DCUN1D4 Human

What is DCUN1D4 and what domains does it contain?

DCUN1D4 (DCN1-like protein 4) is a protein encoded by the DCUN1D4 gene in humans. It contains one DCUN1 domain, which is its primary structural feature. The protein is also known by several aliases including DCN1, defective in cullin neddylation 1 domain containing 4, DCNL4, and DCUN1 domain-containing protein 4. The gene itself may be referred to as KIAA0276. DCUN1D4 is located on chromosome 4 in humans, and despite its characterization, the exact function of this protein remains largely unknown .

What is the sequence conservation of DCUN1D4 across species?

Human DCUN1D4 demonstrates high sequence conservation across mammalian species, with particularly high ortholog identity to mouse and rat proteins. Specifically, human DCUN1D4 shares 96% sequence identity with both mouse and rat orthologs . This high degree of conservation suggests evolutionary importance of the protein and its potential functional significance. For circular RNA derived from DCUN1D4 (circDCUN1D4), sequence mapping between human and mouse revealed 89% identity, further indicating conservation across species .

What post-translational modifications (PTMs) have been identified for DCUN1D4?

Multiple post-translational modifications have been characterized for human DCUN1D4, suggesting complex regulation of this protein:

• Phosphorylation occurs at several serine residues: S53, S71, and S76

• Sumoylation has been detected at lysine K95

• Methylation is present at cysteine C170

• Ubiquitination occurs at lysine K194

These PTMs likely contribute to regulation of DCUN1D4's stability, localization, or function, though specific consequences of each modification require further investigation.

How is DCUN1D4 expressed in different human tissues?

DCUN1D4 demonstrates a complex tissue expression pattern across human tissues. The Human Protein Atlas shows expression across multiple tissues including brain regions (hippocampal formation, amygdala, basal ganglia, midbrain, cerebral cortex, cerebellum), endocrine tissues (thyroid, adrenal, pituitary), and various other organs including lung, gastrointestinal tissues, reproductive organs, and lymphoid tissues . This broad expression pattern suggests DCUN1D4 may have tissue-specific functions that warrant investigation in different physiological contexts.

What recombinant protein tools are available for studying DCUN1D4?

Researchers can access recombinant protein fragments of human DCUN1D4 for experimental use. For instance, a control fragment comprising amino acids 57-112 is commercially available. This fragment is particularly useful for blocking experiments with corresponding antibodies (e.g., PA5-57681). For effective blocking in immunohistochemistry, immunocytochemistry, or western blot experiments, it's recommended to use a 100x molar excess of the protein fragment control based on antibody concentration and molecular weight. The blocking protocol typically involves pre-incubation of the antibody-protein control fragment mixture for 30 minutes at room temperature .

How can researchers validate the circular RNA form of DCUN1D4 (circDCUN1D4)?

Validation of circDCUN1D4 requires multiple complementary approaches:

• RT-PCR with divergent primers followed by Sanger sequencing to confirm the back-splice junction

• RNase R treatment (circRNAs are resistant while linear RNAs degrade)

• Actinomycin D treatment to assess stability (circRNAs typically show greater stability)

• Comparison of random hexamer vs. oligo(dT)18 primers in RT experiments (circRNAs lack poly-A tails, leading to decreased detection with oligo(dT) primers)

In published research, circDCUN1D4 (hsa_circ_0007928) has been confirmed to comprise exons 2-6 of the DCUN1D4 gene, with a total length of 389 nucleotides .

What methodological approaches can be used to study circDCUN1D4's interaction with proteins?

Several complementary techniques have proven effective for studying circDCUN1D4's interactions with proteins like HuR:

• Computational methods:

  • Optimal folding prediction using tools like RNAfold

  • 3D structure generation with RNA Composer

  • Molecular docking simulations (e.g., using NPDock)

• Experimental validation:

  • RNA immunoprecipitation (RIP) assays to show enrichment of circDCUN1D4 in protein complexes

  • Biotin-labeled RNA pull-down followed by western blotting

  • Construction of protein domain truncation mutants to identify specific binding domains

  • Dual-luciferase reporter assays containing canonical binding sites to assess functional activity

This multi-tiered approach provides robust evidence for RNA-protein interactions.

How can researchers manipulate DCUN1D4 expression for functional studies?

