Recombinant Human Uncharacterized protein C2orf62 (C2orf62)

What is C2orf62 and why is it classified as an uncharacterized protein?

C2orf62 is a 387 amino acid protein encoded on human chromosome 2 that remains functionally uncharacterized despite its high conservation across metazoans. It lacks annotated functional domains and has little homology to characterized proteins. Until recently, its existence had only been validated at transcript level in the brain (BC052750), with no comprehensive functional studies available . C2orf62 is part of the "dark proteome" - proteins with experimentally validated expression but unknown function - making it a target for the neXt-CP50 challenge that aims to characterize uncharacterized PE1 proteins (uPE1) .

What is the expression pattern of C2orf62 across different human tissues?

C2orf62 shows tissue-specific expression patterns in humans. RT-PCR analysis demonstrates that C2orf62 is:

• Highly expressed in testis, placenta, prostate, and lung

• Moderately expressed in ovary and brain

• Undetectable in other tissues examined

This restricted expression pattern suggests potential tissue-specific functions, particularly in reproductive and ciliated tissues.

How is C2orf62 expression regulated during development?

In zebrafish embryos, zC2orf62 exhibits dynamic expression patterns:

• Expression starts around 12 hours post-fertilization (hpf) in Kupffer's vesicle

• At 24 hpf, expression is observed in the extremity of neural tube formation inside the tail, brain, and pronephric ducts

• At 28 hpf, expression expands to the olfactory placode and eye

• By 48 hpf, expression becomes restricted to brain, olfactory pits, and various ciliated cells (olfactory placode, ear, neuromasts, pronephric ducts)

• At 96 hpf, expression is observed in neuromasts, olfactory sensory neurons, and ear sensory patches

This developmental expression pattern, particularly in structures containing ciliated cells, supports C2orf62's potential role in ciliogenesis.

Which cell lines express C2orf62 and how does this correlate with cellular phenotypes?

C2orf62 expression correlates strongly with a cell's ability to form cilia:

• Expressed in cilia-forming cell lines: HEK293T, PANC-1, hTERT-RPE1, and HepG2

• Not expressed in cell lines lacking cilia: HeLa, Huh-7, and HOS

This expression pattern provides further evidence supporting C2orf62's involvement in cilia-related processes.

What protein interactions have been identified for C2orf62?

Yeast two-hybrid screening of a human fetal brain cDNA library using full-length C2orf62 as bait identified several potential interacting partners. Three proteins were selected as particularly relevant:

• TTC17 (aa 945-1041): Confirmed to interact with C2orf62 through GST pull-down assays

• PRKRA: Known to play a role in ciliogenesis, but interaction not confirmed in GST pull-down

• CEP192 (aa 1501-1941): Involved in cell cycle and cilia formation, but interaction not confirmed in GST pull-down

The TTC17 interaction is particularly significant as it was validated through multiple methods including FRET analysis, confirming colocalization in ciliated cells.

What is known about the C2orf62-TTC17 interaction and its functional significance?

The C2orf62-TTC17 interaction appears to form a functional axis that regulates both actin polymerization and ciliogenesis:

• Both proteins colocalize in discrete parts of the cytoplasm and in cell protrusions

• Depletion of either protein in human ciliated cells interferes with actin polymerization

• This depletion reduces the number of primary cilia without changing their length

• The interaction was confirmed through multiple methods: yeast two-hybrid, GST pull-down, and FRET analysis

This evidence suggests that C2orf62 and TTC17 function together in a pathway that coordinates cytoskeletal organization with cilia formation.

What functional domains have been identified in C2orf62?

Despite lacking annotated functional domains in major databases, structural analysis of C2orf62 has revealed:

• A C-terminal region containing an RII dimerization/docking (R2D2)-like domain

• This domain is functionally significant, as overexpression of just the C2orf62 C-terminal part in zebrafish embryos produces morphological defects consistent with imperfect ciliogenesis

The presence of this R2D2-like domain suggests a possible role in protein-protein interactions or regulatory functions.

What expression systems and tags are recommended for producing recombinant C2orf62?

Based on published research, successful recombinant C2orf62 production has been achieved using:

• V5-tagged C2orf62 expressed in HEK293T cells for subcellular localization studies and pull-down assays

• EGFP-C2orf62 fusion proteins for live imaging and colocalization studies with TTC17

• GST-fusion proteins for interaction studies

These systems allow for different experimental applications while maintaining protein functionality.

What methods have been effective for studying C2orf62 subcellular localization?

Researchers have successfully determined C2orf62 subcellular localization using:

• Fluorescent protein fusions (EGFP-C2orf62) for live-cell imaging

• V5-tagged C2orf62 with immunofluorescence for fixed-cell imaging

• Co-staining with markers like acetylated tubulin (for cilia) to determine spatial relationships

• FRET analysis to confirm protein-protein interactions in specific cellular compartments

These approaches revealed that C2orf62 localizes to the cytoplasm, nucleus, and F-actin rich zones of the plasma membrane, but is notably excluded from cilia.

