Recombinant Human Transmembrane protein C14orf180 (C14orf180)

What is C14orf180 and what are its alternative names?

C14orf180 is a transmembrane protein encoded by a gene located on chromosome 14 (14q32.33) in humans. It is also known as NRAC (Nutritionally-regulated adipose and cardiac enriched protein), and C14orf77. The protein consists of 160 amino acids post-translation and has a total of 6 exons. It was identified as a novel gene with specific expression patterns in adipose and cardiac tissues and shows regulation in response to nutritional status .

What is the molecular structure and general properties of C14orf180?

C14orf180 is a 160-amino acid protein with a predicted molecular weight of 18.1 kDa and an unmodified isoelectric point of 11 pI. Secondary structure analysis reveals three alpha-helices and two beta-sheets, a pattern that appears to be conserved across some mammals. The protein has two known isoforms: Isoform X1 (177 amino acids) and Isoform X2 (160 amino acids) . The full amino acid sequence is: MRTAAGAVSPDSRPETRRQTRKNEEAAWGPRVCRAEREDNRKCPPSILKRSRPEHHRPEAKPQRTSRRVWFREPPAVTVHYIADKNATATVRVPGRPRPHGGSLLLQLCVCVLLVLALGLYCGRAKPVATALEDLRARLLGLVLHLRHVALTCWRGLLRL .

What is the predicted cellular localization of C14orf180?

C14orf180 is predicted to be active in the plasma membrane according to recent annotations. This localization is consistent with its identification as a transmembrane protein and its enrichment in adipose tissues, suggesting potential roles in cellular signaling or metabolic processes at the cell surface . Experimental evidence from studies on mouse orthologs confirms its localization to the plasma membrane, particularly in adipocyte cell models .

What considerations are important when designing experiments to study C14orf180 expression?

When designing experiments to study C14orf180 expression, researchers should consider:

• Appropriate tissue selection: Focus on adipose tissue (both white and brown) and heart tissue, where C14orf180 is predominantly expressed .

• Nutritional intervention design: Since C14orf180 is nutritionally regulated, experiments should control for feeding status (fed vs. fasted) and diet composition (standard chow vs. high-fat diet) .

• Replication: As with all experiments, biological triplicates are the minimum requirement for statistical validity .

• Randomization: Samples, plots, and experimental groups should be randomized to avoid biases .

• Sample preparation consistency: All preparation stages should occur across samples at the same time and be performed by the same person to minimize technical variation .

• Quality control: RNA quality should be established (RIN > 7.0) prior to expression analysis, and DNA/RNA should not be degraded .

What methods are recommended for analyzing C14orf180 expression in response to nutritional changes?

Based on existing research protocols, the following methods are recommended:

• Quantitative Real-time PCR: For C14orf180 gene expression analysis, researchers can use specific primers (forward: 5′-TCTCTCGCTCTAATTCCCACC-3′; reverse: 5′-CACTTCCTGTTACCATCCCTCT-3′) with β-actin as an internal control .

• RNA-Seq: This approach has been successfully used to identify differential expression of C14orf180 in response to nutritional interventions such as high-fat diet or fasting .

• Nutritional intervention models: Studies should include both acute interventions (24-hour fasting) and chronic interventions (extended high-fat diet feeding, typically 3 months) to capture different regulatory mechanisms .

• Tissue collection: Tissues should be immediately placed in RNAlater solution to preserve RNA integrity before extraction with appropriate kits including DNase treatment .

• Statistical analysis: Researchers should determine their statistical model and planned comparisons before beginning experiments to ensure proper design and power calculations .

What are the recommended protocols for handling recombinant C14orf180 protein?

For researchers working with recombinant C14orf180 protein:

• Reconstitution: The lyophilized protein should be briefly centrifuged prior to opening. Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Storage: Add glycerol to a final concentration of 5-50% (recommended 50%) and aliquot for long-term storage at -20°C/-80°C. Store working aliquots at 4°C for up to one week .

• Avoid freeze-thaw cycles: Repeated freezing and thawing is not recommended as it may compromise protein integrity .

• Buffer considerations: The protein is typically provided in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 .

• Quality assessment: Verify protein purity, which should be greater than 90% as determined by SDS-PAGE before experimental use .

How is C14orf180 expression affected by nutritional status in animal models?

C14orf180 (NRAC) expression shows significant regulation by nutritional status:

• Fasting effects: 24-hour fasting reduces NRAC expression in both white adipose tissue and brown adipose tissue in mouse models .

• Obesity effects: Chronic high-fat diet induces obesity and leads to reduced NRAC expression in white adipose tissue, suggesting a potential role in metabolic adaptation .

• Tissue specificity: The nutritional regulation of NRAC appears to be tissue-specific, with the most pronounced effects observed in adipose tissues .

This nutritional regulation pattern suggests that NRAC may play a role in energy homeostasis and metabolic adaptation, potentially contributing to the physiological responses to different nutritional states.

What is known about C14orf180 expression patterns across tissues and development?

C14orf180 shows distinct tissue-specific expression patterns:

• Tissue distribution: C14orf180 is expressed specifically and abundantly in adipose tissue (both white and brown) and heart tissue, with moderate expression also observed in skeletal muscle .

• Developmental expression: In human fetal development, C14orf180 expression is highest in heart tissue between 11-20 weeks of gestation, with peak expression at approximately 18 weeks .

