Recombinant Mouse Uncharacterized membrane protein C3orf80 homolog

What is C3orf80 and where is it encoded in the genome?

C3orf80 (chromosome 3 open reading frame 80) is a single-pass membrane protein encoded by the C3orf80 gene. In humans, this gene is located on chromosome 3 at position 3q25.33, specifically from base pair 160,225,422 to 160,228,213 on the plus strand. The gene spans 2,792 bases and contains only a single exon, which is relatively uncommon for protein-coding genes . Neighboring genes include IFT80 (on the minus strand), BRD7P2 (on the plus strand), and SMC4 (on the plus strand), which function in intraflagellar transport, as a pseudogene of BRD7, and as part of the condensin complex, respectively .

What is the protein structure and cellular localization of C3orf80?

The human C3orf80 protein consists of 247 amino acids with a predicted molecular weight of 25.6 kDa before any post-translational modifications. The tertiary structure remains uncharacterized, though the protein has been confirmed to exist at the protein level . C3orf80 is annotated as a single-pass membrane protein that localizes to the cell membrane. The amino acid sequence contains:

• A signal peptide

• A transmembrane region

• A disordered region

• Sites for glycosylation

• A domain of unknown function 4719 (DUF4719)

Immunochemical staining has demonstrated that the protein localizes specifically in the cilia of glandular cells in the human fallopian tube, suggesting potential roles in ciliary function .

How conserved is the C3orf80 protein across species?

C3orf80 is highly conserved among mammals but shows varying degrees of conservation across vertebrates. The mouse homolog shares 92% sequence identity and 94% sequence similarity with the human protein . Conservation gradually decreases with evolutionary distance:

• Marsupials (e.g., Tasmanian devil): ~58-59% identity

• Reptiles: ~50-54% identity

• Amphibians: ~41-48% identity

• Fish: ~31-39% identity

• Cartilaginous fish: ~29-30% identity

Notably, avian orthologs (birds) show particularly divergent sequences, with only 20-27% identity to the human protein, suggesting potential functional adaptation or relaxed selection in this lineage . The evolutionary conservation pattern indicates that C3orf80 likely emerged at least 462 million years ago, with the most distant ortholog found in the Australian ghostshark (Callorhinchus milii) .

Where is C3orf80 primarily expressed in tissues?

Expression analysis indicates that C3orf80 is most abundant in the cerebral cortex, esophagus, and colon . The protein concentration in humans is approximately 0.02 parts per million (ppm), which is relatively low compared to other proteins in the human proteome . This restricted expression pattern suggests tissue-specific functions that may be related to specialized cellular processes in these organs. Researchers studying this protein should consider focusing their investigations on these tissues for optimal detection and functional analysis.

What are the known disease associations of C3orf80?

Several disease associations have been reported for C3orf80:

• Multiple sclerosis: Higher expression of C3orf80 has been observed in multiple sclerosis brain lesions

• Cancer associations:

  • Low-grade glioma: A two-fold increase in expression based on CMTM3 expression status

  • Esophageal squamous cell carcinoma: A remarkable 107.61-fold increase in expression following CLIC1 inhibition

  • Invasive carcinoma: C3orf80 was identified as one of three genes whose expression levels created the best machine learning model for predicting invasive carcinoma

  • Chemotherapy response: C3orf80 was included in a 34-gene signature used to predict patient response to FOLFIRI chemotherapy

These associations suggest potential roles in neuroinflammation and cancer pathways, though causative relationships remain to be established.

How do expression patterns of mouse C3orf80 homolog differ from human C3orf80?

When comparing expression data, researchers should account for potential confounding factors such as:

• Differences in tissue sampling techniques

• Varying sensitivity of detection methods

• Age and sex-specific expression patterns

• Environmental influences on gene expression

For accurate cross-species comparisons, identical experimental conditions and standardized quantification methods are essential. RT-qPCR with species-specific primers designed from conserved regions allows for direct expression level comparisons while western blotting with antibodies recognizing conserved epitopes can verify protein-level differences.

What are the technical challenges in working with recombinant C3orf80 protein?