For studying DCUN1D4 function, researchers can employ several gene expression manipulation strategies:

• For overexpression of circDCUN1D4:

  • Use of positive control vectors containing the full circRNA sequence

• For knockdown of circDCUN1D4:

  • Design of small hairpin RNAs (shRNAs) specifically targeting the back-splice junction of circDCUN1D4

Importantly, when targeting the circular form, researchers should verify that linear mRNA expression and protein levels remain unchanged to confirm specificity. In published studies, successful manipulation has been confirmed using qRT-PCR for circRNA levels and western blotting for protein levels .

What is the relationship between circDCUN1D4 and lung adenocarcinoma (LUAD)?

CircDCUN1D4 functions as a tumor suppressor in lung adenocarcinoma. Research has established that:

Functionally, circDCUN1D4 overexpression suppresses LUAD cell migration and invasion, while knockdown promotes these processes. In vivo studies have demonstrated that circDCUN1D4 overexpression significantly reduces lung metastatic colonies in mouse models .

How does circDCUN1D4 influence tumor metastasis and glycolysis at the molecular level?

CircDCUN1D4 suppresses tumor metastasis and glycolysis through a complex molecular mechanism:

• CircDCUN1D4 interacts with the RNA-binding protein HuR through its RNA recognition motif 1 (RRM1)

• This interaction increases HuR translocation from nucleus to cytoplasm

• CircDCUN1D4 acts as a scaffold to facilitate interaction between HuR protein and TXNIP mRNA

• CircDCUN1D4 also directly interacts with TXNIP mRNA through base complementation

• This forms a circDCUN1D4/HuR/TXNIP RNA-protein ternary complex

• The complex enhances stability of TXNIP mRNA

• Increased TXNIP expression ultimately suppresses metastasis and glycolysis in lung cancer cells

This mechanism has been validated through multiple approaches including RIP assays, luciferase reporter assays, and functional studies showing that circDCUN1D4's effects are TXNIP-dependent .

What evidence suggests DCUN1D4's involvement in breast cancer?

Recent research indicates that DCUN1D4 may be implicated in breast cancer biology, particularly in the context of the tumor microenvironment (TME). In a study examining invasive ductal breast carcinoma-associated stroma (IDBCS), DCUN1D4 was identified among a list of genes being investigated for potential roles in cancer progression. While not identified as one of the 12 key risk genes that segregated patients into high-risk and low-risk groups, DCUN1D4 appears in the broader gene set being evaluated for its impact on the stromal component of breast cancer .

What variants affecting DCUN1D4 PTM sites have been identified in cancer samples?

Several cancer-associated variants affecting post-translational modification sites in DCUN1D4 have been identified:

These variants typically involve substitution of the modified residue, which would prevent the respective post-translational modification, potentially altering protein function and contributing to cancer pathogenesis .

How does the structure of circDCUN1D4 relate to its functional properties?

The structure-function relationship of circDCUN1D4 is complex and multi-faceted:

• Secondary structure: Using RNAfold for secondary structure prediction reveals optimal folding patterns that create specific binding domains

• Tertiary structure: 3D modeling with RNA Composer illuminates spatial arrangements critical for protein interactions

• Functional domains: CircDCUN1D4 contains specific binding regions that interact with:

  • HuR protein (specifically through the RRM1 domain)

  • TXNIP mRNA through complementary base pairing

Research indicates that circDCUN1D4 derived from exons 2-6 of the DCUN1D4 gene forms a stable circular structure of 389 nucleotides. This conformation enables its function as a molecular scaffold, facilitating the formation of RNA-protein complexes that ultimately regulate gene expression. In silico molecular docking analyses have demonstrated that circDCUN1D4 can "perfectly dock" with HuR protein, suggesting evolutionary optimization of this interaction .

What is the proposed mechanism for circDCUN1D4's effect on HuR cellular localization?

CircDCUN1D4 regulates HuR cellular localization through a specialized mechanism:

• CircDCUN1D4 binds specifically to HuR's RNA recognition motif 1 (RRM1)

• This interaction appears to mask or alter nuclear localization signals or nuclear export signals associated with HuR

• The binding promotes translocation of HuR from the nucleus to the cytoplasm

• Cytoplasmic retention of HuR is increased in the presence of circDCUN1D4

• This relocation affects HuR's ability to regulate its target mRNAs in the cytoplasm

Functional validation of this mechanism has been demonstrated through dual-luciferase reporter assays, showing that overexpression of circDCUN1D4 improves HuR activity while knockdown suppresses it. This indicates that circDCUN1D4 not only alters HuR's localization but also enhances its functional activity .

