Recombinant Human Protein APCDD1-like (APCDD1L)

Basic Research Questions

• What is APCDD1L and what are its fundamental characteristics?

  APCDD1L (APC Down-Regulated 1 Like) is a protein-coding gene with a molecular mass of approximately 82 kDa. It consists of 501 amino acids in humans and contains a signal peptide and transmembrane domain. An important paralog of this gene is APCDD1 . Current research suggests APCDD1L may function similarly to APCDD1, which acts as a dual inhibitor of both BMP and Wnt signaling pathways . These pathways are crucial for animal development and homeostasis, and their dysregulation is associated with various pathological conditions including cancer.

• How does APCDD1L relate to APCDD1 in terms of structure and function?

  APCDD1 is a known dual inhibitor of both BMP and Wnt signaling pathways, with experimental evidence showing it can bind to the BmprIa receptor and inhibit nuclear localization of Smad1 (the main BMP effector) . Given that APCDD1L is a paralog of APCDD1, researchers hypothesize similar functions, though with potentially different expression patterns or regulatory mechanisms. The amino acid sequence alignment between these proteins shows conserved domains that may be crucial for their signaling inhibitory functions, particularly in the extracellular region that mediates interaction with pathway components.

• What is currently known about APCDD1L-AS1 and its role in cellular processes?

  APCDD1L-AS1 is a long non-coding RNA (lncRNA) transcribed from the antisense strand of the APCDD1L gene locus. Research indicates it has tissue-specific expression patterns and functions. In clear cell renal cell carcinoma (ccRCC), APCDD1L-AS1 appears to function as a tumor suppressor, with decreased expression associated with higher tumor stage, histological grade, and shorter relapse-free survival . Conversely, in oral squamous cell carcinoma (OSCC), high APCDD1L-AS1 expression is associated with 5-fluorouracil (5-FU) resistance and worse prognosis . This suggests context-dependent roles for this lncRNA in different cancer types.

Advanced Research Questions

• What mechanisms regulate APCDD1L-AS1 expression in clear cell renal cell carcinoma (ccRCC)?

  Research has identified two primary mechanisms responsible for APCDD1L-AS1 downregulation in ccRCC:

  a) DNA hypermethylation: TCGA-KIRC data, GEO datasets, qRT-PCR and pyrosequencing results from clinical specimens demonstrate that hypermethylation of the APCDD1L-AS1 promoter region contributes to its reduced expression .

  b) Loss of von Hippel Lindau (VHL) protein expression: VHL, frequently mutated in ccRCC, appears to regulate APCDD1L-AS1 expression. Western blot and Tandem mass tags (TMT) analyses have established this relationship .

  The dysregulation of histone expression caused by APCDD1L-AS1 overexpression may be one of the important mechanisms through which it suppresses ccRCC progression .

• How does APCDD1L-AS1 influence cancer progression and metastasis?

  In ccRCC, APCDD1L-AS1 functions as a tumor suppressor. Experimental data shows that APCDD1L-AS1 overexpression restrains the growth and metastasis of ccRCC cells both in vitro and in vivo . The functional experiments demonstrated:

  Experimental Condition	Effect on Cell Growth	Effect on Metastasis
  Control	Baseline growth	Baseline metastatic potential
  APCDD1L-AS1 overexpression	Reduced proliferation	Decreased metastatic capacity
  APCDD1L-AS1 knockdown	Enhanced proliferation	Increased metastatic capacity

  These findings suggest that APCDD1L-AS1 could serve as a new therapeutic target in ccRCC treatment .

• What role does APCDD1L-AS1 play in chemoresistance mechanisms?

  In OSCC, APCDD1L-AS1 contributes to 5-FU resistance through several mechanisms:

  a) Exosomal transfer: Extracellular APCDD1L-AS1 can be packaged into exosomes and transferred to sensitive cells, thereby transmitting 5-FU resistance .

  b) miR-1224-5p/NSD2 axis: APCDD1L-AS1 functions as a molecular sponge for miR-1224-5p, preventing it from targeting NSD2. This was confirmed using dual-luciferase reporter assay and RIP assay .

