Recombinant UPF0468 protein C16orf80 homolog (C54C6.6)

What is UPF0468 protein C16orf80 homolog and what are its known functions?

UPF0468 protein C16orf80 homolog, commonly known as CFAP20 (cilia and flagella associated protein 20), is a cilium- and flagellum-specific protein that plays critical roles in axonemal structure organization and motility. Research has demonstrated that CFAP20 is involved in regulating cilia size and morphology, and is required for axonemal microtubule polyglutamylation . In C. elegans, CFAP-20 maintains the structural integrity of non-motile cilia inner junctions, influencing sensory-dependent signaling and development . The protein functions within a structural/functional hub centered on the inner junction that is shared between motile and non-motile cilia, and this hub is distinct from other ciliopathy-associated domains or macromolecular complexes .

How is CFAP20 conserved across species?

CFAP20 shows remarkable evolutionary conservation across species. The protein has been studied in various organisms including humans, zebrafish, C. elegans, and Drosophila. In C. elegans, it is identified as C54C6.6 . The conservation extends to birds as well, with the protein identified in Cariama cristata (Red-legged seriema) . In Drosophila, CFAP20 is expressed in both testes and chordotonal (Ch) neurons, showing functional conservation across different ciliated cell types . This high degree of conservation suggests fundamental roles in ciliary function that have been maintained throughout evolution.

What are the structural domains and key features of CFAP20?

CFAP20 contains highly conserved residues that are essential for its function. In zebrafish studies, a frameshift mutation leading to a premature stop codon (p.Asn29*) disrupts the majority of the highly conserved CFAP20 residues . The protein is located at the inner junction of cilia, a critical structural component that connects the A and B tubules of the microtubule doublets. TEM analysis in C. elegans has shown that disruption of CFAP-20 leads to "open" or "unzipped" B-tubules that are separated from the A tubules in both the transition zone and proximal segments of different classes of cilia . This indicates that CFAP20 is essential for maintaining the structural integrity of the axoneme inner junction.

What expression systems are optimal for producing recombinant CFAP20?

Recombinant UPF0468 protein C16orf80 homolog (CFAP20) can be expressed and purified from multiple host systems. According to current research, the optimal expression systems include:

What purification strategy yields the highest purity of recombinant CFAP20?

Based on methodologies used for similar proteins, a multi-step purification strategy is recommended for obtaining high-purity recombinant CFAP20. After expression in the chosen host system, initial capture can be performed using affinity chromatography, typically with a His-tag or GST-tag system . For recombinant proteins expressed in E. coli, purities greater than 80% as determined by SDS-PAGE can be achieved . Following affinity purification, additional polishing steps such as ion exchange chromatography and size exclusion chromatography may be necessary to achieve higher purity. Storage in a Tris-based buffer with 50% glycerol helps maintain protein stability . It is crucial to validate the purity using SDS-PAGE and to assess protein folding using techniques such as circular dichroism or limited proteolysis.

How can researchers develop an efficient screening system for CFAP20 expression?

An effective screening system for CFAP20 expression can be developed using a semi-solid medium combined with a fluorescent reporter system. Based on methodologies for other recombinant proteins, researchers can construct an expression vector containing CFAP20 followed by red fluorescent protein (RFP) . This approach allows for:

• Early identification of high-expressing clones based on fluorescence intensity

• Direct correlation between fluorescence intensity and upstream target gene expression

• Screening of high-yielding and stable cell lines within approximately 3 weeks

• Improved screening efficiency compared to traditional methods

This approach overcomes limitations of conventional methods like limited dilution, cell sorting, and semi-solid medium screening, which are time-consuming, labor-intensive, and often result in low clone survival rates .

How can CFAP20's role in ciliary function be experimentally assessed?

To assess CFAP20's role in ciliary function, researchers have employed multiple complementary approaches:

• Genetic knockdown/knockout studies: RNAi knockdown or CRISPR-Cas9 mediated knockout in model organisms such as zebrafish and C. elegans can be used to assess phenotypic changes. In zebrafish, cfap20 mutants display ventral body curvature, cardiac/gut situs defects, and severe spinal curvature in adults .

• Transmission electron microscopy (TEM): TEM analysis of ciliary ultrastructure can reveal structural abnormalities. In C. elegans cfap-20 mutants, TEM showed "open" or "unzipped" B-tubules separated from A tubules in the transition zone and proximal segments of cilia .

• Immunohistochemistry/immunofluorescence: These techniques can be used to visualize CFAP20 localization in tissues. In zebrafish, CFAP20 localizes to tissues bearing motile cilia and plays an essential role in photoreceptor outer segments (non-motile cilia) .