How might the DCUN1 domain in DCUN1D4 contribute to its cellular function?

While the exact function of DCUN1D4 remains unknown, the presence of a DCUN1 domain provides important clues:

• DCUN1 domains are typically associated with cullin neddylation processes

• Cullins are scaffold proteins in cullin-RING E3 ubiquitin ligase (CRL) complexes

• Neddylation (the addition of NEDD8) activates cullins and subsequently CRL activity

• DCUN1 domain-containing proteins often function as neddylation co-E3 ligases

Based on this domain homology, DCUN1D4 may participate in protein degradation pathways through the ubiquitin-proteasome system, potentially regulating turnover of specific substrate proteins. This connection to protein degradation could explain its observed roles in cancer biology, as dysregulation of protein degradation is a hallmark of many cancers .

What is the significance of DCUN1D4's conservation across species for functional studies?

The high conservation of DCUN1D4 across species (96% identity between human and mouse/rat) has several important implications for functional studies:

• Model organism relevance: The high homology suggests mouse and rat models would be appropriate for studying DCUN1D4 function

• Evolutionary pressure: The conservation indicates important biological functions that have been maintained throughout evolution

• Functional domains: Highly conserved regions likely represent critical functional domains

• Cross-species validation: Findings in one species may be applicable across others

For circDCUN1D4 specifically, the 89% sequence identity between human and mouse forms suggests conservation of this circular RNA structure, further supporting its functional importance. This conservation enables researchers to conduct preliminary studies in model organisms with higher confidence that findings will translate to human biology .

How can researchers differentiate between functions of circular and linear forms of DCUN1D4?

Distinguishing the functions of circDCUN1D4 from linear DCUN1D4 mRNA requires specialized methodological approaches:

• Expression manipulation strategies:

  • For circRNA: Target the back-splice junction using shRNAs

  • For linear mRNA: Target regions unique to the linear form

  • Verification that manipulating one form doesn't affect the other is critical

• Functional validation methods:

  • Rescue experiments using constructs expressing only one form

  • Protein-binding studies to identify form-specific interactions

  • Subcellular localization analysis to determine compartment-specific functions

In published research, circDCUN1D4 overexpression or knockdown was confirmed not to affect mDCUN1D4 (linear mRNA) or DCUN1D4 protein levels, confirming independent functions. This careful validation is essential to attribute observed phenotypes to the correct molecular entity .

What methodological considerations are important when analyzing DCUN1D4 in cancer studies?

When investigating DCUN1D4 in cancer contexts, several methodological considerations are critical:

• Tissue heterogeneity:

  • Differentiate between expression in tumor cells vs. stromal components

  • Consider tumor microenvironment influences on expression patterns

• Isoform specificity:

  • Distinguish between circular RNA (circDCUN1D4) and linear mRNA effects

  • Account for potential tissue-specific isoforms

• Functional redundancy:

  • Consider compensation by related family members

  • Evaluate broader pathway effects rather than isolated gene effects

• Clinical correlation:

  • Correlate expression with specific cancer subtypes

  • Analyze association with clinical parameters (stage, grade, survival)

How should researchers interpret contradictory findings regarding DCUN1D4's role in different cancer types?

When encountering seemingly contradictory results about DCUN1D4 across cancer types, researchers should consider:

• Tissue context specificity:

  • DCUN1D4 may have opposing functions in different tissues

  • Interaction partners may vary between tissue types

• Circular vs. linear form predominance:

  • CircDCUN1D4 has established tumor-suppressive functions in lung adenocarcinoma

  • Linear DCUN1D4 may have different effects

  • The balance between forms may differ by cancer type

• Upstream regulatory differences:

  • Regulatory factors controlling DCUN1D4 expression may vary

  • Epigenetic regulation could differ across tissues

• Integration with broader pathways:

  • Position within signaling networks may vary by cancer type

  • Secondary effects could outweigh primary functions in certain contexts

Current evidence shows circDCUN1D4 suppresses metastasis and glycolysis in lung cancer, while DCUN1D4 (unspecified form) appears in broader gene lists being investigated in breast cancer .