  Knockdown of APCDD1L-AS1 in 5-FU-resistant OSCC cells (HSC-3/5-FU and HN-4/5-FU) resulted in:

  • Reduced IC50 value for 5-FU

  • Suppressed cell viability

  • Accelerated cell apoptosis

  These findings suggest that targeting APCDD1L-AS1 could potentially reverse chemoresistance in OSCC.

• How might the dual inhibition of BMP and Wnt signaling by Apcdd1 inform research on APCDD1L?

  Given the paralogous relationship between APCDD1 and APCDD1L, investigating whether APCDD1L also functions as a dual pathway inhibitor is a promising research direction. Studies on Apcdd1 have shown that:

  a) It inhibits BMP signaling by binding to the BmprIa receptor and preventing nuclear localization of Smad1 .

  b) It simultaneously inhibits Wnt signaling, providing a mechanism for coordinating these two critical developmental pathways .

  c) Immunoprecipitation studies confirmed direct interaction between Apcdd1 and BmprIa, but not with the type II receptor BMPR2 .

  Researchers should explore whether APCDD1L exhibits similar binding properties and pathway inhibition capabilities, potentially using the same experimental approaches as those used for Apcdd1.

• What is the relationship between APCDD1L-AS1 and microRNAs in cancer progression?

  Research on OSCC has identified miR-1224-5p as a molecular target of APCDD1L-AS1 . This microRNA exhibits significantly lower expression in 5-FU-resistant tissues compared to sensitive ones. When overexpressed, miR-1224-5p increases 5-FU sensitivity in resistant OSCC cells.

  The interaction pathway appears to be:
  APCDD1L-AS1 → miR-1224-5p → NSD2

  APCDD1L-AS1 acts as a competing endogenous RNA (ceRNA) that sponges miR-1224-5p, preventing it from suppressing NSD2 expression. NSD2 upregulation then neutralizes the effects of blocking APCDD1L-AS1 on 5-FU resistance in OSCC cells .

  This suggests that microRNA-mediated mechanisms are crucial for understanding APCDD1L-AS1's role in cancer biology.

• How do exosomes contribute to APCDD1L-AS1-mediated drug resistance?

  Exosomes play a critical role in the intercellular transfer of APCDD1L-AS1 and the spread of chemoresistance in OSCC:

  a) Exosomes derived from 5-FU-resistant OSCC cells contain high levels of APCDD1L-AS1 .

  b) These exosomes can be internalized by drug-sensitive OSCC cells, delivering APCDD1L-AS1 and conferring 5-FU resistance .

  c) This exosome-mediated transfer represents a novel mechanism of acquired drug resistance that doesn't require genetic alterations in the recipient cells .

  Understanding the packaging, secretion, and uptake of APCDD1L-AS1-containing exosomes could provide opportunities for therapeutic intervention to prevent the spread of chemoresistance.

• What genomic and epigenetic factors influence APCDD1L expression in different tissues?

  Based on research findings, several factors appear to regulate APCDD1L and APCDD1L-AS1 expression:

  a) DNA methylation: Hypermethylation of the APCDD1L-AS1 promoter region is associated with reduced expression in ccRCC .

  b) Genetic variations: While specific SNPs affecting APCDD1L haven't been detailed in the provided search results, genome-wide association studies have identified various genetic factors influencing disease susceptibility .

  c) Transcription factor binding: VHL protein loss affects APCDD1L-AS1 expression in ccRCC, suggesting transcriptional regulation .

  d) Tissue context: The differential expression patterns and functions of APCDD1L-AS1 in ccRCC versus OSCC indicate tissue-specific regulatory mechanisms .

  These findings suggest that comprehensive genomic and epigenomic profiling could help elucidate the complex regulation of APCDD1L across different tissues and disease states.

Methodological Questions

• What are the optimal conditions for working with recombinant APCDD1L protein?

