NDFIP1 Human

What is NDFIP1 and what is its primary function in human cells?

NDFIP1 functions as an adaptor protein that facilitates interactions between E3 ubiquitin ligases (particularly those of the NEDD4 family, including ITCH) and their target proteins. It contains PPxY motifs that recognize and bind to WW domains in target proteins, thereby mediating protein ubiquitination processes . NDFIP1 is part of a family of integral Golgi membrane proteins that play crucial roles in protein trafficking, degradation pathways, and cellular signaling regulation . This protein is particularly important in immune regulation, where it mediates peripheral tolerance in T cells, and in cancer biology, where evidence suggests it functions as a tumor suppressor through mechanisms including the regulation of oncogenic factors like TAZ .

Where is NDFIP1 primarily expressed in human tissues and cells?

NDFIP1 expression has been detected in various cell types and tissues. Immunofluorescence studies have identified NDFIP1 in neuronal cells, as demonstrated in SH-SY5Y human neuroblastoma cell lines . Western blot analyses have confirmed NDFIP1 expression in multiple cell lines including A172 human glioblastoma and SK-Mel-28 human malignant melanoma cells . In immune contexts, NDFIP1 is expressed in T cells, particularly CD4+ T cells where it plays a critical role in immune tolerance . Cancer studies have detected NDFIP1 in various cell lines including breast cancer (MCF-7) and NSCLC cell lines (A549, SPC-A1, SPC-A1-BM, H520, HCC95, H2170) . Subcellular localization studies show that NDFIP1 is primarily found in the cytoplasm, with particular enrichment in the Golgi membrane system .

What are the key protein interactions of NDFIP1?

NDFIP1 primarily interacts with E3 ubiquitin ligases of the NEDD4 family, including ITCH and NEDD4 itself, serving as an adaptor that brings these enzymes together with their substrate proteins . A particularly significant interaction identified in recent research is between NDFIP1 and TAZ (WWTR1), a transcriptional co-activator with known oncogenic functions . This interaction occurs specifically through TAZ's WW domain binding to the PPxY motifs in NDFIP1, as demonstrated by co-immunoprecipitation studies showing that TAZ mutants lacking the WW domain failed to interact with NDFIP1 . Interestingly, NDFIP1 shows selectivity in its interactions - it does not bind to YAP (a paralog of TAZ) or to cTAZ (a short variant of TAZ lacking an intact WW domain) . This selectivity suggests that NDFIP1 has highly specific regulatory effects on particular signaling pathways, which may be important for both its normal physiological functions and its role in disease contexts.

How does NDFIP1 mediate peripheral T-cell tolerance and what are the implications for autoimmune disease research?

NDFIP1 plays a crucial role in peripheral T-cell tolerance through a distinct mechanism that controls T-cell responses to both self and exogenous antigens. Studies using Ndfip1-deficient mice have revealed that Ndfip1 is specifically required for tolerogen-reactive T cells to exit the cell cycle after one to five divisions and to prevent inappropriate Th2 effector differentiation . This represents a unique step in peripheral tolerance that is separate from the better-characterized Bcl2-interacting mediator of cell death-mediated apoptosis pathway .

The importance of this mechanism is underscored by the finding that Ndfip1 deficiency can precipitate autoimmune pancreatic destruction, suggesting that compromised NDFIP1 function may contribute directly to autoimmune pathology . Human genetic studies have established links between NDFIP1/ITCH and various allergic and autoimmune diseases, indicating that this pathway has clinical relevance .

For autoimmune disease researchers, these findings suggest several important experimental approaches:

• Examining NDFIP1 expression profiles in patient T-cell subsets across different autoimmune conditions

• Investigating whether specific NDFIP1 variants correlate with disease severity or treatment response

• Developing experimental models to test whether enhancing NDFIP1 activity could restore tolerance in autoimmune contexts

• Exploring the downstream molecular mechanisms by which NDFIP1 controls T-cell cycle exit and differentiation

Understanding these aspects could potentially lead to novel therapeutic strategies that target the NDFIP1 pathway to reinforce peripheral tolerance in autoimmune conditions.

What is the mechanism by which NDFIP1 exerts its tumor-suppressive effects in cancer?

NDFIP1 functions as a tumor suppressor through a novel mechanism involving the regulation of oncogenic protein trafficking to exosomes. The most well-characterized example of this mechanism involves TAZ, a transcriptional co-activator that drives tumor formation, progression, and metastasis .

NDFIP1 directly interacts with TAZ through TAZ's WW domain, facilitating the recruitment of TAZ into exosomes . In cells with higher NDFIP1 levels, more TAZ is bound by NDFIP1 and subsequently sorted into exosomes, effectively reducing the amount of TAZ available within the cell to drive oncogenic programs . This leads to lower levels of TAZ in both the cytoplasm and nucleus, ultimately resulting in reduced cell proliferation .

