CDKN2AIPNL Human

What post-translational modifications occur in CDKN2AIPNL?

CDKN2AIPNL undergoes several post-translational modifications that likely regulate its function and stability:

These modifications suggest dynamic regulation of CDKN2AIPNL through various cellular signaling pathways . The phosphorylation at S15 is particularly notable as this site has been found to be affected in thyroid gland cancer, indicating potential relevance to carcinogenesis .

What cellular functions is CDKN2AIPNL associated with?

CDKN2AIPNL is implicated in several cellular processes:

• Signal transduction pathways, as indicated by its biological process classification .

• Cell cycle regulation, particularly in checkpoint mechanisms .

• Tumor suppressor functions, suggesting a role in preventing uncontrolled cell proliferation .

• Response to oxidative stress, indicating involvement in cellular stress response pathways .

• Apoptosis and cellular proliferation regulation .

Its localization in the nucleolus and nucleoplasm suggests roles in nuclear processes, potentially including transcriptional regulation or ribosome biogenesis .

How does CDKN2AIPNL contribute to cancer biology and tumor progression?

CDKN2AIPNL has significant implications in cancer biology through several mechanisms:

• The protein appears to function in tumor suppressor pathways, with its deletion or mutation potentially contributing to oncogenesis .

• Variants affecting the S15 phosphorylation site have been associated with thyroid gland cancer, suggesting a functional role in this malignancy .

• CDKN2AIPNL may influence cancer-related cellular processes including cell cycle checkpoint regulation, apoptosis, and response to cellular stress .

• Its interaction with other tumor suppressor pathways may create complex networks affecting cancer initiation and progression .

Research using knockout models has provided insights into how CDKN2AIPNL deletion affects oncogenic pathways, though the exact molecular mechanisms require further elucidation. The protein's role in various cancer types beyond thyroid cancer remains an active area of investigation .

What experimental models and systems are available for studying CDKN2AIPNL?

Researchers have several models and systems available for CDKN2AIPNL studies:

• CDKN2AIPNL Knockout cell lines: HEK293-based knockout lines are commercially available and serve as valuable tools for loss-of-function studies .

• Recombinant protein systems: Full-length human CDKN2AIPNL (1-116 aa) expressed in E. coli systems is available for in vitro studies with >85% purity .

• Expression systems: The protein can be expressed with tags (such as His-tags) for purification and detection purposes .

• CRISPR/Cas9 and siRNA systems: These are particularly useful for targeted gene editing and knockdown experiments to study CDKN2AIPNL function .

The HEK293 knockout cell line background is especially valuable due to its robust cloning efficiency, transfection capability, and suitability for protein expression studies .

How can contradictory findings in CDKN2AIPNL research be reconciled?

Reconciling contradictory findings in CDKN2AIPNL research requires several methodological considerations:

• Cell type specificity: CDKN2AIPNL may have different functions in different cell types. Researchers should carefully consider cellular context when interpreting results.

• Isoform differences: The existence of two isoforms produced by alternative splicing suggests functional diversity that could explain seemingly contradictory findings .

• PTM status: The various post-translational modifications (acetylation, phosphorylation, ubiquitination) may cause the protein to behave differently under different experimental conditions .

• Experimental approach standardization: Using standardized experimental approaches, particularly for knockout studies, can help resolve contradictions.

• Interaction partners: CDKN2AIPNL may interact with different proteins in different cellular environments, leading to context-dependent functions.

Researchers should develop comprehensive experimental designs that account for these variables, and clearly report the specific conditions used to enable proper comparison across studies.

What are optimal protocols for working with recombinant CDKN2AIPNL protein?

When working with recombinant CDKN2AIPNL, researchers should consider the following optimized protocols:

• Storage and stability:

  • Store at 4°C if the entire vial will be used within 2-4 weeks

  • For longer periods, store frozen at -20°C

  • For long-term storage, add a carrier protein (0.1% HSA or BSA)

  • Avoid multiple freeze-thaw cycles

• Buffer conditions:

  • Optimal formulation: 20mM Tris-HCl buffer (pH 8.0), 0.15M NaCl, 20% glycerol, and 1mM DTT

  • Typical concentration: 0.25mg/ml

• Analysis methods:

  • SDS-PAGE (15%) is effective for purity assessment

  • Mass spectrometry can be used for verification

• Applications:

  • Suitable for protein-protein interaction studies

  • Can be used as a positive control in Western blotting

  • Applicable in enzymatic assays studying PTM mechanisms

When designing experiments, it's essential to account for the His-tag or other fusion tags that may be present in the recombinant protein, as these could affect protein function or interaction studies .

