Recombinant Chicken UPF0454 protein C12orf49 homolog (RCJMB04_18o22)

What is the Recombinant Chicken UPF0454 protein C12orf49 homolog?

The Recombinant Chicken UPF0454 protein C12orf49 homolog (RCJMB04_18o22) is a protein expressed in chicken tissue that shares homology with the human C12orf49 protein. Based on comparative genomics, this protein contains the evolutionarily conserved DUF2054 domain that is present across multiple species including humans, zebrafish, and potentially some plants. The recombinant form is produced using DNA technology where the chicken gene encoding this protein is inserted into expression vectors and introduced into host systems (typically bacterial, insect, or mammalian cells) to produce the protein in larger quantities for research purposes .

What are the key structural features of Chicken C12orf49 homolog?

The chicken C12orf49 homolog likely shares structural similarities with its human counterpart, featuring:

• An N-terminal region that serves as a Golgi localization signal

• The highly conserved DUF2054 (Domain of Unknown Function 2054) domain, which is essential for its biological activity

• Potential N-glycosylation sites that may affect protein-protein interactions

• Subcellular localization primarily to the Golgi apparatus

Unlike some plant homologs (such as in A. thaliana) that possess additional C-terminal glycosyltransferase domains, the chicken version likely maintains a structure more similar to that of other vertebrates, focused on the core regulatory functions associated with lipid metabolism .

How is the function of Chicken C12orf49 homolog related to its mammalian counterparts?

Based on evolutionary conservation patterns, the chicken C12orf49 homolog likely serves similar functions to its mammalian counterparts. In mammals, C12orf49 plays a crucial role in cholesterol and fatty acid metabolism by:

• Localizing to the Golgi apparatus

• Binding to site 1 protease (MBTPS1)

• Facilitating the cleavage of SREBP transcription factors and other MBTPS1 substrates

• Promoting cell proliferation under cholesterol-depleted conditions

Given the high level of conservation of the DUF2054 domain across species, the chicken homolog can be expected to perform similar functions in avian cholesterol and lipid homeostasis, though species-specific regulatory mechanisms may exist .

What expression systems are typically used for recombinant production of this protein?

For the recombinant production of chicken C12orf49 homolog, researchers typically employ several expression systems, each with advantages for different research applications:

The choice of expression system should be guided by the specific research objectives. For studies focusing on protein-protein interactions, particularly with MBTPS1, mammalian expression systems may provide the most physiologically relevant results as they can properly localize the protein to the Golgi and maintain necessary post-translational modifications .

How does the Chicken C12orf49 homolog contribute to SREBP processing in avian systems?

The chicken C12orf49 homolog likely plays a role in SREBP processing similar to its human counterpart, but with avian-specific adaptations. In the SREBP processing pathway:

• The protein likely localizes to the Golgi apparatus through its N-terminal targeting sequence

• It interacts with and modulates the activity of avian MBTPS1 (site 1 protease)

• This interaction facilitates the proteolytic processing of SREBPs and other MBTPS1 substrates

• Processed SREBPs then translocate to the nucleus to regulate genes involved in lipid metabolism

Experimental evidence from mammalian systems indicates that C12orf49 functions downstream of SCAP (SREBP cleavage-activating protein) localization but upstream of mature SREBP function. Knockdown or knockout of C12orf49 in mammalian systems prevents SREBP cleavage even upon brefeldin A treatment, suggesting the chicken homolog may similarly be essential for SREBP processing rather than affecting the nuclear function of mature SREBPs .

To fully characterize this pathway in chicken cells, researchers should design experiments comparing wild-type and C12orf49-depleted chicken hepatocytes or fibroblasts under various sterol conditions to examine SREBP processing efficiency.

What methodological approaches are most effective for studying protein-protein interactions involving Chicken C12orf49 homolog?

For investigating protein-protein interactions of chicken C12orf49 homolog, particularly with MBTPS1 and other potential binding partners, several complementary approaches are recommended:

Research on human C12orf49 has shown that it specifically immunoprecipitates with an N-glycosylated form of S1P (MBTPS1), with this interaction being sensitive to PNGase F treatment. This interaction requires both the proper Golgi localization of the protein and the presence of the DUF2054 domain. Similar experimental approaches should be effective for the chicken homolog, with attention to potential species-specific differences in glycosylation patterns .

How does the chicken C12orf49 homolog compare to orthologs in other species in terms of functional conservation?

The functional conservation of C12orf49 across species appears significant but with some taxonomic variations:

What are the challenges in crystallizing the Chicken C12orf49a homolog for structural studies?