What are effective loss-of-function approaches for studying C2orf62?

Research has employed several strategies to investigate C2orf62 function through loss-of-function approaches:

• Morpholino-mediated knockdown in zebrafish embryos, which produced morphological defects consistent with ciliogenesis impairment

• Depletion of C2orf62 in human ciliated cells, which interfered with actin polymerization and reduced primary cilia numbers

These complementary approaches in different model systems strengthen the evidence for C2orf62's role in ciliogenesis.

How can researchers effectively investigate the C2orf62-TTC17 interaction?

Multiple complementary approaches have proven effective for investigating the C2orf62-TTC17 interaction:

• Yeast two-hybrid screening to identify initial interaction

• GST pull-down assays to confirm direct binding

• Co-expression of mCherry-TTC17 and EGFP-C2orf62 for colocalization studies

• FRET analysis to confirm physical proximity in live cells

• Parallel knockdown of both proteins to assess functional cooperation

This multi-method approach provides robust evidence for both physical interaction and functional cooperation between these proteins.

What developmental processes might involve C2orf62 based on current research?

Based on expression patterns and functional studies, C2orf62 likely participates in several developmental processes:

• Formation and function of ciliated structures, particularly in sensory organs (olfactory placode, ear, neuromasts)

• Development of the nervous system, as suggested by expression in brain and neural tube

• Spermatogenesis, given its strong expression in testis and localization in spermatocytes and spermatids

• Kidney development, based on expression in pronephric ducts (future kidney)

These developmental roles align with the protein's expression in ciliated tissues and its involvement in ciliogenesis.

What is the evidence for C2orf62's role in spermatogenesis?

Several lines of evidence suggest C2orf62 plays a role in spermatogenesis:

• High expression in testis across multiple species (zebrafish, rat, human)

• In rat testis, C2orf62 is highly expressed in pachytene spermatocytes and round spermatids compared to spermatogonia and somatic cells

• Immunohistochemistry shows C2orf62 protein enriched in the cytoplasm of spermatocytes at the pachytene stage and concentrated in elongating spermatids

• The Drosophila ortholog CG13243 is specifically expressed in adult testis and the protein has been identified in sperm

This conserved expression pattern in sperm development across diverse species strongly suggests a fundamental role in spermatogenesis.

What are the potential implications of C2orf62 dysfunction for human diseases?

Though not directly established in the research, C2orf62 dysfunction may contribute to several disease categories:

• Ciliopathies: Given its role in ciliogenesis, mutations might contribute to disorders characterized by cilia dysfunction

• Male infertility: Strong expression in spermatogenesis suggests potential involvement in fertility issues

• Neurodevelopmental disorders: Expression in brain and neural development may implicate it in brain development disorders

• Cancer: The COSMIC database reports three somatic mutations (R133W, I168V, and S162Y) associated with rectal adenocarcinoma samples

These potential disease associations warrant further investigation in clinical contexts.

What approaches are recommended for further functional characterization of C2orf62?

To advance understanding of C2orf62 function, researchers should consider:

• CRISPR/Cas9 knockout models in relevant cell lines and organisms

• Structure-function analysis of the R2D2-like domain

• Proteomic approaches to identify the complete interactome

• Phosphoproteomics to investigate potential post-translational modifications

• Investigating the relationship between C2orf62 and actin dynamics in more detail

• Examining potential involvement in intraflagellar transport or other cilia-related processes

These approaches could help resolve C2orf62's function as part of the neXt-CP50 challenge for dark protein characterization .

How might researchers approach studying C2orf62 in the context of human disease?

To investigate C2orf62's potential role in human diseases, researchers could:

• Screen for C2orf62 mutations in patients with ciliopathies of unknown genetic cause

• Examine C2orf62 expression and localization in tissue samples from relevant disease states

• Develop animal models with C2orf62 mutations that mimic potential human pathogenic variants

• Investigate whether the somatic mutations reported in rectal adenocarcinoma affect protein function

• Examine potential correlations between C2orf62 expression/mutations and male infertility

These approaches could help connect basic research findings to clinical relevance.

What methodological challenges should researchers anticipate when studying C2orf62?

Based on published research, several challenges may arise when studying C2orf62:

• Detection difficulties: Antibodies against C2orf62 work well in tissues with strong expression (e.g., testis) but may fail to detect specific signals in cell lines with lower expression levels

• Functional redundancy: C2orf62 may have redundant functions with other proteins, masking phenotypes in single-gene perturbation studies

• Context-dependent functions: C2orf62's function may vary across different cell types and developmental stages

• Limited prior characterization: As an uncharacterized protein, researchers lack established protocols and reagents

Addressing these challenges requires careful experimental design and validation of research tools.