• Functional assays: These include measuring ciliary beat frequency, flow sensing, and sensory perception. In C. elegans, CFAP20 participates in several non-motile cilia-mediated processes, including gustatory plasticity, lifespan control, locomotion, and body size determination .

• mRNA rescue experiments: Reintroducing wild-type mRNA can confirm phenotypic specificity in mutants .

What experimental design is optimal for studying interactions between CFAP20 and other ciliary proteins?

An optimal experimental design for studying CFAP20 interactions with other ciliary proteins should include multiple complementary approaches:

• Co-immunoprecipitation (Co-IP): This technique can identify physical interactions between CFAP20 and candidate proteins. Based on STRING database analysis, promising interaction candidates include PACRG, TBP, EFHC1, and EFHB .

• Proximity labeling techniques: BioID or APEX2-based proximity labeling can identify proteins that are in close physical proximity to CFAP20 within the cellular context.

• Yeast two-hybrid screening: This can be used to identify novel interaction partners.

• FRET or BRET assays: These techniques can determine if interactions occur in living cells.

• Comparative analysis across species: Examining conserved interactions across humans, zebrafish, and C. elegans can highlight evolutionarily important functional relationships.

The experimental design should include:

• Appropriate controls (positive and negative)

• Multiple biological and technical replicates

• Validation of interactions using at least two independent methods

• Analysis of the functional consequences of disrupting identified interactions

How can researchers analyze contradictory data regarding CFAP20's tissue expression patterns?

When confronted with contradictory data regarding CFAP20's tissue expression patterns, researchers should employ a systematic approach:

• Multiple detection methods: Use complementary techniques including RT-PCR, in situ hybridization, and immunohistochemistry. In Drosophila, RT-PCR confirmed that expression patterns varied between cell types, with some CFAP20 homologs expressed exclusively in testes while others were expressed in Ch neurons .

• Cell-type specific analysis: Employ techniques like fluorescence-activated cell sorting (FACS) followed by RNA analysis to determine expression in specific cell populations. This approach was successfully used to analyze expression in Drosophila Ch neurons .

• Functional validation through cell-type specific knockdown: In Drosophila, RNAi knockdown confirmed that different CFAP20 homologs yield phenotypes in only one cell type or another, correlating with their expression patterns .

• Quantitative analysis: Use qPCR to quantify expression levels across tissues, as was done to show ~90% reduction in cfap20 transcript abundance in zebrafish mutants .

• Cross-species comparison: Examine expression patterns across multiple model organisms to identify conserved expression patterns versus species-specific variations.

By integrating data from multiple approaches and considering developmental timing and experimental conditions, researchers can resolve apparent contradictions in expression data.

What is the evidence linking CFAP20 mutations to retinal dystrophy and other ciliopathies?

The evidence linking CFAP20 mutations to retinal dystrophy and other ciliopathies comes from multiple sources:

• Human genetic studies: Rare biallelic CFAP20 missense and canonical splice-site variants have been identified in 8 human patients from four unrelated families affected by inherited retinal dystrophy .

• Animal model studies: Zebrafish with CFAP20 mutations exhibit retinal dystrophy similar to human patients . Additionally, these mutants display ventral body curvature and cardiac/gut situs defects at 48 hours post-fertilization (hpf), as well as severe spinal curvature in adults .

• Mechanistic studies: CFAP20 functions within a structural/functional hub centered within the cilia inner junction. This hub is distinct from other ciliary modules or macromolecular complexes commonly associated with ciliopathies, representing a previously uncharacterized pathomechanism for retinal dystrophy .

• Dual functionality: CFAP20 functions in both motile and non-motile cilia, demonstrating that its disruption can break the traditional dichotomy between motile and non-motile ciliopathies .

Collectively, these findings establish CFAP20 as a ciliopathy candidate gene and suggest that analysis of CFAP20 mutations should be included in the genetic screening of patients with retinal dystrophies and potentially other ciliopathies.

How can researchers design experimental models to study CFAP20-related diseases?

Researchers can design effective experimental models to study CFAP20-related diseases using the following approach:

• Selection of appropriate model organisms:

  • Zebrafish: Useful for studying both motile cilia function and retinal development. CFAP20 mutants in zebrafish display retinal dystrophy and motile cilia defects .

  • C. elegans: Valuable for studying non-motile cilia functions. CFAP-20 mutants show defects in cilia structure and sensory functions .

  • Mouse models: For more complex physiological systems closer to humans.