Working with recombinant C3orf80 presents several technical challenges:

• Membrane protein expression: As a single-pass membrane protein, C3orf80 may form insoluble aggregates during recombinant expression. Consider using specialized expression systems like mammalian cells or insect cells rather than bacterial systems.

• Protein folding: The presence of a transmembrane domain requires appropriate detergents or membrane mimetics to maintain proper folding during purification.

• Low natural abundance: With only 0.02 ppm in human tissues, detection of native protein may require highly sensitive methods .

• Post-translational modifications: The protein contains glycosylation sites that may be critical for function but difficult to reproduce in recombinant systems .

• Antibody validation: Commercial antibodies should be rigorously validated using recombinant protein controls. The control fragment available (human C3orf80 aa 135-162) can be used for antibody blocking experiments to confirm specificity .

To overcome these challenges, researchers might consider using tagged constructs (e.g., FLAG, His, or GFP) for easier detection and purification while verifying that tags don't interfere with localization or function.

What functional genomics approaches are recommended to elucidate C3orf80 function?

To determine the function of the poorly characterized C3orf80 protein, a multi-faceted functional genomics approach is recommended:

• CRISPR-Cas9 gene editing:

  • Generate knockout mouse models

  • Create cell line knockouts in relevant tissues (cerebral cortex, esophagus, or colon-derived)

  • Develop conditional knockouts to avoid potential developmental effects

• Proteomics approaches:

  • Proximity labeling (BioID or APEX) to identify interaction partners

  • Co-immunoprecipitation followed by mass spectrometry

  • Phosphoproteomics to identify signaling pathways affected by C3orf80 deletion

• Transcriptomics:

  • RNA-seq in knockout vs. wild-type tissues

  • Single-cell RNA-seq to identify cell-type specific effects

  • Ribosome profiling to assess translational impacts

• Phenotypic analysis:

  • Focus on tissues with high expression (cerebral cortex, esophagus, colon)

  • Examine ciliary structures in fallopian tube and other ciliated tissues

  • Investigate cancer-related phenotypes based on the associations with multiple cancer types

• Evolutionary analysis:

  • Functional complementation studies across species

  • Analysis of selective pressures on different domains

  • Investigation of the divergent avian orthologs to understand functional adaptation

How might the unique characteristics of avian C3orf80 orthologs inform research approaches?

The striking divergence of avian C3orf80 orthologs (only 20-27% sequence identity compared to human) presents a unique opportunity for comparative functional studies . This evolutionary pattern suggests either functional adaptation or relaxed selection in birds.

Research approaches leveraging this divergence could include:

• Domain swapping experiments: Replace portions of mammalian C3orf80 with avian sequences to identify functional domains.

• Comparative expression studies: Determine if avian orthologs maintain similar tissue expression patterns despite sequence divergence.

• Structural analysis: Compare predicted structural features across species using advanced protein structure prediction algorithms (AlphaFold, RoseTTAFold) to identify conserved structural elements despite sequence differences.

• Bird-specific functional assays: Investigate whether avian C3orf80 has acquired novel functions related to bird-specific physiology (e.g., flight, unique metabolism, or reproductive biology).

• Selection analysis: Conduct detailed evolutionary rate analysis on specific protein domains to identify regions under positive selection in birds.

This evolutionary divergence might provide key insights into the protein's core function versus adaptable features and could reveal unexpected biological roles in different vertebrate lineages.

What is known about the regulation of C3orf80 expression?

The regulation of C3orf80 expression remains largely unexplored, representing a significant knowledge gap. Based on the available data, several regulatory relationships can be inferred:

• Potential interaction with CMTM3: A two-fold increase in C3orf80 expression was observed in low-grade glioma depending on CMTM3 expression status .

• Regulation by CLIC1: Inhibition of CLIC1 in esophageal squamous cell carcinoma led to a dramatic 107.61-fold increase in C3orf80 expression, suggesting strong negative regulation by CLIC1 .

• Tissue-specific regulation: The preferential expression in cerebral cortex, esophagus, and colon indicates tissue-specific regulatory mechanisms .

• Single-exon structure implications: The lack of introns in C3orf80 suggests it may be regulated differently from typical multi-exon genes, potentially bypassing splicing-related regulatory mechanisms .

