl Transcript: ENSRNOT00000044171

How should recombinant rat Msmo1 be stored and handled for optimal stability?

For optimal stability of recombinant rat Msmo1, researchers should adhere to the following protocols:

• Storage conditions: Store the protein at -20°C for routine use and at -80°C for extended storage periods.

• Buffer composition: The protein is typically supplied in a Tris-based buffer containing 50% glycerol, specifically optimized for Msmo1 stability.

• Working protocols: When working with the protein, maintain working aliquots at 4°C for up to one week.

• Stability considerations: Avoid repeated freezing and thawing cycles as this can compromise protein integrity and enzymatic activity .

These handling precautions are essential for maintaining the structural and functional integrity of the recombinant protein during experimental procedures.

What methodological approaches can be used to study Msmo1 expression and function in disease models?

Researchers investigating Msmo1 can employ several methodological approaches:

• Expression analysis techniques:

  • Wilcoxon rank sum test for comparing expression between normal and cancer tissues

  • Kruskal-Wallis test for correlation between expression and clinical features

  • Logistic and Cox regression analyses for prognostic evaluations

• Functional assessment strategies:

  • Knockdown experiments using siRNAs or CRISPR-Cas9 to evaluate phenotypic consequences

  • Overexpression studies to assess gain-of-function effects

  • Metabolite profiling to analyze changes in C4-methyl sterol levels when Msmo1 function is altered

• Pathway interaction analyses:

  • Investigation of interactions with signaling pathways (e.g., Wnt/β-catenin and RTK pathways)

  • Assessment of effects on downstream targets following Msmo1 modulation

How can researchers accurately measure changes in sterol metabolism related to Msmo1 activity?

To accurately measure changes in sterol metabolism related to Msmo1 activity, researchers should consider these methodological approaches:

• Targeted metabolomics: Quantify specific C4-methyl sterols that are substrates or products of Msmo1 activity. For example, analysis of lophenol and dihydro-TMAS levels can provide insights into flux through the cholesterol biosynthesis pathway .

• Comparative analysis following genetic manipulation: Compare sterol profiles in Msmo1 knockdown/knockout models versus controls. The accumulation of upstream C4-methyl sterols would be expected when Msmo1 activity is reduced .

• Pharmacological interventions: Use inhibitors like ketoconazole (a CYP51A1 inhibitor) to deplete cholesterol biosynthesis intermediates downstream of lanosterol, and measure the impact on sterol profiles and signaling pathways .

• Pathway-specific analyses: Distinguish between effects on the Bloch pathway (where Msmo1 predominantly functions) versus the Kandutsch-Russell pathway (where FAXDC2 appears to function) .

What is the relationship between Msmo1 expression and cancer outcomes?

Research has identified significant correlations between Msmo1 expression and cancer outcomes:

• Expression pattern: Msmo1 is upregulated in cervical cancer tissues compared to normal cervical tissues, with statistical significance demonstrated using the Wilcoxon rank sum test .

• Prognostic significance: High expression of Msmo1 correlates with poor prognosis in cervical squamous cell carcinoma (CESC), as confirmed through both univariate and multivariate analyses .

• Clinical potential: Msmo1 has shown potential as both a diagnostic marker and prognostic indicator in cervical cancer. Statistical analyses suggest strong diagnostic power for Msmo1 expression levels in distinguishing cancer from normal tissue .

The table below summarizes T-stage distribution between low and high Msmo1 expression groups in cervical cancer patients:

Although the p-value didn't reach statistical significance (p=0.14), there was a trend toward more advanced T stages in the high Msmo1 expression group .

What disease associations have been identified for Msmo1 mutations or dysregulation?

Msmo1 has been associated with several pathological conditions:

• Genetic disorders: In humans, MSMO1 mutations have been linked to a syndrome characterized by Microcephaly, Congenital Cataract, and Psoriasiform Dermatitis, as documented in the Online Mendelian Inheritance in Man (OMIM) database .

• Cancer associations:

  • Upregulation of Msmo1 has been strongly associated with poor survival in cervical cancer .

  • Altered sterol metabolism due to Msmo1 dysregulation may contribute to cancer pathogenesis through effects on membrane properties and signaling pathway regulation .

