Recombinant Human UPF0308 protein C9orf21 (C9orf21)

What is UPF0308 protein C9orf21 and what are its alternative designations?

UPF0308 protein C9orf21 is a 226 amino acid protein encoded by a gene mapped to human chromosome 9q22.33. It belongs to the UPF0308 protein family and is also known by several alternative designations:

• AAED1 (AhpC/TSA antioxidant enzyme domain containing 1)

• PRXL2C (Peroxiredoxin-like 2C)

• Thioredoxin-like protein AAED1

The protein has subcellular localization in both the cytoplasm and nucleus. Chromosome 9, where C9orf21 is located, consists of approximately 145 million bases, representing 4% of the human genome and encoding nearly 900 genes .

How evolutionarily conserved is C9orf21 across different species?

C9orf21 demonstrates significant evolutionary conservation across mammalian species. Research using recombinant proteins and comparative analysis has shown:

• The human and mouse orthologs share approximately 93% sequence identity

• The human and rat orthologs share approximately 93% sequence identity

• The bovine homolog (Q148E0) has been fully sequenced and characterized

This high degree of conservation suggests important functional roles that have been maintained throughout mammalian evolution. Researchers should consider this conservation when designing experiments using animal models, as findings may have translational relevance to human biology .

What are the recommended methods for detecting endogenous C9orf21/AAED1 in cellular and tissue samples?

For detecting endogenous C9orf21/AAED1 in experimental settings, the following methodologies are recommended:

Western Blot:

• Recommended antibody dilution: 1:300-5000

• Sample preparation: Standard protein extraction with protease inhibitors

• Expected molecular weight: Approximately 24.7 kDa

Immunohistochemistry (IHC):

• IHC-Paraffin (IHC-P): Antibody dilution 1:50-200

• IHC-Frozen (IHC-F): Antibody dilution 1:50-200

• Antigen retrieval: Heat-mediated citrate buffer pH 6.0

Immunofluorescence (IF):

• IF in cell culture (ICC): Antibody dilution 1:50-200

• Counterstaining: DAPI for nuclear visualization

• Fixation: 4% paraformaldehyde followed by permeabilization

For validation of antibody specificity, blocking experiments using recombinant protein fragments (such as Human AAED1 aa 84-142) at 100× molar excess compared to the antibody concentration is recommended .

How should researchers design experiments to study C9orf21 function in vitro?

When designing experiments to investigate C9orf21 function in vitro:

Gain-of-function approaches:

• Transfection with expression plasmids containing tagged C9orf21 (options include Myc/DDK-tagged or GFP-tagged constructs)

• Lentiviral transduction using vectors such as pLenti-C-mGFP-P2A-Puro for stable expression

• Doxycycline-inducible expression systems for temporal control

Loss-of-function approaches:

• siRNA or shRNA targeting multiple regions of C9orf21 mRNA

• CRISPR/Cas9-mediated knockout using guide RNAs targeting early exons

• Dominant-negative constructs based on critical functional domains

Functional readouts to consider:

• Cell proliferation and viability assays (particularly relevant given association with cancer)

• Metabolic analysis (glycolysis measurements, given evidence of metabolic modulation)

• Oxidative stress response (given peroxiredoxin-like domain)

• Protein-protein interaction studies to identify binding partners

When designing primers for qPCR validation, researchers should be aware of potential splice variants and ensure primers span exon-exon junctions .

What are the best storage and handling conditions for recombinant C9orf21/AAED1 protein?

For optimal storage and handling of recombinant C9orf21/AAED1 protein:

Storage conditions:

• Store at -20°C to -80°C for long-term storage

• For lyophilized form: 12 months stability at -20°C/-80°C

• For liquid form: 6 months stability at -20°C/-80°C

• Avoid repeated freeze-thaw cycles

• For working aliquots: Store at 4°C for up to one week

Reconstitution protocol:

• Centrifuge vial at 5,000×g for 5 minutes before opening

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% for long-term storage

• Aliquot to minimize freeze-thaw cycles

Buffer considerations:

• Typical storage buffers include:

  • 100 mM glycine, 25 mM Tris-HCl, pH 7.3

  • TBS (pH 7.4) with 1% BSA, 0.02-0.03% Proclin300, and 50% Glycerol

For critical experiments, validation of protein activity after storage is recommended, especially if the protein is used for functional studies rather than as a control or standard .

How can researchers investigate the potential role of C9orf21/AAED1 in regulation of cellular metabolism?

Recent evidence suggests that AAED1 (C9orf21) modulates cellular metabolism, particularly glycolysis in cancer models. To investigate this role:

Experimental approaches:

• Metabolic flux analysis: Use Seahorse XF Analyzer to measure extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in cells with modulated C9orf21 expression

• Glucose uptake assays: Measure uptake of labeled glucose (2-NBDG or tritiated 2-deoxyglucose) in control versus C9orf21-overexpressing or knockdown cells

• Expression analysis of glycolytic enzymes: Perform qPCR and Western blot analyses of key glycolytic enzymes (HK2, PFKP, PKM2, LDHA) to determine if C9orf21 affects their expression

• Metabolomics profiling: Conduct LC-MS-based metabolomics to comprehensively analyze changes in glycolytic intermediates and related metabolic pathways

A comprehensive experimental design should include multiple cell types and correlation with clinical samples to validate findings across biological contexts. The peroxiredoxin-like domain suggests potential involvement in redox regulation, which may link to metabolic functions through redox-sensitive metabolic enzymes .