• Adipocyte differentiation: C14orf180 is highly induced during adipocyte differentiation, suggesting a potential role in adipocyte development or function .

• Evolutionary conservation: The expression pattern appears to be conserved across species, indicating functional importance of this tissue-specific expression .

This restricted expression pattern suggests that C14orf180 may have specialized functions in adipose and cardiac tissues, potentially related to metabolism or tissue-specific signaling.

What is the evolutionary conservation status of C14orf180?

C14orf180 shows significant evolutionary conservation:

• Phylogenetic distribution: The gene is found in jawed vertebrates and is highly conserved in mammals .

• Sequence conservation: Sequence identity across species shows varying degrees of conservation:

• Structure conservation: The predicted secondary structure (three alpha-helices and two beta-sheets) appears to be conserved across mammalian species, suggesting functional importance .

• No paralogs: There are no known paralogs of C14orf180, which may indicate unique, non-redundant functions .

The significant conservation across species, particularly in mammals, suggests an important biological role that has been maintained throughout evolution.

What approaches can be used to investigate the functional role of C14orf180 in adipocyte metabolism?

To investigate C14orf180's functional role in adipocyte metabolism, researchers could consider:

• Gene knockdown/knockout studies: Using siRNA, CRISPR-Cas9, or other gene editing technologies to reduce or eliminate C14orf180 expression in adipocyte cell models or animal models .

• Overexpression studies: Introducing recombinant C14orf180 to examine gain-of-function effects on adipocyte differentiation, lipid metabolism, or insulin signaling .

• Nutritional challenge experiments: Examining how C14orf180 expression changes during metabolic challenges (fasting, refeeding, high-fat diet) and correlating these changes with metabolic parameters .

• Protein interaction studies: Identifying potential binding partners using techniques such as co-immunoprecipitation, yeast two-hybrid, or proximity labeling methods .

• Metabolomic analysis: Comparing metabolite profiles in tissues or cells with normal versus altered C14orf180 expression to identify affected metabolic pathways .

These approaches can provide insights into the physiological role of C14orf180 in adipose tissue metabolism and potentially reveal its contribution to metabolic disorders.

How can researchers investigate potential interactions between C14orf180 and other metabolic pathways?

To investigate interactions between C14orf180 and other metabolic pathways:

• Transcriptomic analysis: Perform RNA-Seq on tissues or cells with manipulated C14orf180 expression to identify co-regulated genes and affected pathways .

• Pathway analysis: Use bioinformatic tools to predict potential interactions between C14orf180 and known metabolic pathways based on expression patterns or structural motifs .

• Metabolic flux analysis: Measure changes in metabolic flux rates in response to C14orf180 manipulation to determine which metabolic processes are affected .

• Signaling pathway inhibition studies: Use specific inhibitors of key metabolic signaling pathways (insulin, AMPK, mTOR) to determine if C14orf180 regulation is dependent on these pathways .

• Co-expression network analysis: Identify genes whose expression patterns correlate with C14orf180 across different metabolic conditions to build potential functional networks .

These approaches can help place C14orf180 within the broader context of cellular metabolism and identify its role in specific metabolic processes.

What are the challenges in studying C14orf180 function and how can they be addressed?

Studying C14orf180 function presents several challenges:

These strategies can help overcome the challenges associated with studying this nutritionally-regulated protein.

What statistical considerations are important when analyzing C14orf180 expression data?

When analyzing C14orf180 expression data, researchers should consider:

Following these statistical best practices ensures robust and reproducible findings when studying C14orf180 expression.

How should researchers interpret changes in C14orf180 expression in the context of metabolic disorders?

When interpreting changes in C14orf180 expression in metabolic disorders:

• Consider tissue context: Changes in expression may have different implications in adipose tissue versus cardiac tissue .

• Correlate with metabolic parameters: Analyze relationships between C14orf180 expression and metabolic measurements (glucose levels, insulin sensitivity, lipid profiles) .

• Temporal dynamics: Distinguish between acute changes (e.g., in response to fasting) and chronic adaptations (e.g., in obesity) .

• Direction of change: Both decreased expression (as seen in obesity and fasting) and increased expression may have functional implications that should be interpreted in the specific metabolic context .

• Species differences: Be cautious when extrapolating findings between species, as there are differences in sequence conservation that may affect function .

• Mechanism versus consequence: Determine whether altered C14orf180 expression is driving metabolic changes or is a consequence of altered metabolism .

These considerations help researchers place C14orf180 expression changes within the broader context of metabolic regulation and disease pathophysiology.

What approaches can be used to link C14orf180 sequence variants to potential functional outcomes?

To link C14orf180 sequence variants to functional outcomes:

• In silico prediction tools: Use computational tools to predict the impact of variants on protein structure, stability, or function .

• Evolutionary conservation analysis: Assess whether variants occur in evolutionarily conserved regions, which may indicate functional importance .

• Structure-function analysis: Map variants onto the predicted secondary structure (three alpha-helices and two beta-sheets) to assess potential structural disruption .

• Functional assays: Develop cell-based assays to compare the activity of wild-type and variant forms of C14orf180 in relevant contexts (adipocyte differentiation, metabolic regulation) .

• Population correlation studies: Analyze associations between C14orf180 variants and metabolic phenotypes in human populations .

• Animal models: Generate animal models expressing specific variants to assess in vivo functional consequences .

These approaches can help determine whether C14orf180 variants contribute to metabolic disease risk or phenotypic variation in adipose and cardiac function.