Recombinant Monopterus albus Osteoclast-stimulating factor 1 (ostf1)

What is Monopterus albus Osteoclast-stimulating Factor 1 and what is its functional significance?

Osteoclast-stimulating Factor 1 (ostf1) is an SH3-domain containing protein originally identified for its role in the indirect activation of osteoclasts, which are cells responsible for bone resorption. In mammalian models, ostf1 has been linked to bone development, as evidenced by increased trabecular mass in the long bones of ostf1 knockout mice . While specific research on Monopterus albus ostf1 is emerging, its study is valuable for comparative analysis with mammalian systems.

The protein plays significant roles beyond bone metabolism, as indicated by its widespread expression in the vasculature of most organs and in various cell types in both adult and embryonic tissues . Its functional importance is further underscored by its association with human conditions - ostf1 has been linked to spinal muscular atrophy through its interaction with SMN1 and is one of six genes deleted in a human developmental microdeletion syndrome at 9q21.13 .

How do environmental factors influence ostf1 expression in Monopterus albus?

Temperature is a critical environmental factor affecting gene expression in Monopterus albus. Although specific data on ostf1 response to temperature is still being developed, research on other genes provides a framework for investigating ostf1 regulation.

Studies show that high-temperature stimulation significantly affects gene expression patterns in M. albus, with many immune-related genes showing temperature-dependent expression profiles. For instance, genes like TLR1, C3, TNF-α, and IL-β display similar patterns following exposure to high temperatures, initially increasing in expression before declining, with peak expression observed at 34°C . In contrast, other genes like IRAK3 show an inverse pattern, initially decreasing in expression before increasing .

Research methodologies examining ostf1 expression should therefore include carefully controlled temperature conditions, preferably testing at multiple temperature points between 30°C and 38°C to capture the complete response profile.

What expression systems are optimal for producing recombinant Monopterus albus ostf1?

When expressing recombinant M. albus ostf1, researchers should consider both prokaryotic and eukaryotic expression systems, each offering distinct advantages depending on research objectives.

For functional studies, mammalian or fish cell lines may provide more physiologically relevant expression conditions. Given that temperature significantly affects gene expression in M. albus, with optimal physiological responses observed at 32°C-34°C , expression systems should be maintained within this temperature range to ensure proper protein folding and modification.

What approaches can be used to characterize protein-protein interactions of recombinant Monopterus albus ostf1?

Characterizing protein-protein interactions of M. albus ostf1 requires multiple complementary approaches to ensure robust results. Based on established methodologies for mammalian ostf1, researchers should consider:

• Co-immunoprecipitation assays: This technique has successfully identified ostf1 interactions with F-actin, c-Src, and Cbl in mammalian systems . When applying this method to M. albus ostf1, use species-specific antibodies or epitope tags to ensure specificity.

• Peptide array analysis: This high-throughput approach can systematically map interaction domains within ostf1 and its binding partners.

• Yeast two-hybrid screening: This method has previously identified ostf1 interactions with SMN1 and SMN2 and can be adapted to screen M. albus cDNA libraries for novel binding partners.

• Proximity labeling techniques: BioID or APEX2-based approaches can identify proximal proteins in the native cellular environment, capturing both stable and transient interactions.

When investigating whether M. albus ostf1 maintains the same interactions observed in mammalian systems, particular attention should be paid to interactions with bone metabolism regulators, as ostf1 knockout in mice leads to increased trabecular mass in long bones .

How can CRISPR-Cas9 technology be optimized for ostf1 knockout in Monopterus albus?

Developing effective CRISPR-Cas9 strategies for ostf1 knockout in M. albus requires careful consideration of several technical factors:

• Guide RNA design: Multiple sgRNAs targeting different exons of ostf1 should be designed and validated. Focus on early exons (such as exons 3 and 4, as targeted in mouse models) to ensure complete functional disruption.

• Delivery method optimization: For M. albus embryos, microinjection at the one-cell stage offers the highest efficiency. Electroporation may be considered for juvenile specimens.

• Temperature considerations: Since M. albus shows optimal physiological responses at 32°C-34°C , maintain embryos and cell cultures within this range during and after CRISPR-Cas9 delivery to ensure optimal cellular repair mechanisms.

• Verification strategies: Employ T7 endonuclease assays for initial screening, followed by sequencing and Western blot confirmation of protein knockout. RT-PCR analysis should verify the absence of alternative splicing that might rescue function.

• Off-target analysis: Conduct whole-genome sequencing on a subset of knockout individuals to assess off-target effects, which can be particularly problematic in fish genomes with high repetitive content.