To investigate regulatory mechanisms, researchers should consider:

• Promoter analysis and reporter assays

• ChIP-seq for transcription factor binding

• DNA methylation and histone modification profiling

• Analysis of potential microRNA binding sites

• Investigation of long non-coding RNA interactions

What are the recommended protocols for expressing recombinant mouse C3orf80 homolog?

For successful expression of recombinant mouse C3orf80 homolog, consider the following optimized protocol:

Expression System Selection:

• Mammalian expression system (HEK293T or CHO cells) is recommended for proper folding and post-translational modifications

• Insect cell systems (Sf9 or Hi5) offer an alternative with potentially higher yields

• Avoid bacterial expression systems due to the transmembrane domain unless using specialized strains designed for membrane proteins

Expression Vector Design:

• Include a cleavable N-terminal signal sequence

• Add purification tag (His6, FLAG, or Strep-tag) preferably at the C-terminus to avoid interference with the signal peptide

• Consider a fluorescent protein fusion for localization studies

• Include a TEV or PreScission protease site for tag removal

Optimization Parameters:

• Expression temperature: 30-32°C often yields better folding than 37°C

• Induction duration: 48-72 hours for mammalian cells

• Cell density at transfection: Aim for 70-80% confluence

• Consider using chemical chaperones (e.g., 4-phenylbutyric acid) to improve folding

Purification Strategy:

• Membrane fraction isolation using differential centrifugation

• Solubilization with mild detergents (DDM, LMNG, or digitonin)

• Affinity chromatography using tag-specific resin

• Size exclusion chromatography for final purification

Quality Control:

• Western blot confirmation using anti-tag antibodies and/or anti-C3orf80 antibodies

• Mass spectrometry for protein identity confirmation

• Circular dichroism to verify secondary structure content

• Dynamic light scattering to assess homogeneity

What antibody validation strategies should be employed for mouse C3orf80 homolog detection?

Proper antibody validation is critical for reliable C3orf80 detection. A comprehensive validation strategy should include:

Positive and Negative Controls:

• Recombinant protein positive control: Use purified mouse C3orf80 homolog

• Tissue positive control: Mouse cerebral cortex, esophagus, or colon samples

• Negative control: Tissue from C3orf80 knockout mouse or CRISPR-edited cell lines

• Blocking peptide control: Pre-incubate antibody with recombinant C3orf80 fragment (similar to the human control fragment aa 135-162)

Multi-technique Validation:

• Western blot:

  • Expected MW: ~25.6 kDa (verify mobility on SDS-PAGE)

  • Evaluate multiple tissue types with varying expression levels

  • Test under reducing and non-reducing conditions

• Immunoprecipitation:

  • Pull-down from tissues with known expression

  • Verify with mass spectrometry

• Immunohistochemistry/Immunofluorescence:

  • Expected localization: Cell membrane and possibly cilia

  • Compare to mRNA expression data

  • Perform dual staining with established membrane or ciliary markers

• Flow cytometry:

  • Compare staining in transfected vs. non-transfected cells

  • Analyze surface vs. intracellular staining patterns

Cross-reactivity Assessment:

• Test against human C3orf80 (96% homology to mouse)

• Evaluate specificity in cells overexpressing related proteins

• For polyclonal antibodies, consider affinity purification against the immunogen

Reporting Standards:

• Document all validation steps according to the Antibody Validation Guidelines

• Provide complete details of antibody source, catalog number, lot, dilutions, and protocols

How should researchers design experiments to study the relationship between C3orf80 and multiple sclerosis?

To investigate the observed association between C3orf80 and multiple sclerosis (MS) , a methodical experimental approach is recommended:

Patient Sample Analysis:

• Tissue collection:

  • MS lesions (acute, chronic, active, and inactive)

  • Normal-appearing white matter from MS patients

  • Control white matter from non-MS individuals

  • Consider CSF samples for protein detection

• Expression profiling:

  • RT-qPCR for mRNA quantification

  • Western blot for protein level analysis

  • Single-cell RNA-seq to identify cell types with altered expression

  • Spatial transcriptomics to map expression changes relative to lesion boundaries

• Immunohistochemical analysis:

  • Co-staining with cell type-specific markers (oligodendrocytes, astrocytes, microglia)