Conversely, in cancer cells with downregulated NDFIP1 (as commonly observed across multiple cancer types), less TAZ is packaged into exosomes, allowing more TAZ to remain within the cell and promote proliferation . This mechanism has been validated through multiple experimental approaches:

• Co-immunoprecipitation studies demonstrating direct physical interaction between NDFIP1 and TAZ

• Domain mapping showing that this interaction requires the WW domain of TAZ

• Subcellular co-localization studies confirming the spatial proximity of these proteins

• Functional assays demonstrating that NDFIP1 knockout increases cancer cell proliferation while NDFIP1 overexpression reduces it

• In vivo tumor xenograft models showing that NDFIP1 modulation affects tumor growth

Importantly, silencing TAZ eliminated the increased proliferation caused by NDFIP1 knockout, confirming that NDFIP1's tumor-suppressive effects operate at least partly through TAZ regulation . This mechanism represents a previously unrecognized mode of tumor suppression that operates through the control of protein trafficking rather than direct effects on gene expression or classical cell cycle regulation.

How does NDFIP1 contribute to exosomal protein sorting and what are the implications for cancer biomarker research?

NDFIP1 functions in the loading of specific proteins into exosomes through a selective mechanism involving the recognition of WW domains in target proteins by the PPxY motifs in NDFIP1 . This function has particular significance in cancer, where NDFIP1 facilitates the sorting of oncogenic proteins like TAZ into exosomes, thereby reducing their intracellular levels and suppressing their pro-tumorigenic activities .

This exosomal sorting mechanism has significant implications for cancer biomarker research. The research indicates that low NDFIP1 levels in tumor tissues combined with low TAZ levels in serum exosomes may serve as complementary diagnostic indicators for NSCLC and potentially other cancers . This suggests a novel dual biomarker approach where tissue NDFIP1 expression and exosomal protein content could be assessed in combination to improve diagnostic accuracy.

For biomarker researchers, several methodological approaches could exploit this finding:

• Development of standardized assays to quantify both tumor NDFIP1 expression and exosomal TAZ levels

• Investigation of whether the NDFIP1-exosome axis affects the levels of other clinically relevant proteins in circulation

• Longitudinal studies to determine if changes in these markers correlate with disease progression or treatment response

• Exploration of whether this biomarker approach could enable earlier detection of malignancies or recurrence

Furthermore, the exosomal sorting function of NDFIP1 raises interesting questions about the broader role of selective protein trafficking in cancer biology and whether targeting this pathway might have therapeutic potential beyond its diagnostic applications.

What is the relationship between NDFIP1 expression and immune infiltrates in the tumor microenvironment?

Research indicates a significant relationship between NDFIP1 expression and immune cell infiltration in the tumor microenvironment, particularly in breast cancer . NDFIP1 has been investigated as a potential prognostic marker that can be used to determine immune infiltration patterns and disease progression in breast cancer .

The research has specifically examined NDFIP1 in relation to different immune cell subsets characterized by their expression of immune checkpoint molecules, including CTLA4-PD1-, CTLA4-PD1+, CTLA4+PD1-, and CTLA4+PD1+ populations . This suggests that NDFIP1 may play a role in shaping the immune landscape within tumors and potentially influence responses to immunotherapy.

This connection between NDFIP1 and tumor immune infiltrates likely reflects NDFIP1's established role in T-cell regulation and tolerance . Given that NDFIP1 is necessary for appropriate T-cell responses to antigens, its altered expression in the tumor microenvironment could affect how immune cells recognize and respond to cancer cells.

For immunotherapy researchers, these findings suggest several important research directions:

• Characterizing how NDFIP1 expression correlates with specific immune cell populations in different cancer types

• Investigating whether NDFIP1 status predicts response to immune checkpoint inhibitors

• Exploring whether modulation of NDFIP1 could enhance anti-tumor immune responses

• Examining how NDFIP1-mediated exosomal sorting might influence communication between cancer cells and immune cells

Understanding these relationships could potentially help stratify patients for immunotherapy and might suggest combination therapeutic approaches targeting both NDFIP1 and immune checkpoints.

What are the recommended methods for detecting and quantifying NDFIP1 expression in human samples?

Based on published research, several complementary approaches have been validated for detecting and quantifying NDFIP1 in human samples:

Western Blot Analysis:
Western blot has been successfully used to detect NDFIP1 in various human cell lines, including A172 glioblastoma and SK-Mel-28 malignant melanoma cells . NDFIP1 appears as a specific band at approximately 26-28 kDa under reducing conditions . Validated antibodies include Sheep Anti-Human/Mouse/Rat NDFIP1 Antigen Affinity-purified Polyclonal Antibody . Researchers should optimize reducing conditions and use appropriate positive controls when establishing this assay.

Immunohistochemistry (IHC):
IHC has been effectively applied to detect NDFIP1 in paraffin-embedded tumor tissues, including those arranged in tissue microarrays . Published protocols have used NDFIP1 antibody (1:100, ab236892, Abcam) with goat anti-rabbit IgG H&L (HRP) (1:1000, ab6721, Abcam) for immunostaining . IHC provides valuable information about both expression levels and spatial distribution of NDFIP1 within tissue architecture.

