Recombinant Pongo abelii Methylsterol monooxygenase 1 (MSMO1)

What is the molecular structure and function of Pongo abelii MSMO1?

Pongo abelii MSMO1 (Uniprot NO.: Q5R574) is an enzyme that catalyzes the removal of methyl groups from C4-methylsterols during post-squalene cholesterol biosynthesis . The protein contains distinctive metal binding motifs similar to those found in membrane desaturases-hydroxylases family . It is localized to the endoplasmic reticulum membrane, which is consistent with its role in cholesterol synthesis . The full amino acid sequence of Pongo abelii MSMO1 is: MATNESVSIFSASLAVEY VDSLLPENPLQEPFKNA WNYMLNNYT KFQIATWGSLIVHEALYFLFCLPGFLFQFIPYMKKYKIQKDKPETWENQWKCFKVLLFNHFCIQLPLICGTYYFTEYFNIPYDWERMPRWYFLLARCFGCAVIEDTWHYFLHRLLHHKRIYKYIHKVHHEFQAPFGMEAEYAHPLETLILGTGFFIGIVLLCDHVILLWAWVTIRLLETIDVHSGYDIPLNPLNLIPFYAGSRHHDFHHMNFIGNYASTFTWWDRIFGTDSQYNAYNEKRKKFEKKTE . The enzyme is classified under EC 1.14.13.72, indicating its role as an oxidoreductase acting on paired donors with incorporation of molecular oxygen .

How is MSMO1 conserved across species, and what insights does Pongo abelii MSMO1 provide?

MSMO1 shows evolutionary conservation across species, with the Pongo abelii version sharing significant homology with human and other mammalian counterparts. Studies in zebrafish have demonstrated that MSMO1 function is essential for survival, as loss of Msmo1 is lethal at larval stages . The conservation of this enzyme across evolutionary diverse species suggests its fundamental role in cholesterol metabolism. Pongo abelii MSMO1 serves as a valuable comparative model for understanding primate-specific aspects of cholesterol biosynthesis. The zebrafish models have shown that MSMO1 expression is particularly important in pre-hypertrophic chondrocytes, with its downregulation resulting in defective chondrocyte differentiation, irregular bone formation, and ectopic ossification within growth plates . This suggests that MSMO1 has maintained critical developmental functions throughout vertebrate evolution.

What experimental approaches are typically used to study recombinant MSMO1 expression and activity?

Studying recombinant MSMO1 typically involves expression in heterologous systems, protein purification, and enzymatic activity assays. For Pongo abelii MSMO1, recombinant protein can be produced with various tags to facilitate purification . Activity assays often measure the conversion of C4-methylsterols to their demethylated products. ELISA-based methods can be used to quantify protein levels and assess binding properties . In zebrafish models, researchers have employed genetic approaches including targeted mutations and transgenic rescue experiments to study MSMO1 function . For instance, researchers created mutations eliminating MSMO1 activity and then performed liver-specific restoration of the enzyme to assess its tissue-specific roles . RNA isolation and quantification from different tissues can be used to measure expression levels, as was done in zebrafish studies comparing gene expression in bone-enriched tissues versus other body regions .

How can tissue-specific expression of MSMO1 be manipulated in experimental models?

Advanced manipulation of MSMO1 expression in specific tissues can be achieved through several methodological approaches. In zebrafish models, researchers successfully employed partial endoderm replacement to create chimeric endodermal organs with restored MSMO1 function . This was accomplished by overexpressing sox32 at the one-cell stage to push donor embryo cells toward an endodermal fate, followed by transplantation into host embryos . Tissue-specific transgenic rescue is another effective approach, where MSMO1 expression is driven by tissue-specific promoters. For example, liver-specific restoration of MSMO1 in corresponding mutant backgrounds has been demonstrated to suppress larval lethality in zebrafish . Inducible expression systems, such as heat-shock promoters (hsp70l), have also been used to control the timing and level of MSMO1 expression, as demonstrated by the ability to suppress early lethality in msmo1 homozygotes through controlled overexpression . These methodologies provide valuable tools for dissecting the tissue-specific roles of MSMO1 in development and disease.

