Recombinant Human Uncharacterized membrane protein C3orf80 (C3orf80)

What is the genomic organization of the C3orf80 gene?

C3orf80 is located on chromosome 3 at position 160,225,422 to 160,228,213 bp (spanning 2,792 bases) on the plus strand. Notably, the gene contains only one exon, which is unusual compared to most human genes that typically contain multiple exons . This single-exon structure has implications for both its expression regulation and evolutionary history. When designing experiments to study C3orf80, researchers should consider this unusual genomic organization, particularly when developing primers for amplification or when planning gene editing approaches.

What are the neighboring genes of C3orf80 and how might this inform functional studies?

The genomic neighborhood of C3orf80 includes several genes that may provide contextual clues for functional studies:

Researchers should consider potential functional relationships or co-expression patterns with these neighboring genes, particularly IFT80 which functions in intraflagellar transport . This genomic proximity could suggest involvement in similar biological processes, especially considering the localization of C3orf80 in cilia structures.

What is the basic structural organization of the C3orf80 protein?

C3orf80 protein consists of 247 amino acids with a molecular weight of approximately 25.6 kDa before post-translational modifications. The protein has been experimentally confirmed to exist at the protein level, though its tertiary structure remains undetermined . Key structural features include:

• A signal peptide at the N-terminus

• A single transmembrane region

• Disordered regions

• Glycosylation sites

• Domain of unknown function 4719 (DUF4719)

For experimental design, researchers should consider the membrane-associated nature of this protein when planning isolation, purification, and functional studies. Standard detergent-based methods for membrane protein solubilization would be appropriate starting points.

In which tissues is C3orf80 most highly expressed?

C3orf80 shows highest expression in the cerebral cortex, esophagus, and colon . This tissue-specific expression pattern suggests potential roles in neural function and digestive tract biology. When designing experiments to study C3orf80, researchers should consider using cell lines or primary cells derived from these tissues to maximize detection sensitivity. The pronounced expression in cerebral cortex indicates potential neurological functions that warrant investigation, particularly in the context of neurological disorders.

How should researchers optimize qPCR protocols for studying C3orf80 expression?

When studying C3orf80 expression via qPCR, researchers should adhere to the MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) to ensure reproducibility . Key methodological considerations include:

• Sample acquisition and handling: Preserve RNA integrity by rapid processing and storage

• RNA extraction: Use methods optimized for the specific tissue type being studied

• RNA quality assessment: Verify RNA integrity number (RIN) > 7 before proceeding

• cDNA synthesis: Select reverse transcriptase enzymes with high fidelity and consistency

• Primer design: Target the single exon of C3orf80 with carefully designed primers that avoid potential SNPs

• Reference gene selection: Use multiple reference genes appropriate for the tissue being studied

• Biological replicates: Include sufficient biological replicates (minimum n=3) to account for natural variation

Poor RNA quality or suboptimal reaction conditions can lead to inaccurate results when studying low-abundance transcripts like C3orf80, making rigorous quality control essential throughout the experimental workflow.

What is known about the subcellular localization of C3orf80?

Immunochemical staining has revealed that C3orf80 localizes specifically to the cilia of glandular cells in the human fallopian tube . This ciliary localization is particularly noteworthy given that one of its neighboring genes, IFT80, functions in intraflagellar transport. For studying C3orf80 localization in other cell types, researchers should consider:

• Using cell lines with prominent ciliary structures (e.g., hTERT-RPE1)

• Examining co-localization with established ciliary markers

• Employing super-resolution microscopy techniques to precisely visualize ciliary structures

• Investigating potential interactions with other ciliary proteins, particularly IFT complex components

The protein concentration in humans is relatively low at approximately 0.02 ppm, indicating that sensitive detection methods will be required for most localization studies .

What are the key considerations for producing recombinant C3orf80 for structural and functional studies?

Producing recombinant C3orf80 presents several challenges due to its membrane protein nature. Researchers should consider the following methodological approaches:

• Expression systems: Mammalian expression systems (HEK293 or CHO cells) are recommended over bacterial systems to maintain proper post-translational modifications, particularly glycosylation

• Tags and fusion partners: Include purification tags (His, FLAG) positioned to avoid interference with the transmembrane domain or signal peptide

• Solubilization: Test multiple detergent conditions (DDM, CHAPS, digitonin) to identify optimal solubilization conditions

• Purification strategy: Use two-step purification approaches to enhance purity

• Functional validation: Confirm proper folding through binding or activity assays once established

For structural studies, researchers might consider nanobody stabilization approaches which have proven successful for other membrane proteins with similar characteristics.

How can researchers effectively design experiments to study the function of the DUF4719 domain in C3orf80?