Crystallizing chicken C12orf49 homolog presents several challenges that researchers must address:

• Membrane association: The protein's Golgi localization suggests it may have membrane-interacting regions that reduce solubility and homogeneity

• Post-translational modifications: N-glycosylation sites may create heterogeneity in the protein sample

• Flexible regions: The protein likely contains disordered regions that impede crystal formation

• Protein-protein interactions: Its function in binding MBTPS1 suggests it may not be stable in isolation

To overcome these challenges, researchers might consider:

• Creating truncated constructs focusing on the DUF2054 domain

• Using deglycosylation enzymes like PNGase F to reduce glycan heterogeneity

• Employing co-crystallization with stabilizing binding partners

• Exploring alternative structural biology techniques such as cryo-electron microscopy or NMR for flexible regions

How should one design CRISPR-Cas9 knockout experiments for studying Chicken C12orf49 homolog function?

When designing CRISPR-Cas9 knockout experiments for the chicken C12orf49 homolog:

• Target selection:

  • Target the conserved DUF2054 domain for maximum disruption

  • Design at least 3-4 guide RNAs (gRNAs) targeting different exons

  • Avoid regions with potential off-target effects using predictive algorithms

• Cell line selection:

  • Use chicken hepatocyte cell lines (such as LMH) for metabolic studies

  • Consider chicken fibroblast lines for general cellular functions

  • Include appropriate wild-type and negative control cell lines

• Validation approaches:

  • Confirm gene disruption by sequencing the targeted locus

  • Verify protein loss by Western blotting with specific antibodies

  • Perform rescue experiments with wild-type chicken C12orf49 and mutant variants

• Phenotypic characterization:

  • Assess SREBP processing under normal and sterol-depleted conditions

  • Examine lipid droplet formation using fluorescent dyes

  • Measure expression of SREBP target genes using qRT-PCR

  • Analyze cellular cholesterol and fatty acid levels

What expression vector systems are optimal for recombinant production of the Chicken C12orf49 homolog?

The optimal expression vector systems for recombinant production of chicken C12orf49 homolog depend on the intended application:

For studies involving Golgi localization and MBTPS1 interaction, mammalian or avian expression systems would be most appropriate as they maintain the cellular compartmentalization and post-translational modifications necessary for authentic function. For biochemical characterization of the DUF2054 domain, bacterial systems may suffice if protein solubility can be achieved .

How can researchers establish a reliable cholesterol metabolism assay using the Chicken C12orf49 homolog?

To establish a reliable cholesterol metabolism assay using chicken C12orf49 homolog:

• Cell system preparation:

  • Generate C12orf49-knockout chicken cell lines using CRISPR-Cas9

  • Create stable lines expressing wild-type or mutant versions of chicken C12orf49

  • Include appropriate controls (wild-type cells, empty vector transfected)

• Cholesterol depletion protocol:

  • Culture cells in lipoprotein-deficient serum

  • Add cholesterol synthesis inhibitors (e.g., statins)

  • Include sterol regulatory elements (25-hydroxycholesterol) in control conditions

• Validation experiments:

  • Rescue experiments with human C12orf49 to confirm functional conservation

  • Brefeldin A treatment to assess Golgi-dependent SREBP processing

  • Overexpression of mature SREBP forms to bypass processing requirements

What are the key considerations for generating specific antibodies against the Chicken C12orf49 homolog?

Generating specific antibodies against chicken C12orf49 homolog requires careful consideration of several factors:

• Antigen design strategy:

  • Full-length protein: Provides comprehensive epitope coverage but may include conserved regions

  • DUF2054 domain: Targets the functional region but may cross-react with homologs

  • N-terminal region: Potentially more species-specific but may be less accessible in native protein

  • Synthetic peptides: High specificity but potentially lower affinity

• Production considerations:

  • Recombinant protein expression should maintain native conformation

  • Consider using chicken-specific sequences that differ from mammalian homologs

  • Ensure high purity (>90%) of immunogen

• Antibody validation protocols:

  • Western blot against recombinant protein and chicken tissue lysates

  • Immunoprecipitation to confirm specific binding

  • Immunofluorescence to verify Golgi localization

  • Testing in C12orf49-knockout cells as negative controls

  • Cross-reactivity assessment with human/mouse homologs

• Application-specific considerations:

  Application	Antibody Type	Key Considerations
  Western blotting	Polyclonal/Monoclonal	Linear epitope recognition
  Immunoprecipitation	Monoclonal	High affinity, specific binding
  Immunohistochemistry	Monoclonal	Fixation-resistant epitopes
  Proximity ligation	Paired antibodies	Non-overlapping epitopes
  ELISA	Matched pair	Capture/detection optimization

Researchers should prioritize antibodies that can distinguish between chicken C12orf49 and mammalian homologs when working in mixed systems .

How should researchers interpret differences in SREBP processing patterns between chicken and mammalian systems?