  • Evaluation of C3orf80 localization in different lesion types

  • Correlation with inflammatory markers

Functional Studies:

• In vitro models:

  • Primary oligodendrocyte cultures with C3orf80 manipulation

  • Myelinating co-cultures to assess impact on myelination

  • Microglial activation assays to evaluate inflammatory responses

• Animal models:

  • C3orf80 knockout in EAE (Experimental Autoimmune Encephalomyelitis) mouse model

  • Conditional knockout in specific cell types (oligodendrocytes, microglia)

  • Viral overexpression of C3orf80 in EAE models

• Mechanistic investigations:

  • RNA-seq of manipulated cells to identify pathways

  • Phosphoproteomics to detect altered signaling

  • Assessment of blood-brain barrier integrity

  • Analysis of immune cell infiltration and activation

Statistical Considerations:

• Power analysis to determine appropriate sample sizes

• Adjustment for covariates (age, sex, disease duration, treatment)

• Multiple testing correction for omics analyses

• Longitudinal study design where possible to track disease progression

What experimental approaches can determine the subcellular localization and trafficking of C3orf80?

To precisely determine subcellular localization and trafficking pathways of C3orf80, combine the following complementary approaches:

Fixed-Cell Imaging Techniques:

• Confocal microscopy:

  • Immunofluorescence with validated anti-C3orf80 antibodies

  • Co-staining with organelle markers:

    • Plasma membrane: Na⁺/K⁺-ATPase, WGA

    • ER: Calnexin, KDEL

    • Golgi: GM130, TGN46

    • Endosomes: EEA1, Rab5, Rab7

    • Cilia: Acetylated tubulin, Arl13b

  • Super-resolution microscopy (STORM, PALM) for nanoscale localization

• Immunoelectron microscopy:

  • Gold-labeled antibodies for ultrastructural localization

  • Correlative light and electron microscopy (CLEM)

Live-Cell Imaging Approaches:

• Fluorescent protein fusions:

  • C-terminal GFP or mCherry tags

  • Photoactivatable or photoconvertible tags for pulse-chase

  • Split fluorescent protein complementation to study protein interactions

• Trafficking dynamics:

  • FRAP (Fluorescence Recovery After Photobleaching) for mobility assessment

  • Photoactivation for directional trafficking analysis

  • Time-lapse imaging during cell stimulation or drug treatment

Biochemical Fractionation:

• Differential centrifugation:

  • Separate nuclear, cytosolic, membrane, and organelle fractions

  • Density gradient separation of membrane compartments

• Selective permeabilization:

  • Digitonin for plasma membrane versus saponin for all membranes

  • Protease protection assays to determine membrane topology

Inducible Targeting Systems:

• RUSH system (Retention Using Selective Hooks):

  • Monitor synchronized protein trafficking from ER

  • Quantify kinetics of transport to final destination

• Self-labeling protein tags:

  • SNAP-tag or HaloTag for pulse-chase labeling

  • Enables quantitative analysis of protein turnover

• Optogenetic approaches:

  • Light-inducible clustering or translocation

  • Assess impact of forced relocalization on function

Based on immunochemical staining data showing C3orf80 localization in cilia of glandular cells in the human fallopian tube , special attention should be paid to ciliary targeting sequences and trafficking pathways.

How can researchers investigate the potential role of C3orf80 in cancer pathways?

Given the multiple associations between C3orf80 and cancer , a systematic investigation of its role in oncogenic pathways is warranted:

Expression Analysis in Cancer:

• Multi-cancer screening:

  • Analysis across cancer types using public databases (TCGA, ICGC)

  • Stratification by cancer subtypes and stages

  • Correlation with patient outcomes and treatment responses

• Detailed profiling in specific cancers:

  • Focus on cancers with known associations:

    • Low-grade glioma

    • Esophageal squamous cell carcinoma

    • Invasive carcinoma

  • Compare primary tumors, matched normal tissue, and metastases

  • Single-cell analysis to identify relevant cell populations

Functional Genomics:

• Loss-of-function studies:

  • CRISPR knockout in cancer cell lines

  • shRNA/siRNA knockdown for temporal control

  • Assess effects on:

    • Proliferation and cell cycle progression

    • Migration and invasion

    • Apoptosis resistance

    • Colony formation and 3D growth

• Gain-of-function approaches:

  • Stable overexpression of wild-type C3orf80

  • Structure-function analysis with domain mutants

  • Inducible expression systems

Mechanistic Studies:

• Pathway analysis:

  • Investigate relationship with CLIC1 (given 107.61-fold expression change after CLIC1 inhibition)

  • Examine connection to CMTM3 pathway in glioma

  • Assess impact on canonical cancer pathways (MAPK, PI3K/AKT, WNT, etc.)