What are the molecular mechanisms by which MSMO1 dysfunction affects chondrocyte differentiation and skeletal development?

MSMO1 dysfunction severely impacts chondrocyte differentiation and skeletal development through complex molecular mechanisms. Research in zebrafish demonstrates that loss of MSMO1 expression in pre-hypertrophic chondrocytes results in a near-complete loss of hypertrophic chondrocytes, which are normally marked by col10a1a expression . The molecular basis appears to involve both cholesterol deprivation and accumulation of sterol intermediates. Comparative studies between single msmo1 mutants and double mutants also lacking Lanosterol synthase (Lss) revealed that the combined deficiencies altered survival outcomes, suggesting interacting pathways . Importantly, this skeletal phenotype does not appear to result from disruption of Indian hedgehog (Ihh) signaling within growth plates, indicating alternative mechanistic pathways . The relationship between cholesterol metabolism and chondrocyte differentiation likely involves multiple signaling pathways and cellular processes, including potential effects on membrane properties, steroid hormone production, or direct regulatory roles of specific sterol intermediates in gene expression and cellular differentiation.

What bioinformatic approaches can be used to analyze MSMO1 expression patterns across different disease states?

Comprehensive bioinformatic analysis of MSMO1 expression across disease states requires sophisticated methodological approaches. Researchers studying cervical cancer employed R code and Perl language tools to analyze gene expression profiles from The Cancer Genome Atlas (TCGA) database, comparing cancer tissues with para-cancerous tissues to identify differential expression . Multiple database validations enhance reliability—researchers verified MSMO1 expression patterns using Oncomine, GEPIA2, and UALCAN databases . Heat maps generated from GSE7803 expression profile data can visually represent expression patterns across different sample types . ROC curve analysis using "pROC" package in R software quantifies the diagnostic potential of MSMO1, with researchers finding high area under the curve (AUC) values (AUC = 0.720 for T stage, AUC = 0.751 for independent prognostic analysis) . Correlation between gene expression and clinical data requires linking expression datasets with patient information through programming tools, followed by statistical analyses including logistic regression, Cox regression, and Kaplan-Meier survival analysis . These methodological approaches provide a framework for researchers investigating MSMO1 in various disease contexts.

How does Pongo abelii MSMO1 differ structurally and functionally from human MSMO1?

Analyzing the structural and functional differences between Pongo abelii and human MSMO1 requires detailed comparative biochemistry and molecular biology approaches. While specific comparative data between these species is limited in the provided search results, systematic approaches would include sequence alignment analysis to identify conserved domains and species-specific variations. The metal binding motifs and active site residues would be of particular interest for functional comparison . Recombinant protein expression of both variants, followed by enzymatic assays measuring substrate specificity, reaction kinetics, and inhibitor sensitivity would provide functional insights. Crystal structure determination or homology modeling could reveal three-dimensional structural differences that might impact enzyme activity or regulation. Additionally, cellular localization studies using fluorescently tagged proteins could identify any differences in subcellular targeting or membrane association. Given the evolutionary closeness of orangutans and humans, any significant differences identified could highlight functionally important adaptations in cholesterol metabolism pathways during primate evolution.

What insights can phylogenetic analysis of MSMO1 across primates provide about evolutionary conservation of cholesterol metabolism?