  Based on manufacturer specifications for recombinant Human APCDD1L protein with GST-tag:

  Parameter	Recommended Condition
  Storage temperature	-80°C
  Storage buffer	50 mM Tris-HCI, 10 mM reduced Glutathione, pH=8.0
  Sample preparation	Aliquot to avoid repeated freezing and thawing
  Shelf life	Best use within three months from receipt
  Applications	ELISA, Western Blot, Antibody Production, Protein Array

  The recombinant protein typically consists of the full-length human APCDD1L ORF (BAC11111.1, 1 a.a. - 501 a.a.) with GST-tag at the N-terminal .

• What experimental approaches are recommended for studying APCDD1L-AS1 function?

  Based on published research methodologies:

  a) Expression analysis:

  • qRT-PCR for quantifying mRNA levels

  • Analysis of TCGA-KIRC and GEO datasets for expression patterns across cancer types

  • Pyrosequencing for DNA methylation status assessment

  b) Functional assays:

  • Overexpression and knockdown experiments to assess effects on cell growth and metastasis

  • Colony forming assays and flow cytometry for cell viability and apoptosis

  • IC50 determination using CCK-8 assays for drug resistance studies

  c) Interaction studies:

  • Dual-luciferase reporter assay for miRNA target validation

  • RIP (RNA Immunoprecipitation) assay for RNA-protein interactions

  • Co-immunoprecipitation for protein-protein interaction studies

  d) In vivo models:

  • Xenograft models for assessing tumor growth and metastasis

  • Development models in chicken, frog, and zebrafish for studying signaling pathway interactions

• How can researchers effectively detect and quantify APCDD1L protein expression?

  For accurate detection and quantification of APCDD1L protein:

  a) Western blotting:

  • Using specific anti-APCDD1L antibodies

  • GST-tagged recombinant APCDD1L can serve as a positive control

  • Expected molecular mass: approximately 82 kDa

  b) Immunohistochemistry/Immunofluorescence:

  • For in situ detection in tissue samples or cultured cells

  • Can provide spatial information about protein localization

  c) ELISA:

  • For quantitative measurement in cell or tissue lysates

  • Can be used for high-throughput screening

  d) Mass spectrometry:

  • Tandem mass tags (TMT) approach has been used to explore relationships between APCDD1L-AS1 and other proteins

  • Provides detailed information about protein modifications and interactions

• What cell models are most appropriate for investigating APCDD1L function?

  Based on existing research approaches:

  a) Cancer cell lines:

  • ccRCC cell lines for investigating tumor suppressor functions

  • OSCC cell lines (HSC-3, HN-4) and their 5-FU-resistant derivatives for studying chemoresistance

  b) Non-cancer cell lines:

  • NIH 3T3 mouse fibroblasts for signaling pathway studies

  • CHO cells for protein interaction studies

  c) Primary cells:

  • Patient-derived primary cells for translational relevance

  • Normal kidney or oral epithelial cells as controls

  d) 3D culture systems:

  • Organoids or spheroids to better recapitulate in vivo conditions

  • Co-culture systems to study cell-cell interactions and exosome transfer

• What strategies can be employed to modulate APCDD1L expression for functional studies?

  Several approaches for manipulating APCDD1L expression include:

  a) Genetic overexpression:

  • Transfection with expression vectors containing APCDD1L cDNA

  • Viral vectors for stable integration and expression

  • Inducible expression systems for temporal control

  b) Gene silencing:

  • siRNA or shRNA targeting APCDD1L mRNA

  • CRISPR/Cas9-mediated knockout for complete gene inactivation

  • Antisense oligonucleotides for transient knockdown

  c) Pharmacological modulation:

  • DNA methyltransferase inhibitors to reverse hypermethylation

  • Histone deacetylase inhibitors to modify epigenetic regulation

  d) Exosome manipulation:

  • Isolation and application of APCDD1L-AS1-rich exosomes

  • Inhibition of exosome biogenesis or uptake to block transfer

  The choice of approach should be tailored to the specific research question, considering factors such as cell type, timeframe, and desired level of modulation.