• Interactome mapping:

  • BioID or APEX proximity labeling

  • Co-immunoprecipitation coupled with mass spectrometry

  • Yeast two-hybrid screening

• Post-translational modifications:

  • Phosphorylation status in response to growth factors

  • Glycosylation profile in normal versus cancer cells

Translational Research:

• Patient-derived models:

  • PDX (patient-derived xenograft) models

  • Organoids from normal and tumor tissue

  • Ex vivo culture of patient samples

• Therapeutic targeting potential:

  • siRNA-based approaches

  • Small molecule screening

  • Antibody-based targeting of extracellular domain

• Biomarker development:

  • Validate as part of gene expression signatures for specific cancer types

  • Evaluate in liquid biopsy approaches (if secreted forms exist)

What can phylogenetic analysis tell us about the function of C3orf80?

Phylogenetic analysis provides valuable insights into the potential function and evolutionary constraints on C3orf80:

Evolutionary Conservation Pattern:
The presence of C3orf80 orthologs exclusively in vertebrates, with the most distant ortholog in the Australian ghostshark (Callorhinchus milii), suggests the gene emerged approximately 462 million years ago during early vertebrate evolution . This coincides with several major evolutionary innovations:

The conservation pattern suggests a vertebrate-specific function that may relate to specialized tissues or developmental processes unique to vertebrates.

Domain Conservation Analysis:
The domain of unknown function 4719 (DUF4719) in C3orf80 represents a conserved functional unit. Comparative analysis of this domain across species could reveal:

• Critical amino acid residues maintained across all vertebrates

• Lineage-specific adaptations

• Structural predictions based on conservation patterns

Synteny Analysis:
Examining the genomic context of C3orf80 across species could provide functional clues:

• The neighboring genes in humans (IFT80, BRD7P2, SMC4) should be evaluated for conservation of synteny

• Maintained gene neighbors might suggest functional relationships or co-regulation

• Breaks in synteny could indicate adaptive reorganization

Rate of Evolution Analysis:
The unusually divergent avian orthologs (20-27% identity) represent a particularly interesting case that might indicate:

• Relaxed functional constraints in birds

• Adaptive evolution for bird-specific functions

• Potential functional shifts or specialization

How should differences between mouse and human C3orf80 impact experimental design?

Despite the high sequence similarity between mouse and human C3orf80 (92% identity, 94% similarity) , researchers should carefully consider the following differences when designing experiments:

Sequence Divergence Considerations:

• Identify non-conserved regions:

  • Map the 8% sequence differences to specific domains

  • Determine if differences affect functional motifs

  • Consider impacts on antibody epitopes and cross-reactivity

• Post-translational modification sites:

  • Compare predicted glycosylation, phosphorylation sites

  • Validate conservation of the signal peptide and transmembrane domain

  • Assess potential differences in protein processing

Expression Pattern Differences:

• Tissue-specific expression:

  • Validate if mouse expression matches human pattern (cerebral cortex, esophagus, colon)

  • Identify any mouse-specific expression sites

  • Consider developmental timing differences

• Quantitative expression:

  • Compare relative abundance across tissues

  • Assess cell-type specificity of expression

  • Evaluate response to physiological stimuli

Functional Considerations:

• Protein interactions:

  • Validate if mouse orthologs of human interaction partners exist

  • Test conservation of binding interfaces

  • Consider species-specific adaptor proteins

• Subcellular localization:

  • Confirm ciliary localization in mouse tissues

  • Assess membrane distribution patterns

  • Evaluate trafficking pathways

Experimental Controls:

• Antibody validation:

  • Test human antibodies on mouse tissues with appropriate controls

  • Consider developing mouse-specific antibodies if needed

  • Use recombinant proteins as positive controls

• Knockout models:

  • Design targeting strategies accounting for species differences

  • Include rescue experiments with both human and mouse cDNAs

  • Monitor for compensatory mechanisms

• Cross-species complementation:

  • Test if human C3orf80 can functionally replace mouse protein

  • Identify domains responsible for any functional differences

  • Create chimeric proteins to map functional regions

The high sequence similarity suggests functional conservation, but the 8% sequence divergence could impact specific interactions or regulatory mechanisms that should be experimentally validated.

What is the potential of C3orf80 as a biomarker for multiple sclerosis or cancer?

Based on its associations with multiple sclerosis brain lesions and various cancers , C3orf80 warrants investigation as a potential biomarker:

Multiple Sclerosis Biomarker Potential:

• Tissue-specific expression:

  • Elevated expression in MS lesions suggests potential as a disease activity marker

  • Could differentiate lesion types (active vs. chronic inactive)

  • May correlate with specific MS phenotypes (relapsing-remitting vs. progressive)

• Accessibility considerations:

  • Determine if C3orf80 or fragments are detectable in CSF

  • Evaluate potential as a blood-based biomarker

  • Consider exosomal C3orf80 as a liquid biopsy target

• Clinical correlation studies needed:

  • Association with disease progression

  • Predictive value for treatment response

  • Correlation with MRI findings

Cancer Biomarker Applications:

• Diagnostic potential:

  • Part of a three-gene signature for invasive carcinoma detection

  • Significant expression changes in esophageal squamous cell carcinoma

  • Potential role in low-grade glioma classification

• Predictive biomarker:

  • Component of a 34-gene signature predicting FOLFIRI chemotherapy response

  • Could guide treatment selection

  • May indicate tumor aggressiveness

• Implementation approaches:

  • qPCR-based gene expression panels

  • Immunohistochemistry scoring systems

  • Inclusion in multiparameter predictive algorithms

Biomarker Development Roadmap:

• Discovery phase:

  • Expanded patient cohort validation

  • Comparison with existing biomarkers

  • Determination of sensitivity and specificity

• Analytical validation:

  • Assay development and standardization

  • Reproducibility across laboratories

  • Reference range establishment

• Clinical validation:

  • Prospective clinical trials

  • Evaluation in diverse patient populations

  • Assessment of added value over standard markers

The 107.61-fold increase in C3orf80 expression following CLIC1 inhibition in esophageal squamous cell carcinoma represents a particularly promising lead for pharmacodynamic biomarker development that should be prioritized for further study.

How should researchers approach targeting C3orf80 for therapeutic development?

Developing therapeutics targeting C3orf80 requires a strategic approach given its membrane localization and limited functional characterization:

Target Validation:

• Disease relevance confirmation:

  • Strengthen causal relationship in MS and cancer

  • Determine if altered expression is driver or passenger

  • Identify patient subgroups most likely to benefit

• Mechanism of action studies:

  • Delineate signaling pathways impacted

  • Identify critical protein-protein interactions

  • Determine if function is pro- or anti-disease

• Model system development:

  • Generate relevant in vitro and in vivo models

  • Establish clear phenotypic readouts

  • Develop target engagement assays

Therapeutic Modality Selection:

• Small molecule approaches:

  • Target potential ligand binding pockets

  • Disrupt protein-protein interactions

  • Modulate trafficking or degradation

• Biologics strategy:

  • Antibody development against extracellular epitopes

  • ADC (antibody-drug conjugate) targeting for cancer

  • Protein replacement for loss-of-function contexts

• Genetic medicine options:

  • Antisense oligonucleotides for expression modulation

  • mRNA therapeutics for supplementation

  • Gene editing for permanent modification

Drug Development Considerations:

• Target site accessibility:

  • Blood-brain barrier penetration for MS applications

  • Tumor penetration for cancer therapeutics

  • Membrane protein targeting challenges

• Safety assessment:

  • Impact on normal tissues with high expression

  • Potential on-target toxicity in cerebral cortex, esophagus, colon

  • Off-target effects on related proteins

• Combination approaches:

  • Synergy with CLIC1 inhibitors in esophageal cancer

  • Complement to existing MS therapies

  • Integration with personalized medicine approaches

Biomarker Integration:

• Develop companion diagnostics to identify responders

• Utilize C3orf80 expression as pharmacodynamic marker

• Monitor target engagement and biological response

The single transmembrane domain architecture and potential ciliary localization present both challenges and opportunities for therapeutic targeting that should be carefully considered in development strategies.