How should researchers design and utilize CDKN2AIPNL knockout cell models?

Effective utilization of CDKN2AIPNL knockout models requires careful experimental design:

• Model selection:

  • HEK293-based knockout lines are well-established for CDKN2AIPNL research

  • Consider the specific research question when choosing between electric rotation method vs. viral method knockout approaches

• Cultivation conditions:

  • Optimal media: 90% DMEM + 10% FBS

  • Passage ratio: 1:3~1:6

  • Storage: Dry ice preservation for transport; liquid nitrogen for long-term storage

• Functional validation:

  • Confirm knockout by Western blot or PCR

  • Assess phenotypic changes in cell proliferation, differentiation, and apoptosis

  • Compare with wild-type controls under identical conditions

• Experimental applications:

  • Particularly effective for studying cellular responses to oxidative stress

  • Valuable for evaluating impact on apoptosis and proliferation

  • Can be incorporated into CRISPR/Cas9 and siRNA knockdown experiments

• Controls and considerations:

  • Always include appropriate wild-type controls

  • Consider potential compensatory mechanisms when interpreting results

  • Test for mycoplasma contamination regularly

What techniques are recommended for analyzing CDKN2AIPNL post-translational modifications?

Analyzing CDKN2AIPNL post-translational modifications requires specific techniques optimized for each modification type:

• For phosphorylation (e.g., at S15):

  • Phospho-specific antibodies for Western blotting

  • Mass spectrometry following phosphopeptide enrichment

  • Phos-tag gel electrophoresis for mobility shift detection

  • In vitro kinase assays to identify responsible enzymes

• For acetylation (e.g., at M1):

  • Acetylation-specific antibodies

  • Mass spectrometry with acetylpeptide enrichment

  • Deacetylase inhibitor treatments to enhance detection

• For ubiquitination (e.g., at K35, K38):

  • Ubiquitination-specific antibodies

  • Proteasome inhibitor treatments prior to analysis

  • Tandem ubiquitin binding entity (TUBE) assays

  • Immunoprecipitation under denaturing conditions

• General considerations:

  • Combine multiple techniques for confirmation

  • Include appropriate positive and negative controls

  • Consider dynamic nature of PTMs and their potential interdependence

  • When possible, correlate PTM status with functional outcomes

Understanding these modifications is crucial as they may directly impact CDKN2AIPNL function, particularly in disease contexts such as thyroid cancer .

What are the most promising therapeutic approaches targeting CDKN2AIPNL?

Several therapeutic approaches targeting CDKN2AIPNL show promise for future development:

• Restoration of function: For cancers where CDKN2AIPNL function is lost, gene therapy approaches to restore expression could have therapeutic potential.

• PTM modulation: Targeting the enzymes responsible for CDKN2AIPNL phosphorylation, acetylation, or ubiquitination could regulate its function in disease contexts .

• Synthetic lethality: Identifying genes that, when inhibited, cause selective death in cells with CDKN2AIPNL alterations could provide novel therapeutic targets.

• Protein-protein interaction disruption: Small molecules that modulate CDKN2AIPNL interactions with other proteins in the CARF family could have therapeutic applications .

• Combination approaches: Using CDKN2AIPNL status as a biomarker to guide selection of other therapeutics, particularly in cancers where its expression is altered.

Research using knockout models has already begun elucidating potential therapeutic windows, though translation to clinical applications requires further preclinical validation and mechanistic understanding .

What emerging technologies could advance CDKN2AIPNL research?

Emerging technologies that could significantly advance CDKN2AIPNL research include:

• Single-cell analysis: Techniques like single-cell RNA-seq and proteomics could reveal cell-type specific functions and expression patterns of CDKN2AIPNL .

• Cryo-EM and structural biology: Determining the three-dimensional structure of CDKN2AIPNL alone and in complexes would provide crucial insights into its function.

• Proximity labeling techniques: BioID or APEX2-based methods could identify previously unknown interaction partners in living cells.

• CRISPR base editing: More precise genetic manipulation to introduce specific mutations found in diseases could help understand their functional consequences.

• Organoid models: Patient-derived organoids could better recapitulate the in vivo context of CDKN2AIPNL function compared to traditional cell lines.

• Artificial intelligence approaches: Machine learning algorithms could identify patterns in large datasets to predict CDKN2AIPNL functions or interactions not evident through conventional analysis.

• Proteomics-based interactome mapping: Advanced mass spectrometry techniques could uncover the complete CDKN2AIPNL interactome under various cellular conditions .