The domain of unknown function 4719 (DUF4719) presents an intriguing target for functional characterization. Consider these methodological approaches:

• Structure prediction: Utilize AlphaFold2 or similar advanced protein structure prediction algorithms to generate structural models

• Domain truncation: Create constructs with systematic truncations or mutations within the DUF4719 domain

• Interactome analysis: Employ BioID or APEX2 proximity labeling to identify interaction partners of the domain

• Cross-species complementation: Test functional conservation by expressing C3orf80 from different species in knockout models

• Domain-swapping experiments: Replace DUF4719 with similar domains from other proteins to assess functional specificity

The relatively high conservation of this domain across vertebrates suggests functional importance, making comparative approaches particularly valuable.

How conserved is C3orf80 across vertebrate species?

C3orf80 shows a clear pattern of evolutionary conservation across vertebrates, with orthologs dating back approximately 462 million years to cartilaginous fish . The following table illustrates the conservation patterns:

The high conservation among mammals (92% identity between human and mouse) suggests important functional constraints. When designing animal models for C3orf80 research, mouse models would likely maintain functional relevance, while more distantly related models might require careful validation .

What explains the unusual divergence pattern of C3orf80 in avian species?

Avian orthologs of C3orf80 show remarkably low sequence identity (20-27%) compared to other vertebrate groups at similar evolutionary distances . This unusual divergence pattern may indicate:

• Relaxed selective constraints in birds

• Acquisition of bird-specific functions

• Compensatory changes due to avian-specific physiological adaptations

• Possible gene duplication events in the avian lineage

When using comparative genomics approaches to study C3orf80, researchers should account for this avian divergence by:

• Analyzing avian sequences separately from other vertebrates

• Investigating potential functional divergence through targeted experiments

• Examining expression patterns in avian tissues compared to mammals

• Considering bird-specific anatomical differences, particularly in ciliated structures

What evidence links C3orf80 to multiple sclerosis and other neurological conditions?

Higher expression of C3orf80 has been observed in multiple sclerosis brain lesions, suggesting potential involvement in neuroinflammatory processes . When designing experiments to investigate this association, researchers should consider:

• Cell models: Using microglia or astrocyte cultures to examine C3orf80 expression under inflammatory conditions

• Human samples: Analyzing C3orf80 expression in cerebrospinal fluid or brain tissue from MS patients versus controls

• Functional assays: Examining effects of C3orf80 overexpression or knockdown on neuroinflammatory markers

• Pathway analysis: Investigating interactions with established MS risk genes or inflammatory pathways

The predominant expression of C3orf80 in the cerebral cortex further supports potential neurological functions that warrant detailed investigation.

How is C3orf80 expression altered in different cancer types?

C3orf80 expression changes have been associated with several cancer types through various mechanistic relationships:

• Low-grade glioma: Two-fold increase in expression associated with CMTM3 expression status

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

• Invasive carcinoma: One of three genes identified in a machine learning model for predicting invasiveness

• Chemotherapy response: Part of a 34-gene signature predictive of response to FOLFIRI chemotherapy

When designing cancer-related studies with C3orf80, researchers should:

• Stratify samples based on molecular subtypes of the cancer being studied

• Correlate C3orf80 expression with clinical outcomes and treatment responses

• Investigate mechanistic relationships with known cancer-associated genes (CMTM3, CLIC1)

• Consider C3orf80 as part of multi-gene signatures rather than in isolation

The substantial expression increase (107.61-fold) following CLIC1 inhibition in esophageal cancer suggests a potential compensatory relationship worth investigating further.

What are the critical quality control steps when studying a low-abundance protein like C3orf80?

When studying C3orf80, which has a relatively low abundance (0.02 ppm in humans), implementing rigorous quality control measures becomes essential:

• Antibody validation: Thoroughly validate antibodies using knockout/knockdown controls and multiple detection methods

• Expression verification: Confirm expression levels through multiple methodologies (qPCR, western blot, immunofluorescence)

• Subcellular fractionation: Enrich membrane and ciliary fractions to concentrate the protein

• Signal amplification: Consider using proximity ligation assays or similar techniques for detecting low-abundance proteins

• Statistical power: Calculate appropriate sample sizes to detect small changes in expression

• Technical replicates: Include sufficient technical replicates to confirm experimental accuracy

The MIQE guidelines provide a framework for ensuring reproducibility in gene expression studies that is particularly relevant for low-abundance targets like C3orf80 .

How should researchers address biological variability when studying C3orf80 expression patterns?

Biological variability presents significant challenges when studying genes with tissue-specific expression like C3orf80. To address this:

• Include sufficient biological replicates (minimum n=3, preferably n≥5) to capture natural variation

• Account for demographic factors (age, sex, ethnicity) that may influence expression

• Consider tissue heterogeneity, particularly in complex tissues like brain

• Use appropriate statistical approaches that account for non-normal distributions

• Validate findings across independent cohorts or sample sets

As noted by experts in gene expression analysis, "analyzing one sample once can indicate a certain process is occurring but doesn't show trends or validate that process for that sample type. Running sufficient replicates to get statistically correct information verifies an observed change in expression levels."