What are the most pressing knowledge gaps regarding C3orf80 function?

Despite progress in characterizing C3orf80, significant knowledge gaps remain that should guide future research priorities:

• Molecular function: The fundamental biochemical activity of C3orf80 remains unknown. Does it function as a receptor, transporter, scaffold protein, or have enzymatic activity? The presence of DUF4719 (Domain of Unknown Function) highlights this critical knowledge gap.

• Signaling pathways: How C3orf80 interacts with established cellular signaling networks remains undefined, though associations with CLIC1 and CMTM3 provide initial leads .

• Physiological role: The normal function in tissues with high expression (cerebral cortex, esophagus, colon) requires elucidation through careful phenotypic analysis of knockout models.

• Ciliary function: Despite localization to cilia in fallopian tube glandular cells , the specific role in ciliary biology remains undefined, particularly whether it affects ciliary assembly, signaling, or specialized functions.

• Disease mechanisms: While associations with multiple sclerosis and cancer have been identified , the causal relationships and mechanistic underpinnings remain to be established.

• Regulation: The factors controlling C3orf80 expression, trafficking, and turnover are largely unknown, though the dramatic response to CLIC1 inhibition provides one regulatory connection .

• Structural information: The three-dimensional structure remains uncharacterized, limiting structure-based functional predictions and drug design approaches.

• Evolutionary adaptation: The basis for the unusual divergence in avian orthologs represents an evolutionary puzzle that could provide functional insights.

Addressing these knowledge gaps requires integrated approaches combining biochemical, cellular, physiological, and computational methods within a collaborative research framework.

What resources and tools are currently available for C3orf80 research?

Researchers investigating C3orf80 can leverage several existing resources and tools:

Molecular Tools:

• Recombinant proteins:

  • Human C3orf80 (aa 135-162) control fragment is commercially available

  • Can be used for antibody validation and blocking experiments

  • Useful as positive control in various assays

• Genetic constructs:

  • C3orf80 sequence data is available in genomic databases (NCBI, Ensembl)

  • Mammalian expression vectors can be generated from available sequence information

  • CRISPR targeting constructs for gene editing

Bioinformatics Resources:

• Sequence databases:

  • Complete gene and protein sequences are available

  • Orthologs across multiple species identified

  • Conserved domain information (DUF4719)

• Expression databases:

  • Tissue-specific expression data in public repositories

  • Single-cell RNA-seq datasets for cell-type specific analysis

  • Cancer expression databases (TCGA, ICGC)

• Variant information:

  • ClinVar and dbVar contain variant data

  • SNP information for genetic association studies

  • Genome viewers for contextual genomic analysis

Reference Data:

• Protein characteristics:

  • 247 amino acids, 25.6 kDa molecular weight

  • Contains signal peptide, transmembrane region, disordered region, glycosylation sites

  • Associated with single-pass membrane proteins

• Genomic context:

  • Chromosome 3 location: 160,225,422-160,228,213

  • Single-exon structure

  • Neighboring genes (IFT80, BRD7P2, SMC4)

• Evolutionary conservation:

  • Comprehensive ortholog table across vertebrate lineages

  • Sequence identity and similarity metrics for comparative analysis

  • Divergence timing estimates for major taxonomic groups

Experiment Models:

• Cell systems:

  • Cancer cell lines with variable C3orf80 expression

  • Cerebral cortex, esophagus, and colon cell lines

  • Primary ciliated cell cultures

• Model organisms:

  • Mouse models (92% protein sequence identity to human)

  • Potential for zebrafish models for developmental studies

  • Consideration of avian models for divergent function analysis

These resources provide a foundation for advancing C3orf80 research while highlighting the need for development of additional tools and reagents to address remaining knowledge gaps.