• Generation of mutant models:

  • CRISPR-Cas9 gene editing to create null alleles or to introduce specific patient mutations

  • Morpholino knockdown for rapid assessment of phenotypes (as was done in zebrafish )

  • Conditional knockout models to study tissue-specific effects

• Comprehensive phenotypic analysis:

  • Retinal imaging and electroretinography for visual function

  • TEM analysis of ciliary ultrastructure

  • Behavioral assays to assess sensory functions

  • Developmental monitoring for systemic effects

• Rescue experiments:

  • mRNA rescue to confirm specificity of phenotypes

  • Testing potential therapeutic interventions

• Patient-derived models:

  • iPSC-derived organoids from patients with CFAP20 mutations

  • CRISPR correction of mutations in patient cells to demonstrate causality

These experimental models should be designed to incorporate controls that distinguish between CFAP20-specific effects and secondary consequences of ciliary dysfunction.

What protein interaction networks involve CFAP20 and how do these inform function?

CFAP20 participates in several protein interaction networks that provide insight into its functional roles:

• Inner Junction Hub proteins: CFAP20 interacts with PACRG (Parkin Co-Regulated Gene protein), which is located adjacent to CFAP20 at the inner junction hub. Both proteins share similar biological functions in non-motile cilia-mediated processes, including gustatory plasticity, lifespan control, locomotion, and body size determination .

• EF-hand domain-containing proteins: CFAP20 shows strong interaction scores with EFHC1 (EF-hand domain-containing protein 1) and EFHB (EF-hand domain-containing family member B) . These proteins are involved in calcium sensing and microtubule regulation, suggesting CFAP20 may have roles in calcium-dependent ciliary functions.

• Transcription factors: Interestingly, CFAP20 shows a high interaction score with TBP (TATA-box-binding protein) , suggesting potential non-ciliary roles or regulatory mechanisms that have not been fully explored.

• Axonemal components: In Drosophila, homologs of CFAP20 are expressed alongside various outer dynein arm (ODA) and inner dynein arm (IDA) components in a cell-type specific manner, suggesting functional interactions with these axonemal complexes .

These interaction networks suggest that CFAP20 functions as part of larger protein complexes that maintain ciliary structure and regulate ciliary functions in both motile and non-motile cilia.

What are the technical challenges in determining the three-dimensional structure of CFAP20?

Determining the three-dimensional structure of CFAP20 presents several technical challenges:

• Protein expression and purification:

  • While recombinant CFAP20 can be expressed in various systems, obtaining sufficient quantities of properly folded protein for structural studies may be challenging

  • The protein may require specific post-translational modifications for proper folding that are only present in eukaryotic expression systems

  • Purification to the homogeneity required for crystallization (>95%) may require optimization beyond the standard methods that achieve >80% purity

• Structural analysis methods:

  • X-ray crystallography requires the formation of well-ordered crystals, which may be difficult for membrane-associated or flexible proteins

  • NMR spectroscopy is limited by protein size and solubility

  • Cryo-EM may be challenging for smaller proteins like CFAP20 unless it's studied as part of a larger complex

• Protein-protein interactions:

  • CFAP20 may only adopt its native conformation when in complex with other proteins of the inner junction hub

  • Co-expression and co-purification with interaction partners like PACRG may be necessary

• Membrane association:

  • If CFAP20 associates with membranes in its native state, this may complicate structural determination

• Protein stability:

  • Maintaining protein stability during purification and structural analysis requires optimization of buffer conditions, potentially including the use of glycerol as a stabilizing agent

How can researchers investigate the mechanistic role of CFAP20 in maintaining axonemal integrity?

To investigate the mechanistic role of CFAP20 in maintaining axonemal integrity, researchers can employ a multi-faceted approach:

• High-resolution imaging:

  • Super-resolution microscopy to visualize CFAP20 localization within the axoneme at nanometer resolution

  • Cryo-electron tomography to visualize the 3D structure of the inner junction in wild-type and CFAP20 mutant axonemes

  • TEM analysis to characterize structural defects, such as the "open" or "unzipped" B-tubules observed in C. elegans CFAP-20 mutants

• Biochemical analysis:

  • In vitro microtubule binding assays to determine if CFAP20 directly binds to tubulin

  • Microtubule polymerization assays to assess if CFAP20 affects microtubule dynamics

  • Analysis of post-translational modifications of axonemal microtubules, as CFAP20 is required for axonemal microtubule polyglutamylation

• Domain analysis:

  • Structure-function analysis using truncated or mutated versions of CFAP20 to identify domains required for axonemal localization and function

  • Cross-linking mass spectrometry to identify contact sites between CFAP20 and other axonemal proteins

• Live cell imaging:

  • FRAP (Fluorescence Recovery After Photobleaching) analysis to study the dynamics of CFAP20 within the axoneme

  • Live imaging of fluorescently tagged CFAP20 during ciliogenesis to understand its incorporation into the axoneme

• Comparative analysis:

  • Compare the effects of CFAP20 disruption across different types of cilia (motile vs. non-motile) and in different organisms to identify conserved mechanisms