Phylogenetic analysis of MSMO1 across primates can reveal evolutionary patterns in cholesterol metabolism through several methodological approaches. Researchers should begin by collecting MSMO1 sequences from diverse primate species, including great apes (orangutans, chimpanzees, gorillas), Old World monkeys, New World monkeys, and prosimians. Multiple sequence alignment using tools like MUSCLE or CLUSTAL would identify conserved domains and variable regions. Construction of phylogenetic trees using maximum likelihood or Bayesian methods would reveal the evolutionary relationships of MSMO1 across the primate lineage. Selection pressure analysis using dN/dS ratios can identify regions under purifying or positive selection, providing insights into functionally critical domains. Ancestral sequence reconstruction could determine the likely MSMO1 sequence in the primate common ancestor and trace evolutionary changes. Correlation of MSMO1 sequence variations with species-specific metabolic adaptations or disease susceptibilities could reveal functional consequences of evolutionary changes. This comprehensive phylogenetic approach would provide valuable insights into how cholesterol metabolism pathways have evolved within the primate lineage and identify potentially important adaptive changes in Pongo abelii.

How can zebrafish models of MSMO1 deficiency inform our understanding of MSMO1 function in primates?

Zebrafish models provide powerful tools for understanding MSMO1 function with important translational implications for primate biology. Research has shown that zebrafish msmo1 homozygote mutants exhibit developmental defects and die by 9 days post-fertilization (dpf), demonstrating the essential nature of this enzyme . These models reveal tissue-specific requirements for MSMO1 function—while koliber nu7 homozygote fish (with decreased MSMO1 expression in pre-hypertrophic chondrocytes) survive to adulthood with skeletal abnormalities, complete loss of MSMO1 is lethal . Intervention studies in zebrafish show that liver-specific restoration of MSMO1 activity is sufficient for post-larval survival, highlighting the crucial role of hepatic cholesterol synthesis . Transgenic rescue experiments using heat-shock promoter driven wild-type msmo1 expression (Tg(hsp70l:msmo1:IRESnlsEGFP)) successfully suppressed early lethality in msmo1 homozygotes, confirming the specific role of MSMO1 in the observed phenotypes . These methodological approaches in zebrafish can inform experimental design for studying MSMO1 function in primate cell lines and potential development of primate disease models.

What are common challenges in producing and purifying active recombinant Pongo abelii MSMO1?

Producing and purifying active recombinant Pongo abelii MSMO1 presents several technical challenges requiring careful methodological considerations. As a membrane-associated enzyme localized to the endoplasmic reticulum, MSMO1 contains hydrophobic domains that can cause folding issues and aggregation during heterologous expression . Expression systems should be carefully selected—mammalian cell systems may provide more appropriate post-translational modifications and membrane environments than bacterial systems. Codon optimization for the expression host is advisable, as is consideration of expression temperature and induction conditions to maximize proper folding. Purification approaches must account for MSMO1's membrane association—detergent selection is critical, with gentle detergents like digitonin or DDM often preferred for maintaining enzymatic activity. Tag design and placement require careful consideration to avoid interfering with enzyme function—the tag type may need to be determined during the production process for optimal results . Storage conditions are also crucial—the commercial recombinant Pongo abelii MSMO1 is recommended to be stored in Tris-based buffer with 50% glycerol at -20°C, with repeated freeze-thaw cycles not recommended . Working aliquots can be stored at 4°C for up to one week to minimize activity loss .

How can researchers address variability in MSMO1 activity assays across different experimental conditions?

Addressing variability in MSMO1 activity assays requires systematic methodological approaches. Researchers should first standardize enzyme preparations, ensuring consistent protein concentration, purity, and storage conditions. Buffer composition significantly impacts membrane enzyme activity—parameters including pH, ionic strength, and the presence of stabilizing agents should be optimized and strictly controlled across experiments. Substrate preparation and delivery methods must account for the hydrophobic nature of sterol substrates—solubilization methods using appropriate detergents or cyclodextrins should be standardized. Control experiments should include positive controls with known MSMO1 activators and negative controls with specific inhibitors or heat-inactivated enzyme. Verification of activity through multiple complementary assay methods provides more robust results—direct measurement of substrate conversion by chromatographic methods can complement spectrophotometric assays. Statistical approaches to address variability include performing sufficient biological and technical replicates, using appropriate statistical tests, and implementing normalization methods when comparing across experimental conditions. By systematically addressing these factors, researchers can significantly reduce assay variability and increase the reliability of MSMO1 activity measurements.