What are the implications of temperature variation for ostf1 function in Monopterus albus?

Temperature significantly impacts gene expression and protein function in M. albus, with profound implications for ostf1 research. Experimental designs investigating ostf1 function must account for the following temperature-related considerations:

High-temperature stimulation has been shown to enhance immunity and adaptability in M. albus, with optimal responses observed at 32°C-34°C . At this temperature range, several physiological parameters reach their peak:

• Growth performance indices including feed conversion ratio (FCR), final body weight (FBW), and specific growth rate (SGR) show optimal values at 34°C, as demonstrated in the table below:

• Antioxidant enzyme activities (SOD, CAT, and POD) in the liver reach peak levels at 34°C .

• Expression of immune-related genes follows temperature-dependent patterns, with many showing peak expression at 34°C .

Given that ostf1 plays roles in both bone metabolism and immune function through its interactions with various proteins, temperature fluctuations likely modulate these functions in M. albus. Researchers investigating ostf1 should design experiments with precise temperature controls and consider examining ostf1 expression and function across a temperature gradient of 30°C-38°C to capture potential temperature-dependent effects.

How does Monopterus albus ostf1 compare structurally and functionally to its mammalian counterparts?

Comparative analysis of M. albus ostf1 with mammalian homologs provides important evolutionary insights. While specific structural comparisons await further characterization, functional patterns can be inferred from what is known about mammalian ostf1:

In mammals, ostf1 contains an SH3 domain crucial for protein-protein interactions . This domain enables ostf1 to interact with partners including F-actin, c-Src, and Cbl, with the latter interaction being particularly important for bone-resorption properties in osteoclasts . The interaction with Cbl is strengthened by co-localization in the podosomes of osteoclast-like cells .

Functional studies in mammals demonstrate that ostf1 influences bone density, as evidenced by increased trabecular mass in ostf1 knockout mice . Additionally, mammalian ostf1 interacts with Survival of Motor Neuron proteins (SMN1 and SMN2) , suggesting roles beyond bone metabolism.

Researchers investigating M. albus ostf1 should examine whether these key structural features and functional interactions are conserved. Particular attention should be paid to:

• Domain conservation analysis through bioinformatic approaches

• Yeast two-hybrid or co-immunoprecipitation studies testing interactions with M. albus homologs of known mammalian binding partners

• Tissue expression patterns, with special focus on bone tissue and vascular systems, which show high ostf1 expression in mammals

What insights can Monopterus albus ostf1 research provide for understanding bone disorders in fish and mammals?

Research on M. albus ostf1 offers unique comparative perspectives on bone metabolism across vertebrate lineages:

• Temperature-dependent regulation: Unlike mammals, fish experience significant environmental temperature fluctuations that affect their physiology. M. albus shows optimal growth and immune function at 32°C-34°C , but temperatures above 36°C cause liver cell damage and growth inhibition . Understanding how temperature modulates ostf1 function in fish could reveal adaptive mechanisms relevant to bone homeostasis under varying conditions.

• Evolutionary adaptations: M. albus has unique environmental adaptations, including air-breathing capabilities and extended survival in oxygen-poor conditions. These adaptations may have influenced ostf1 function in ways distinct from mammalian counterparts.

• Translational potential: Given that ostf1 knockout in mice increases trabecular bone mass , comparative studies in M. albus could provide insights into conserved mechanisms of bone density regulation relevant to human conditions like osteoporosis.

Research approaches should include:

• Histological analysis of bone tissue in ostf1-manipulated M. albus specimens

• Molecular characterization of ostf1-mediated signaling pathways in bone cells

• Comparative genomic analysis of regulatory elements controlling ostf1 expression across vertebrate lineages

How can researchers address the challenges of purifying functional recombinant Monopterus albus ostf1?

Purifying functional recombinant M. albus ostf1 presents several technical challenges that researchers can address through methodological optimizations:

• Solubility enhancement: SH3-domain containing proteins like ostf1 may experience solubility issues during recombinant expression. Researchers should consider:

  • Fusion tags that enhance solubility (MBP, SUMO, or Thioredoxin)

  • Expression at lower temperatures (16-20°C) to improve proper folding

  • Co-expression with chaperone proteins

  • Screening multiple detergents for extraction if membrane association occurs

• Temperature considerations: Since M. albus physiology is optimized at 32°C-34°C , expression and purification protocols should be tested within this temperature range. Researchers should avoid temperatures above 36°C, which have been shown to cause cellular damage in M. albus .

• Post-translational modifications: If functional studies are planned, eukaryotic expression systems may be necessary to maintain relevant post-translational modifications. Given ostf1's interactions with proteins like c-Src , phosphorylation status may be critical for function.

