FNDC5 Human, Yeast
Description
Definition and Background
FNDC5 Human, Yeast refers to recombinant human Fibronectin Type III Domain-Containing Protein 5 (FNDC5) produced in yeast expression systems. FNDC5 is a type I transmembrane glycoprotein and precursor of the hormone irisin, which is cleaved from its extracellular domain . Recombinant FNDC5 is engineered for research and therapeutic applications, leveraging yeast platforms (Pichia pastoris) for scalable, glycosylated protein production .
Production Methods in Yeast
Expression System:
Key Steps:
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Transformation: Linearized plasmid integrated into yeast genome via homologous recombination .
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Selection: Zeocin-resistant colonies screened for high-yield expression .
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Induction: Methanol-induced expression in buffered media (BMMY) .
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Purification: Ni-NTA affinity chromatography yields >90% purity .
Yield: ~33 kDa glycosylated protein confirmed via SDS-PAGE .
In Vitro Studies
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Adipocyte Browning: Recombinant FNDC5 (20–50 nM) upregulates UCP-1, PRDM16, and CIDEA in 3T3-L1 cells, promoting white adipose tissue (WAT) browning .
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Lipolysis: Enhances ATGL and HSL expression, increasing lipolysis .
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Activity Comparison: Secretable FNDC5 (sFNDC5) shows stronger effects than irisin due to intact structure .
In Vivo Applications
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Pump Implantation: Osmotic pumps delivering 0.25 µg/µL FNDC5 induce metabolic changes in rodent models .
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Specific Activity: 10 ng/mL induces UCP-1 expression in adipocytes .
Advantages of Yeast-Produced FNDC5 vs. Other Systems
Therapeutic Potential
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Obesity/Metabolic Disorders: Promotes WAT browning, increasing energy expenditure .
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Diabetes: Improves glucose uptake and insulin sensitivity via AMPK/MAPK pathways .
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Neuroprotection: Linked to BDNF upregulation, suggesting roles in cognitive health .
Challenges and Controversies
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Start Codon Mutation: Human FNDC5 has an ATA start codon, reducing translation efficiency .
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Irisin Detection: Commercial antibody assays often misidentify FNDC5 fragments as irisin .
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Species Specificity: Murine and human irisin sequences are identical, but human expression is debated .
Future Directions
Product Specs
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__N__K__D__E__V__T__M__K__E.Q&A
How is FNDC5 structurally and functionally distinct from irisin?
FNDC5 is a transmembrane glycoprotein (212 aa in humans) with a signal peptide, fibronectin type III domain (FNIII), and membrane anchor. Irisin is the soluble cleaved ectodomain (112 aa in humans), retaining the FNIII core . Key distinctions include:
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Membrane localization: FNDC5 remains anchored to cells, while irisin circulates as a hormone .
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Biological activity: Irisin mediates systemic effects (e.g., browning white fat, cardiac protection), while FNDC5 may regulate local cellular processes .
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Processing: The cleavage enzyme for FNDC5 remains unidentified, though proteolytic shedding mechanisms (e.g., ADAM/TACE proteases) are hypothesized .
What are the primary challenges in detecting FNDC5/irisin in human samples?
Recommendation: For human studies, combine mass spectrometry with orthogonal validation (e.g., immunoprecipitation) to resolve discrepancies observed in ELISA-based studies .
How does FNDC5 expression vary across tissues and conditions?
FNDC5 is primarily expressed in skeletal muscle, with lower levels in liver, adipose tissue, and brain . Key regulatory factors include:
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Exercise: PGC-1α upregulates FNDC5 in muscle, linking physical activity to irisin secretion .
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Aging: FNDC5 declines in aging hearts, correlating with cardiac dysfunction .
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Metabolic stress: Overexpression in liver prevents diet-induced obesity in rodents .
How can conflicting data on FNDC5/irisin’s role in human obesity be resolved?
Key Insight: Recent studies using FNDC5 knockouts and recombinant irisin (e.g., in Alzheimer’s models) suggest irisin’s therapeutic potential persists regardless of baseline human levels .
What strategies optimize recombinant FNDC5 expression in yeast systems?
Pichia pastoris systems show promise for producing secretable FNDC5 (sFNDC5) isoforms lacking transmembrane domains . Optimization steps include:
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Vector design: Use methanol-inducible promoters (e.g., AOX1) for scalable production.
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Secretion efficiency: Engineer sFNDC5 with N-terminal signal peptides to bypass ER retention signals.
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Purification: Utilize chromatography (e.g., Ni-NTA for His-tagged proteins) to achieve >90% purity.
Performance Comparison:
| System | Yield | Activity (vs. Irisin) | Advantage |
|---|---|---|---|
| Pichia pastoris | High | Similar or superior | Low-cost, high-volume production |
| Mammalian (HEK293) | Moderate | Standard | Native glycosylation |
How does FNDC5 modulate cardiac function in aging?