What controls and validations are necessary when studying MSMO1 in disease models?

Rigorous controls and validations are essential when investigating MSMO1 in disease models. Expression validation through multiple methodologies strengthens findings—researchers studying MSMO1 in cervical cancer validated expression patterns across multiple databases (TCGA, Oncomine, GEPIA2, UALCAN) and used both computational analysis and experimental validation . Specificity controls must confirm that observed phenotypes are directly attributable to MSMO1—genetic rescue experiments, as performed in zebrafish models using wild-type msmo1 driven by heat-shock promoters, provide compelling evidence of specificity . Dosage effects should be assessed through carefully designed experiments comparing heterozygous and homozygous models or using inducible systems with variable expression levels. Tissue-specific validation is crucial—the zebrafish studies revealed dramatically different consequences of MSMO1 deficiency in different tissues, with a 10-fold downregulation in chondrocytes but only 2-fold in trunks . Temporal considerations must be addressed through time-course studies, as MSMO1 deficiency may have different effects during development versus adulthood. Disease relevance must be established through careful correlation with human patient data when possible—studies in cervical cancer demonstrated this through detailed survival analysis and correlation with clinical parameters . These methodological controls ensure robust and translatable findings when studying MSMO1 in disease contexts.

How might MSMO1 function in immunomodulation be further investigated?

The emerging role of MSMO1 in immunomodulation presents exciting research opportunities requiring sophisticated methodological approaches. Studies in cervical cancer have identified significant correlations between MSMO1 expression and multiple immune cell subsets, with negative correlations observed for activated B cells, eosinophils, immature B cells, mast cells, monocytes, natural killer cells, and plasmacytoid dendritic cells, while CD56bright cells showed positive correlation . Further investigation should employ single-cell RNA sequencing to precisely map MSMO1 expression across immune cell populations and identify cell-specific effects. Co-culture systems with immune cells and MSMO1-modulated cells can assess direct immunomodulatory effects. In vivo models with tissue-specific or inducible MSMO1 knockdown/overexpression in immune compartments would reveal functional consequences. Mechanistic studies should investigate whether MSMO1's immunomodulatory effects stem from altered membrane cholesterol content affecting immune receptor function, changes in steroid hormone production influencing immune cell development, or direct effects of specific sterol intermediates as signaling molecules. The identified associations with 22 immune stimulators and 14 immune inhibitors provide specific targets for mechanistic investigation . These methodological approaches would significantly advance our understanding of MSMO1's role in immune regulation with potential therapeutic implications.

How can advanced imaging techniques enhance our understanding of MSMO1 subcellular localization and dynamics?

Advanced imaging methodologies offer powerful approaches to elucidate MSMO1 subcellular dynamics. Super-resolution microscopy techniques like STORM or PALM can visualize MSMO1 distribution within the endoplasmic reticulum at nanoscale resolution, revealing potential microdomains or associations with other biosynthetic enzymes. Live-cell imaging with fluorescently tagged MSMO1 can track dynamic changes in localization in response to cellular cholesterol levels or during developmental processes. Correlative light and electron microscopy (CLEM) could connect MSMO1 localization with ultrastructural features of the endoplasmic reticulum membrane. Proximity labeling approaches like BioID or APEX2 can identify proteins in the immediate vicinity of MSMO1, revealing potential interaction partners or regulatory complexes. FRET-based biosensors could be developed to monitor MSMO1 activity in real-time within living cells. Multi-color imaging can simultaneously track MSMO1 alongside its substrates, products, or other pathway components to understand spatial relationships. Tissue-clearing techniques combined with light-sheet microscopy could map MSMO1 distribution across entire organs or organisms during development. These advanced imaging approaches would significantly enhance our understanding of how MSMO1 functions within the complex cellular environment and how its dynamics contribute to normal development and disease states.