What is the relationship between C9orf21 and C9orf72 in neurodegenerative disease research?

It is important to note that C9orf21 and C9orf72 are distinct genes, though both are located on chromosome 9. This can lead to confusion in the literature:

Key distinctions:

• C9orf72: Associated with hexanucleotide repeat expansions (GGGGCC) that cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)

• C9orf21 (AAED1/PRXL2C): Not directly implicated in repeat expansion disorders

Research considerations when studying neurodegeneration:

• Differential expression analysis: Compare C9orf21 expression in post-mortem brain tissues from patients with C9orf72 expansions versus those without expansions

• Potential functional interactions: Investigate whether C9orf21 interacts with C9orf72 or modifies pathological processes in C9orf72-related diseases

• Co-expression networks: Use WGCNA (weighted gene co-expression network analysis) to determine if C9orf21 belongs to gene modules associated with neurodegeneration

Recent studies of cerebellar transcriptomes in C9orf72 expansion carriers have revealed widespread transcriptomic alterations. Investigating whether C9orf21 is part of these altered networks could provide insights into disease mechanisms .

What experimental approaches can be used to analyze post-translational modifications of C9orf21/AAED1?

Analysis of post-translational modifications (PTMs) of C9orf21/AAED1 requires systematic approaches:

Mass spectrometry-based strategies:

• Sample preparation: Immunoprecipitate endogenous or tagged C9orf21 from cell lysates under conditions that preserve PTMs (phosphatase/deacetylase inhibitors)

• MS analysis options:

  • Shotgun proteomics for broad PTM identification

  • Targeted MS for specific modification sites

  • TMT-based quantitative proteomics for comparing PTM levels across conditions

• Isotopic labeling: Use of C13 and N15-labeled recombinant protein as an internal standard for accurate quantification

Site-specific mutation analysis:

• Identify putative modification sites through in silico prediction tools

• Generate site-specific mutants (e.g., S→A for phosphorylation, K→R for ubiquitination/acetylation)

• Compare functional outcomes between wild-type and mutant proteins

PTMs of particular interest:

• Phosphorylation (multiple potential Ser/Thr/Tyr sites)

• Redox modifications (given peroxiredoxin domain)

• Ubiquitination (potential regulation of protein stability)

Researchers should consider using C13 and N15-labeled recombinant protein as a standard for mass spectrometry experiments, which allows precise quantification of modifications across experimental conditions .

How is C9orf21/AAED1 expression altered in cancer and what are the functional implications?

Research has demonstrated that C9orf21/AAED1 expression is altered in certain cancers, with particular evidence in gastric cancer:

Expression patterns:

• Increased expression observed in gastric cancer tissues compared to adjacent normal tissues

• Expression levels correlate with clinical parameters including tumor size and stage

Functional implications:

• Proliferation: AAED1 modulates cancer cell proliferation, with knockdown resulting in reduced proliferation rates

• Metabolism: AAED1 affects glycolytic metabolism in cancer cells, potentially contributing to the Warburg effect

• Molecular mechanisms: May involve interaction with key metabolic enzymes or signaling pathways that regulate cell growth

Experimental validation approaches:

• Colony formation assays

• Cell cycle analysis by flow cytometry

• In vivo tumor xenograft models

• Correlation analysis with clinical outcomes in patient cohorts

Researchers investigating C9orf21/AAED1 in cancer should consider both its direct effects on cellular processes and its potential as a biomarker for disease progression or therapeutic response .

What methodological approaches should be considered when analyzing C9orf21/AAED1 in blood-brain barrier studies?

When investigating C9orf21/AAED1 in relation to the blood-brain barrier (BBB), several specialized methodological approaches should be considered:

In vitro BBB models:

• Transwell co-culture systems: Human brain microvascular endothelial cells (hBMECs) with astrocytes and/or pericytes

• Microfluidic organ-on-chip platforms: For dynamic flow conditions that better mimic physiological BBB

• Measurement parameters: TEER (transendothelial electrical resistance), permeability coefficients for model compounds

Ex vivo approaches:

• Isolated brain microvessels: For protein/mRNA extraction and analysis of C9orf21 expression

• Brain microvessel endothelial cell isolation: For proteomic analysis, as demonstrated in studies with C9orf72 models

In vivo approaches:

• In situ transcardiac brain perfusion: Has been successfully used to study BBB transport in C9orf72 models

• Tandem Mass Tag (TMT)-proteomics: For analysis of isolated brain microvascular endothelial cells (BMECs)

Data analysis considerations:

• Comparison between disease models (e.g., C9orf72 expansion models) and wild-type controls

• Correlation with BBB transport markers (e.g., glucose transporter expression)

• Functional enrichment analysis of proteomic data

These methodologies have been successfully applied in C9orf72 studies and could be adapted to investigate C9orf21/AAED1 in BBB function and dysfunction .