FNDC5 attenuates aging-related cardiac dysfunction via:
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Inflammation suppression: Inhibits NLRP3 inflammasome activation, reducing TNF-α and IL-1β .
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AMPKα activation: Blocks lysosomal degradation of GLP-1R, enhancing AMPKα signaling and mitochondrial biogenesis .
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Senescence inhibition: Irisin infusion mitigates TNF-α-induced senescence in cardiomyocytes .
Therapeutic Potential: AAV9-mediated FNDC5 overexpression in young mice delays cardiac dysfunction onset in old age .
What are the implications of alternative FNDC5 transcripts in humans?
Human FNDC5 has two start codons (ATA and ATG), leading to:
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Full-length FNDC5: Rarely expressed due to weak ATA initiation.
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Truncated FNDC5: Lacks signal peptide, potentially intracellular .
Key Findings:
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Mass spectrometry confirms human plasma irisin (~3 ng/mL), supporting functional FNDC5 cleavage .
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The sFNDC5 isoform (lacking transmembrane domain) exhibits enhanced lipolysis in 3T3-L1 cells compared to irisin .
Recommendation: Investigate alternative splicing mechanisms and their impact on irisin secretion in human tissues.
How to design a FNDC5 knockout (KO) study?
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Model selection: CRISPR-Cas9 targeting exons encoding the FNIII domain critical for irisin cleavage.
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Validation: Confirm KO via Western blot (absence of full-length FNDC5 and irisin) and qPCR (reduced FNDC5 mRNA).
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Phenotyping: Assess metabolic (glucose tolerance, fat browning), cardiac (echocardiography), and cognitive (maze testing) outcomes .
What are the limitations of using recombinant irisin in therapeutic trials?
How to analyze FNDC5’s role in bone metabolism?
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In vivo models:
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Use Fndc5 KO mice to assess cortical bone density and osteoclast activity.
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Administer recombinant irisin to evaluate anabolic effects.
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In vitro:
Can FNDC5 serve as an exercise mimetic?
Yes, in rodent models:
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Irisin infusion improves cognitive function and metabolic health in sedentary mice .
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FNDC5 overexpression in muscle recapitulates exercise-induced browning of white fat .
Caveat: Human trials are needed to confirm translatability due to differences in FNDC5 cleavage efficiency .
What is the therapeutic potential of FNDC5 in neurodegenerative diseases?
Irisin promotes neurogenesis and reduces amyloid-β plaques in Alzheimer’s models . Potential strategies include:
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Peripheral administration: Systemic irisin delivery to cross the blood-brain barrier.
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Gene therapy: AAV-mediated FNDC5 expression in hippocampal neurons.
How to resolve discrepancies in FNDC5/irisin quantification?
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Multi-omics approach: Combine proteomics (MS), transcriptomics (FNDC5 mRNA), and functional assays (e.g., UCP1 induction in adipocytes).
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Standardization: Establish reference materials (e.g., recombinant irisin) for ELISA calibration .
What statistical methods are appropriate for FNDC5 KO studies?
Product Science Overview
FNDC5 is a protein-coding gene that encodes a type I transmembrane glycoprotein. The protein contains a fibronectin type III domain, which is a common structural motif found in a variety of proteins involved in cell adhesion, growth, and differentiation . The FNDC5 gene is located on chromosome 1 in humans .
One of the most notable aspects of FNDC5 is its cleaved form, known as irisin. Irisin is released into the bloodstream during physical exercise and has been implicated in the conversion of white adipose tissue to brown adipose tissue, a process known as "browning" . This conversion is significant because brown fat burns energy and generates heat, which can help in weight management and metabolic health.
FNDC5 is expressed in various tissues, but it is most prominently found in muscle tissue. During exercise, the expression of FNDC5 increases, leading to higher levels of irisin in the bloodstream . Irisin has been shown to have several beneficial effects, including improving glucose homeostasis and lipid metabolism .
The recombinant form of FNDC5, produced in yeast, is a glycosylated polypeptide chain containing 110 amino acids and has a molecular mass of 20-25 kDa . This recombinant protein is often used in research to study its biological activity and potential therapeutic applications. The yeast expression system is advantageous because it allows for the production of large quantities of the protein with proper post-translational modifications .
The recombinant FNDC5 protein is typically lyophilized (freeze-dried) for stability and can be stored at room temperature for up to three weeks. For long-term storage, it is recommended to keep the protein desiccated below -18°C. Upon reconstitution, the protein should be stored at 4°C for short-term use and below -18°C for long-term use .
Given its role in metabolic processes, FNDC5/irisin has been studied for its potential therapeutic applications in treating metabolic disorders such as obesity and type 2 diabetes. Additionally, its ability to induce the browning of white adipose tissue makes it a promising candidate for weight management therapies .
