p16-INK4a Human, TAT
Description
1. Introduction to p16-INK4a Human, TAT
p16-INK4a Human, TAT is a recombinant protein fusion combining the tumor suppressor p16-INK4a with the HIV-1 TAT peptide (GYGRKKRRQRRR). This chimeric protein enables efficient intracellular delivery of p16-INK4a, bypassing traditional transfection methods . p16-INK4a is encoded by the CDKN2A gene and functions as a cyclin-dependent kinase (CDK) inhibitor, regulating cell cycle progression, senescence, and apoptosis .
Molecular Structure and Production
The recombinant protein comprises 167 amino acids, including:
| Property | Specification |
|---|---|
| Molecular Weight | 18.0 kDa |
| Expression Host | Escherichia coli (E. coli) |
| Purity | >95% (SDS-PAGE/HPLC) |
| Endotoxin Levels | <1 EU/µg |
The TAT peptide facilitates rapid cellular uptake via lipid raft-mediated macropinocytosis, enabling functional delivery within 5–60 minutes .
Mechanism of Action
p16-INK4a exerts its tumor-suppressive effects through:
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CDK4/6 Inhibition: Binds directly to CDK4/6, blocking cyclin D interaction and preventing retinoblastoma protein (pRb) phosphorylation .
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Cell Cycle Arrest: Induces G1-phase arrest by maintaining pRb in its active, hypophosphorylated state .
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Senescence and Apoptosis: Triggers replicative senescence in aging cells and apoptosis in cancer cells via p53-independent pathways .
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MicroRNA Regulation: Modulates miR-141 and miR-146b-5p through CDK4-Sp1 complex interactions, influencing gene expression .
In Vitro and In Vivo Studies
Post-Translational Modifications
Therapeutic Potential
Product Specs
Q&A
What is the mechanism of TAT-mediated delivery for p16-INK4a in cellular models?
The HIV-1 transactivator of transcription (TAT) peptide enables cell-penetrating capabilities via electrostatic interactions with membrane phospholipids and glycosaminoglycans. Recombinant p16-INK4a-TAT (18 kDa) contains a 13-residue C-terminal TAT sequence (GGYGRKKRRQRRR), allowing direct cytoplasmic and nuclear translocation without transfection reagents . Methodologically, researchers should:
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Optimize concentration: Titrate doses between 50–500 nM based on cell type (e.g., epithelial vs. hematopoietic).
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Validate uptake: Use immunofluorescence with anti-p16 antibodies (e.g., clone JC8) to confirm nuclear localization within 2–4 hours post-treatment .
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Control for off-target effects: Include TAT-only controls to distinguish p16-specific outcomes from peptide-induced artifacts .
How do researchers assess p16-INK4a-TAT’s functional activity in cell cycle arrest assays?
Functional validation requires correlating protein delivery with downstream CDK4/6 inhibition:
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Cell cycle analysis: Perform propidium iodide staining 24–48 hours post-treatment. Expect G1 phase accumulation (>60% in HMEC cells) .
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CDK4/6 activity assays: Use kinase-specific substrates (e.g., Rb C-terminal fragment) with immunoprecipitated CDK4/6 complexes. A ≥50% reduction in phosphorylation indicates effective inhibition .
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Senescence markers: Co-stain for β-galactosidase (SA-β-Gal) and p21WAF1/CIP1 to confirm senescence induction .
How can conflicting data on p16-INK4a-TAT’s senescence induction in primary versus cancer cells be resolved?
Discrepancies often arise from cell-type-specific RB pathway status:
Methodological adjustments:
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Pre-screen for RB1 mutations via sequencing or immunoblotting.
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Combine p16-INK4a-TAT with CDK4/6 inhibitors (e.g., palbociclib) in RB-null models .
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Use single-cell RNA sequencing to identify senescence-resistant subpopulations .
What experimental designs address p16-INK4a-TAT’s crosstalk with p53 in vivo?
The p53-p16 axis exhibits context-dependent antagonism:
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In p53-WT models: p16-INK4a-TAT upregulates p21 via p53 stabilization, amplifying senescence .
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In p53-null models: p16-INK4a-TAT induces DNMT1 degradation, causing CDKN2A demethylation and sustained p16 expression .
Recommended workflow:
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Genotype models: Use p53−/− mice or CRISPR-edited cell lines.
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Monitor DNA damage: Quantify γH2AX foci alongside p16/p53 immunofluorescence.
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Epigenetic profiling: Perform Methyl-Seq on the CDKN2A promoter post-treatment .
How do researchers optimize p16-INK4a-TAT transduction in low-proliferation cell types (e.g., quiescent stem cells)?
Slow-cycling cells resist senescence due to reduced CDK4/6 activity. Strategies include:
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Co-treatment with mitogens: EGF (10 ng/mL) or FGF2 (5 ng/mL) enhances CDK4/6 activation, sensitizing cells to p16-INK4a-TAT .
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Pulse-chase dosing: 2-hour pulses followed by 72-hour recovery improve senescence synchrony .
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Metabolic tracing: Use 2H-glucose labeling to link proliferation resumption to p16 efficacy .
What criteria distinguish p16-INK4a-TAT’s on-target effects from oxidative stress artifacts?
The TAT peptide can induce ROS in sensitive lineages (e.g., neurons). Control experiments should:
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Measure ROS levels: Compare H2O2 production (via Amplex Red) in TAT-only vs. p16-TAT groups.
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Antioxidant rescue: Add 2 mM N-acetylcysteine; senescence should persist if p16-specific .
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Transcriptomic profiling: RNA-Seq should show RB/E2F target repression without NRF2 pathway activation .
Can p16-INK4a-TAT be integrated with CRISPR screens to identify senescence bypass mechanisms?
Yes. A pooled sgRNA library screen with p16-INK4a-TAT treatment revealed DNMT1, EZH2, and BMI1 as critical regulators of senescence maintenance . Key steps:
Product Science Overview
Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A), also known as p16INK4a, is a crucial protein in the regulation of the cell cycle. It is encoded by the CDKN2A gene located on chromosome 9p21.3 in humans . This gene is known for its role in tumor suppression and is frequently mutated or deleted in various types of cancers .
The CDKN2A gene encodes two distinct proteins through alternative splicing: p16INK4a and p14ARF. These proteins are transcribed from the same second and third exons but have different first exons, resulting in different reading frames and amino acid sequences . The p16INK4a protein consists of four ankyrin repeats, each spanning 33 amino acid residues, forming a helix-turn-helix motif .
CDKN2A plays a pivotal role in cell cycle regulation by inhibiting cyclin-dependent kinases 4 and 6 (CDK4 and CDK6). This inhibition prevents the phosphorylation of the retinoblastoma (Rb) protein, thereby blocking the transition from the G1 phase to the S phase of the cell cycle . The p14ARF protein, on the other hand, stabilizes the tumor suppressor protein p53 by interacting with and sequestering the E3 ubiquitin-protein ligase MDM2, which is responsible for p53 degradation .
Mutations and deletions in the CDKN2A gene are common in a wide variety of tumors, making it a significant tumor suppressor gene . Germline mutations in CDKN2A are associated with familial melanoma, glioblastoma, and pancreatic cancer . Additionally, the gene contains single nucleotide polymorphisms (SNPs) associated with an increased risk of coronary artery disease .
The human recombinant form of CDKN2A, often fused with the TAT peptide, is used in research to study its function and potential therapeutic applications. The TAT peptide facilitates the delivery of the recombinant protein into cells, allowing for the investigation of its effects on cell cycle regulation and tumor suppression.