How should researchers interpret contradictory findings regarding C9orf21/AAED1 in different experimental systems?

When encountering contradictory findings regarding C9orf21/AAED1 across different experimental systems, researchers should consider:

Sources of experimental variation:

• Cell/tissue type differences: C9orf21 may have context-dependent functions

• Expression level differences: Overexpression systems may produce artifacts not present at endogenous levels

• Tag interference: Different protein tags (Myc/DDK, GFP, etc.) may affect protein function

• Species differences: Despite high conservation (93% between human and mouse/rat), species-specific functions may exist

Resolution strategies:

• Cross-validation: Employ multiple experimental approaches (e.g., both gain- and loss-of-function)

• Physiological relevance: Prioritize findings from systems with endogenous expression levels

• Rescue experiments: Perform complementation studies to validate specificity

• Dose-response relationships: Determine if effects are dependent on expression levels

• Temporal considerations: Assess acute versus chronic effects of modulating C9orf21

Integration framework:

• Develop a working model that accommodates seemingly contradictory findings

• Consider potential context-specific regulatory mechanisms

• Validate key findings in primary cells or tissues when possible

This systematic approach helps reconcile disparate findings and builds a more comprehensive understanding of C9orf21/AAED1 biology .

What are the critical factors to consider when designing repeat-primed PCR for C9orf21 studies?

While C9orf21 is distinct from C9orf72 (which is known for hexanucleotide repeat expansions), the methodological considerations for repeat-primed PCR are valuable for researchers studying genomic regions:

Primer design considerations:

• Specificity: Design primers that uniquely target C9orf21 with minimal off-target binding

• GC content: Optimize GC content (40-60%) to ensure efficient amplification

• Annealing temperature: Empirically determine optimal temperature for specificity

• Amplicon size: Keep amplicons under 200bp for optimal efficiency

PCR optimization parameters:

• Mastermix components: Consider specialized mixes for difficult templates (high GC content)

• Additives: 5M Betaine can improve amplification of difficult templates

• Cycling conditions:

  • Initial denaturation: 95°C for 10 minutes

  • Two-stage cycling with gradually increasing extension times

  • Final extension at 72°C for 7 minutes

Controls and validation:

• Include positive and negative controls in each experiment

• Verify PCR products by sequencing to confirm target specificity

• Consider alternative methods (qPCR, digital PCR) for validation

The protocols used for C9orf72 repeat expansion detection can provide methodological guidance, though they should be adapted specifically for C9orf21 genomic regions of interest .

How can researchers effectively use recombinant C9orf21/AAED1 as a control in experimental validation?

Recombinant C9orf21/AAED1 protein serves as an important control in various experimental settings:

Antibody validation:

• Blocking experiments: Pre-incubate antibody with recombinant protein (100× molar excess) for 30 minutes at room temperature before application to samples

• Western blot positive control: Load 10-50 ng of recombinant protein alongside experimental samples

• ELISA standard curve: Use serial dilutions (0.1-1000 ng/mL) for quantitative assays

Mass spectrometry applications:

• C13 and N15-labeled protein: Use as internal standard for absolute quantification

• Peptide identification: Generate theoretical peptide maps from recombinant sequence

• Post-translational modification analysis: Compare modification patterns between recombinant and endogenous protein

Functional assays:

• Enzyme activity baseline: Establish baseline activity for comparison with mutant variants

• Protein-protein interaction controls: Use as bait protein in pull-down experiments

• Structural studies: Purified protein for crystallography or NMR studies

When using recombinant protein, researchers should verify protein quality through SDS-PAGE (>85% purity) and consider potential differences from endogenous protein (such as tag effects or lack of post-translational modifications) .

What are the best practices for designing co-expression network analyses to identify functional associations of C9orf21/AAED1?

When implementing co-expression network analyses to identify functional associations of C9orf21/AAED1:

Data preparation and quality control:

• Expression normalization: Use appropriate normalization methods (e.g., TMM, quantile normalization)

• Batch effect correction: Apply ComBat or similar methods if data comes from multiple sources

• Outlier detection: Remove samples with extreme expression patterns

• Covariate adjustment: Generate residual expression values adjusted for relevant covariates (age, sex, RIN)

Network construction parameters:

• Correlation method: Use biweight mid-correlation ("bicor") for robustness against outliers

• Signed hybrid network: Preserves direction of correlations

• Power threshold: Set to maintain scale-free topology (typically 6-12)

• Module detection: Apply dynamic tree cutting with merge height of 0.4 and minimum module size of 30

Functional interpretation:

• Module eigengene analysis: Correlate with phenotypic traits or experimental conditions

• Module membership calculation: Determine strength of association between C9orf21 and each module

• Hub gene identification: Identify central genes in modules containing C9orf21

• Pathway enrichment analysis: Determine biological processes represented in C9orf21-containing modules

Visualization and validation:

• Network visualization: Use Cytoscape (v3.8.2 or newer) to create interactive network graphs

• Cross-validation: Split data into discovery and validation cohorts

• Experimental validation: Test predicted interactions through co-immunoprecipitation or functional assays