When interpreting differences in SREBP processing between chicken and mammalian systems involving C12orf49 homologs, researchers should consider:

• Evolutionary context:

  • Birds and mammals diverged approximately 320 million years ago

  • Avian-specific adaptations in lipid metabolism related to egg production and flight

  • Potential differences in regulatory mechanisms while maintaining core pathway functions

• Interpretation guidelines:

  • Direct comparison requires equivalent cellular contexts (e.g., hepatocytes from both species)

  • Consider differences in baseline cholesterol metabolism between species

  • Account for variations in protein expression levels when comparing knockout phenotypes

  • Distinguish between qualitative (pathway architecture) and quantitative (processing efficiency) differences

• Validation approach:

  • Cross-species complementation experiments to test functional conservation

  • Domain-swapping between chicken and mammalian C12orf49 to identify species-specific functional regions

  • Comparative analysis of C12orf49 binding partners across species

What statistical approaches are most appropriate for analyzing C12orf49 knockout phenotypes in chicken cells?

For analyzing C12orf49 knockout phenotypes in chicken cells, the following statistical approaches are recommended:

• Experimental design considerations:

  • Use multiple independent knockout clones (minimum 3)

  • Include appropriate controls (wild-type, empty vector)

  • Perform rescue experiments to confirm specificity

  • Account for potential off-target effects

• Statistical methods by data type:

  Data Type	Statistical Approach	Considerations
  Gene expression (qPCR)	ANOVA with post-hoc tests	Log-transform data if not normally distributed
  Protein levels (Western blot)	t-tests or ANOVA	Normalize to appropriate loading controls
  Cell viability assays	Survival analysis	Consider time-dependent effects
  Lipid measurements	ANOVA or non-parametric tests	Account for potential non-normal distribution
  High-dimensional data	PCA or clustering analysis	Appropriate for transcriptomics/proteomics

• Multiple testing correction:

  • Apply Benjamini-Hochberg or Bonferroni correction for multiple comparisons

  • Consider false discovery rate control for genome-wide studies

• Effect size reporting:

  • Include fold-change values alongside p-values

  • Report confidence intervals when possible

  • Use standardized effect sizes (Cohen's d) for cross-study comparison

• Power analysis:

  • Conduct a priori power analysis to determine sample size

  • Typically aim for 80% power at α = 0.05

  • Consider biological significance alongside statistical significance

How can researchers differentiate between direct and indirect effects of Chicken C12orf49 homolog on lipid metabolism?

Differentiating between direct and indirect effects of chicken C12orf49 homolog on lipid metabolism requires a multi-faceted experimental approach:

• Temporal analysis:

  • Use inducible knockout/knockdown systems to monitor immediate versus delayed effects

  • Perform time-course experiments after C12orf49 depletion to establish sequence of events

  • Monitor acute changes in SREBP processing versus long-term adaptations

• Mechanistic dissection:

  • Direct effects: Immediate disruption of MBTPS1-dependent SREBP processing

  • Indirect effects: Secondary changes in gene expression or cellular metabolism

• Domain-specific perturbation:

  • Generate mutations in specific domains (e.g., DUF2054) to disrupt particular functions

  • Use domain-swapping experiments to isolate functional regions

• Complementary approaches:

  • Proximity labeling to identify direct interactors

  • Rescue experiments with constitutively active SREBP to bypass processing

  • Comparative analysis with MBTPS1 knockout to identify shared versus unique effects

How might the study of Chicken C12orf49 homolog contribute to understanding avian-specific lipid metabolism?

The study of chicken C12orf49 homolog offers unique insights into avian-specific lipid metabolism:

• Avian-specific metabolic adaptations:

  • Birds have distinct lipid metabolism related to egg production

  • Avian liver is the primary site of lipogenesis (unlike mammals where adipose tissue plays a major role)

  • Unique regulatory mechanisms exist for mobilizing lipids during migration and reproduction

• Research opportunities:

  • Comparative analysis of C12orf49 function in liver versus adipose tissue in chickens

  • Examination of sex-specific differences related to egg production

  • Investigation of seasonal variations in C12orf49 expression and function

  • Analysis of C12orf49's role in yolk formation and deposition

• Potential discoveries:

  Aspect	Research Focus	Potential Impact
  Egg production	C12orf49's role in yolk lipid synthesis	Improved understanding of reproductive biology
  Flight capability	Metabolism during extended migration	Insights into extreme metabolic adaptations
  Species diversity	C12orf49 variation across avian species	Evolutionary insights into metabolic adaptation
  Domestic chicken breeds	C12orf49 variants in different breeds	Agricultural applications

• Translational relevance:

  • Birds as models for understanding metabolic disorders

  • Insights into evolutionary divergence of lipid regulation

  • Agricultural applications for improved egg production

What are the most promising future research directions for understanding the molecular function of C12orf49 homologs?