• Pathway-mediated effects: The cholesterol biosynthesis pathway, in which Msmo1 plays a key role, intersects with oncogenic signaling pathways, including EGFR signaling in breast cancer cells and potentially Wnt/β-catenin signaling in other cancers .

How does Msmo1 interact with the tumor microenvironment?

While the search results don't provide direct evidence on Msmo1's interaction with the tumor microenvironment, several inferences can be made based on its functional role:

• Sterol metabolism influence: As Msmo1 regulates sterol metabolism, it may affect membrane composition in both tumor cells and surrounding stromal cells, potentially influencing cell-cell interactions within the tumor microenvironment .

• Signaling pathway modulation: Msmo1 has been shown to influence receptor tyrosine kinase (RTK) signaling, which plays critical roles in interactions between tumor cells and their microenvironment. For example, altered EGFR levels resulting from changes in sterol metabolism could affect communication between cancer cells and surrounding tissues .

• Immune cell interactions: Although not directly addressed in the search results, sterol metabolism has been shown to influence immune cell function. Changes in Msmo1 activity might therefore affect tumor-immune cell interactions within the microenvironment.

How does Msmo1 interact with signaling pathways in cancer progression?

Msmo1 exhibits complex interactions with several signaling pathways relevant to cancer progression:

• RTK signaling interaction: As a C4-methyl sterol oxidase in the Bloch pathway, Msmo1 has been shown to regulate EGFR signaling in breast cancer cells. Changes in sterol metabolism due to altered Msmo1 activity can affect RTK abundance and downstream signaling .

• Pathway-specific effects: The cholesterol biosynthesis inhibitor ketoconazole increases cell surface levels of EGFR in cancer cells, similar to effects observed following Wnt pathway inhibition, suggesting sterol-dependent regulation of receptor trafficking .

• Differential regulation: Msmo1 and FAXDC2 (another sterol metabolism enzyme) appear to have distinct effects on signaling pathways despite both being involved in sterol metabolism. For example, siRNA knockdown of Msmo1 did not affect ETC-159-mediated changes in EGFR cell surface levels in HPAF-II cells, while FAXDC2 knockdown did prevent these changes .

• Pathway crosstalk: The research suggests complex interplay between the cholesterol biosynthesis pathway (involving Msmo1) and oncogenic signaling networks, potentially creating opportunities for targeting these interactions therapeutically.

What are the differences in function between Msmo1 and other C4-methyl sterol oxidases?

The search results reveal important functional distinctions between Msmo1 and other C4-methyl sterol oxidases, particularly FAXDC2:

• Pathway specificity: Msmo1 functions predominantly in the Bloch pathway of cholesterol biosynthesis, while FAXDC2 appears to be more active in the Kandutsch-Russell (KR) pathway .

• Substrate specificity: FAXDC2 specifically catalyzes steps between dihydro-T-MAS and zymostenol in the KR pathway, parallel to Msmo1's activity in the Bloch pathway .

• Regulation differences: FAXDC2 is repressed by Wnt signaling, leading to C4-methyl sterol accumulation when Wnt is active. When Wnt signaling is inhibited, FAXDC2 expression increases, enhancing flux through the KR pathway and depleting C4-methyl sterols .

• Signaling impact: While both enzymes affect sterol metabolism, they have different impacts on downstream signaling. For example, FAXDC2 knockdown prevents Wnt inhibition-mediated increases in EGFR levels, whereas Msmo1 knockdown does not have this effect in the same cell model .

How can researchers distinguish between direct and indirect effects of Msmo1 on cellular phenotypes?

To distinguish between direct and indirect effects of Msmo1 on cellular phenotypes, researchers should consider these methodological approaches:

• Complementary approaches:

  • Genetic manipulation: Use both knockdown/knockout and rescue experiments. If reintroduction of wild-type Msmo1 restores the phenotype, this suggests a direct effect of Msmo1 activity .

  • Pharmacological intervention: Compare phenotypes resulting from Msmo1 manipulation with those produced by inhibitors of sterol metabolism (e.g., ketoconazole) to identify which effects are sterol-dependent .

• Temporal analyses:

  • Time-course experiments can help establish causality by determining whether changes in Msmo1 activity precede changes in downstream phenotypes.