• Activity verification: Purified ostf1 should be validated through functional assays such as:

  • Binding assays with known interaction partners (F-actin, c-Src, Cbl)

  • Osteoclast activation assays, given ostf1's role in osteoclast stimulation

  • Thermal stability assays to assess proper folding across a temperature range

What experimental designs best address the multifunctional nature of ostf1 in Monopterus albus?

Given ostf1's diverse roles in bone metabolism, immune function, and potentially other physiological processes, comprehensive experimental designs should address its multifunctional nature:

• Tissue-specific expression analysis: Quantitative RT-PCR and in situ hybridization across multiple tissues and developmental stages. In mammals, ostf1 shows widespread expression in vasculature and various cell types , suggesting similar broad distribution may occur in M. albus.

• Conditional knockout/knockdown approaches: Rather than global knockout, consider:

  • Tissue-specific CRISPR interference using tissue-specific promoters

  • Inducible knockout systems that can activate at specific developmental stages

  • Morpholino-based knockdown for transient, dose-dependent functional analysis

• Integrated multi-omics approach:

  • Transcriptomics to identify co-expressed gene networks

  • Proteomics to map interaction partners in different tissues

  • Metabolomics to assess downstream effects on bone metabolism

• Environmental variable testing: Include temperature gradients (30°C-38°C) in experimental designs, as M. albus shows significant physiological differences across this range . Other environmental stressors like hypoxia may also be relevant given M. albus's adaptations to diverse environments.

• Functional redundancy analysis: Investigate potential compensatory mechanisms involving related proteins or pathways that may mask ostf1 phenotypes in certain contexts.

What are promising avenues for translational research involving Monopterus albus ostf1?

Research on M. albus ostf1 offers several promising translational directions:

• Bone density regulation: Given that ostf1 knockout in mice leads to increased trabecular mass in long bones , M. albus ostf1 could provide insights into novel approaches for treating conditions like osteoporosis. The temperature-dependent regulation of gene expression in M. albus offers a unique experimental advantage for studying how environmental factors influence bone metabolism.

• Environmental adaptation mechanisms: M. albus thrives in diverse environments and shows optimal growth and immune function at specific temperatures (32°C-34°C) . Understanding how ostf1 contributes to this adaptive capability could inform research on climate change impacts on vertebrate physiology.

• Comparative immunology: Ostf1 interacts with components of immune signaling pathways. In M. albus, high-temperature stress activates immune-related genes like TLR1, TNF-α, and IL-β , suggesting potential roles for ostf1 in temperature-dependent immune regulation that may be relevant to human inflammatory conditions.

• Neurodegenerative disease insights: Mammalian ostf1 interacts with SMN1 and SMN2, proteins linked to spinal muscular atrophy . Comparative studies in M. albus could reveal evolutionarily conserved mechanisms relevant to neurodegenerative disorders.

How might integrative approaches enhance our understanding of ostf1 function across diverse vertebrate systems?

Advancing our understanding of ostf1 function requires integrative approaches spanning multiple disciplines and model systems:

• Evolutionary functional genomics: Comparing ostf1 sequence, structure, and function across vertebrate lineages can reveal conserved domains crucial for core functions versus lineage-specific adaptations. This approach should include:

  • Phylogenetic analysis of ostf1 across fish and tetrapod lineages

  • Identification of conserved regulatory elements controlling expression

  • Cross-species functional rescue experiments

• Systems biology integration: Developing comprehensive models of ostf1 function through:

  • Network analysis of protein-protein interactions across species

  • Pathway mapping to identify conserved and divergent signaling cascades

  • Mathematical modeling of ostf1's role in bone homeostasis under varying conditions

• Environmental physiology perspectives: M. albus provides an excellent model for studying how environmental factors modulate gene function. Research designs should integrate:

  • Controlled environmental chambers allowing precise manipulation of temperature and other variables

  • Field studies examining ostf1 expression in wild M. albus populations across habitats

  • Epigenetic analysis of environmentally-induced modifications affecting ostf1 regulation

• Interdisciplinary technology application: Emerging technologies that could advance ostf1 research include:

  • Single-cell transcriptomics to resolve cell-type specific expression patterns

  • CRISPR-based epigenome editing to manipulate ostf1 regulation

  • Advanced imaging approaches to visualize ostf1-dependent processes in vivo

By integrating these approaches, researchers can develop a comprehensive understanding of ostf1 function that spans from molecular mechanisms to ecological adaptations, ultimately contributing to both basic science and translational applications.