Several promising research directions for understanding the molecular function of C12orf49 homologs include:

• Structural biology approaches:

  • Crystal or cryo-EM structure of the DUF2054 domain

  • Structural analysis of C12orf49-MBTPS1 complex

  • Molecular dynamics simulations of protein-protein interactions

• Comprehensive interactome mapping:

  • Proximity labeling combined with mass spectrometry

  • Identification of additional binding partners beyond MBTPS1

  • Comparison of interactomes across species

• Detailed mechanistic studies:

  • Precise role in enhancing MBTPS1 activity

  • Potential regulatory post-translational modifications

  • Investigation of potential enzyme activity within the DUF2054 domain

• Evolutionary studies:

  • Functional analysis of C12orf49 in species lacking SREBP

  • Investigation of additional roles in lower organisms

  • Comparative genomics across vertebrate and invertebrate species

How can researchers leverage cross-species conservation of C12orf49 for comparative studies?

Researchers can leverage the cross-species conservation of C12orf49 for comparative studies through several strategic approaches:

• Phylogenetic analysis framework:

  • Construct comprehensive phylogenetic trees of C12orf49 homologs

  • Identify regions under purifying versus diversifying selection

  • Correlate sequence divergence with functional differences

  • Map species-specific insertions/deletions to functional domains

• Complementation studies:

  • Express homologs from different species in knockout cellular models

  • Assess the ability to rescue SREBP processing defects

  • Identify species-specific functional constraints

  • Create chimeric proteins to map functional domains

• Comparative expression analysis:

  Species	Tissue Distribution	Developmental Regulation	Metabolic Responsiveness
  Human	Broad, highest in liver	Stable in adult tissues	Responsive to sterol levels
  Mouse	Similar to human	Developmental changes	Similar to human
  Chicken	Predicted broad expression	Potential egg-laying regulation	Likely sterol-responsive
  Zebrafish	Broad expression	Critical in development	Responsive to dietary lipids

• Model organism comparative advantages:

  • Chickens: Avian-specific metabolism, egg production

  • Zebrafish: Developmental studies, in vivo lipid processing

  • Mice: Mammalian model, genetic tools availability

  • Cell culture: Mechanistic studies, high-throughput approaches

• Evolutionary insights:

  • Investigation of C12orf49 in species lacking SREBP orthologs

  • Analysis of co-evolution with MBTPS1 and other pathway components

  • Examination of C12orf49 in species with specialized lipid metabolism

What are the key considerations for researchers beginning work with Chicken C12orf49 homolog?

Researchers beginning work with chicken C12orf49 homolog should consider several key factors:

• Experimental system selection:

  • Primary chicken hepatocytes provide physiologically relevant context but are challenging to maintain

  • Immortalized chicken cell lines offer convenience but may have altered metabolism

  • In vivo models (embryos, chickens) provide whole-organism context but increase complexity

• Technical considerations:

  • Limited availability of chicken-specific reagents may require validation of cross-reactive tools

  • Need for appropriate controls when using antibodies developed against mammalian proteins

  • Potential requirement for custom tools (antibodies, constructs) specific to chicken C12orf49

• Methodological priorities:

  • Verification of subcellular localization to the Golgi apparatus

  • Confirmation of interaction with chicken MBTPS1

  • Establishment of knockout/knockdown models

  • Characterization of effects on SREBP processing

• Collaborative opportunities:

  • Partner with poultry research institutions

  • Leverage expertise in avian metabolism

  • Combine resources with mammalian C12orf49 researchers

  • Integrate with broader lipid metabolism research community

How should research findings on C12orf49 homologs be integrated into the broader understanding of metabolic regulation?

Research findings on C12orf49 homologs should be integrated into the broader understanding of metabolic regulation through:

• Contextual framework:

  • Position C12orf49 within the established SREBP processing pathway

  • Connect findings to broader lipid homeostasis mechanisms

  • Consider species-specific adaptations versus core conserved functions

  • Relate to other Golgi-localized regulatory proteins

• Translational connections:

  Research Area	Integration Approach	Potential Impact
  Human disease	Link to hyperlipidemia findings	Biomarker or therapeutic target development
  Agricultural science	Application to poultry research	Improved egg production, meat quality
  Comparative metabolism	Cross-species analysis	Evolutionary insights
  Systems biology	Network modeling	Comprehensive pathway understanding

• Knowledge dissemination strategy:

  • Publication in both specialized (protein science, lipid research) and broader (general biochemistry) journals

  • Data sharing through appropriate repositories

  • Integration with existing pathway databases

  • Development of educational resources on lipid metabolism regulation