  • Inducible expression systems allow for precise temporal control of Msmo1 expression or activity.

• Substrate/product manipulation:

  • Supplement with specific sterols to determine if they can rescue phenotypes resulting from Msmo1 inhibition.

  • Analyze whether the accumulation of C4-methyl sterols (Msmo1 substrates) or depletion of downstream metabolites better correlates with observed phenotypes .

• Pathway dissection:

  • Use inhibitors or activators of potential downstream pathways to determine if they can mimic or block Msmo1-dependent phenotypes.

  • Conduct epistasis experiments by simultaneously manipulating Msmo1 and downstream pathway components .

What statistical methods are most appropriate for analyzing Msmo1 expression in clinical samples?

Based on the search results, several statistical methods have been successfully employed for analyzing Msmo1 expression in clinical contexts:

• Comparison of expression levels:

  • Wilcoxon rank sum test: For comparing Msmo1 expression between normal tissues and cancer samples .

  • Kruskal-Wallis test: For assessing correlations between Msmo1 expression and various clinical features .

• Association with clinical characteristics:

  • Logistic regression analysis: For investigating relationships between Msmo1 expression and clinical parameters .

  • Fisher's exact test or Chi-square test: For categorical comparisons, such as T stage distribution between high and low Msmo1 expression groups .

• Survival and prognostic analyses:

  • Cox regression analysis: For both univariate and multivariate analysis of the association between Msmo1 expression and patient prognosis .

  • Kaplan-Meier method with Log-rank test: For comparing survival rates between patient groups with differential Msmo1 expression .

These statistical approaches allow for comprehensive assessment of Msmo1's clinical significance, from basic expression comparisons to complex multivariate analyses accounting for various clinical factors.

How should researchers interpret conflicting data about Msmo1 function across different experimental models?

When faced with conflicting data about Msmo1 function across different experimental models, researchers should consider these interpretation frameworks:

• Model-specific differences:

  • Cell/tissue specificity: Msmo1 may have distinct functions in different cell types or tissues based on the metabolic requirements and signaling context.

  • Pathway redundancy: Alternative enzymes or pathways may compensate for Msmo1 deficiency in some models but not others.

• Methodological considerations:

  • Knockdown efficiency: Variations in the degree of Msmo1 suppression may lead to different phenotypic outcomes.

  • Experimental timing: Acute versus chronic Msmo1 inhibition might yield different results due to adaptive responses.

• Analytical approaches:

  • Integrate multiple data types: Combine gene expression, protein level, metabolite profile, and functional data to develop a more comprehensive understanding.

  • Consider pathway context: Interpret Msmo1 function in the context of the entire cholesterol biosynthesis pathway rather than in isolation .

• Reconciliation strategies:

  • Identify conditional dependencies: Determine what factors (e.g., Wnt pathway activation status) might explain differential Msmo1 functions across models.

  • Perform parallel experiments: When possible, conduct side-by-side comparisons using standardized protocols across different models to minimize technical variability.

What considerations are important when designing experiments to study Msmo1's role in cholesterol biosynthesis?

When designing experiments to study Msmo1's role in cholesterol biosynthesis, researchers should consider these important factors:

• Pathway specificity:

  • Distinguish between the Bloch and Kandutsch-Russell pathways, as Msmo1 appears to function predominantly in the former while FAXDC2 functions in the latter .

  • Include analyses of specific sterol intermediates that distinguish between these pathways.

• Metabolite profiling:

  • Implement comprehensive sterol profiling to detect shifts in multiple intermediates rather than focusing on end-product cholesterol alone.

  • Monitor both C4-methyl sterols (e.g., lophenol, dihydro-TMAS) and downstream metabolites to assess pathway flux .

• Regulatory context:

  • Consider the influence of signaling pathways (e.g., Wnt/β-catenin) that may regulate Msmo1 expression or activity.

  • Evaluate how physiological factors like nutrient availability affect Msmo1 function .

• Functional readouts:

  • Beyond measuring sterol levels, assess functional consequences such as effects on membrane properties, receptor trafficking, and signal transduction.

  • Include both cell-autonomous effects and potential non-cell-autonomous impacts of altered Msmo1 activity .