Immunofluorescence:
Immunofluorescence has been employed to visualize NDFIP1 in cultured cells, such as SH-SY5Y human neuroblastoma cells . This approach is particularly useful for studying subcellular localization, with studies showing that NDFIP1 is primarily localized to the cytoplasm with enrichment in Golgi-like structures .

mRNA Analysis:
Quantitative RT-PCR approaches have been used to assess NDFIP1 transcript levels in both cell lines and tissues . This can be complemented with analysis of public gene expression datasets, such as those available through Oncomine, to examine NDFIP1 expression across different conditions and disease states .

Quantification Methods:
For semi-quantitative analysis of NDFIP1 protein expression in tissues, the IHC Profiler plugin in ImageJ software has been used to qualify staining intensity . For more precise quantification, densitometric analysis of western blot bands or qRT-PCR with appropriate housekeeping gene controls can be employed.

When implementing these methods, researchers should consider using multiple detection approaches in parallel to provide complementary data on NDFIP1 expression at both protein and transcript levels.

How can researchers effectively model NDFIP1 deficiency or overexpression in experimental systems?

The literature describes several validated approaches for modulating NDFIP1 expression in experimental systems:

Knockout/Knockdown Models:

• CRISPR/Cas9 gene editing has been successfully applied to generate NDFIP1 knockout cell lines, as demonstrated in SPC-A1 NSCLC cells . This approach provides complete elimination of NDFIP1 expression for studying loss-of-function effects.

• RNA interference approaches have been employed for transient NDFIP1 knockdown in multiple cell lines including A549 . This method allows for dose-dependent reduction in NDFIP1 levels.

• Transgenic mouse models have been developed, including both Ndfip1-deficient strains for studying loss-of-function effects and Ndfip1-YFP reporter strains for tracking expression . These models are particularly valuable for studying NDFIP1's role in complex physiological processes like T-cell tolerance.

Overexpression Models:

• Transient overexpression of NDFIP1 has been achieved in cell lines through transfection with expression vectors containing the NDFIP1 coding sequence .

• Stable transfection approaches have generated cell lines with consistent NDFIP1 overexpression, which is particularly valuable for long-term studies and in vivo experiments .

In Vivo Models:

• Subcutaneous injection of stably transfected cells (with NDFIP1 knockout or overexpression) into immunodeficient mice has been used to assess the effects of NDFIP1 modulation on tumor growth in vivo .

• These xenograft models have shown that NDFIP1 knockout increases tumor size, weight, and proliferation markers (PCNA, Ki-67), while NDFIP1 overexpression reduces these parameters .

Functional Validation:

• To establish specific mechanistic pathways, researchers have employed approaches like silencing downstream targets (e.g., TAZ) in NDFIP1 knockout/knockdown cells to determine if this rescues the observed phenotypes .

• Combined with protein interaction studies, this approach can confirm that phenotypic effects are specifically due to NDFIP1's regulation of particular target proteins.

When designing experiments, researchers should consider the temporal aspects of NDFIP1 modulation, as acute versus chronic changes may produce different phenotypes, particularly in systems where compensatory mechanisms might develop.

What techniques are most effective for studying NDFIP1 protein interactions and its role in protein trafficking?

Several complementary techniques have proven effective for investigating NDFIP1's protein interactions and trafficking functions:

Co-Immunoprecipitation (Co-IP):
Co-IP has been successfully employed to demonstrate direct protein interactions with NDFIP1, most notably its binding to TAZ . This approach not only confirms physical interactions but also helps establish specificity, as demonstrated by studies showing that NDFIP1 interacts with TAZ but not with the related protein YAP . Bidirectional Co-IP (immunoprecipitating each protein and detecting the other) provides the strongest evidence for genuine interactions.

Domain Mapping:
Structure-function analyses using protein constructs with specific domain deletions have identified the domains required for NDFIP1 interactions. For example, studies demonstrated that NDFIP1 interacts with wild-type TAZ but not with TAZ lacking its WW domain (TAZ ΔWW), confirming the WW domain is required for this interaction . Similar approaches can determine which regions of NDFIP1 participate in specific protein interactions.

Immunofluorescence Co-localization:
Confocal microscopy with fluorescently labeled antibodies or tagged proteins has visualized the subcellular co-localization of NDFIP1 with its binding partners . This approach provides spatial context for protein interactions within intact cells and can reveal in which cellular compartments these interactions occur.

Exosome Isolation and Analysis:
To study NDFIP1's role in exosomal protein sorting, researchers have isolated exosomes using ultracentrifugation or commercial kits, followed by western blot analysis of exosomal proteins compared to cellular fractions . This technique directly demonstrates NDFIP1's function in recruiting specific proteins into exosomes and can be quantified to measure trafficking efficiency.

Proximity Ligation Assays:
Although not explicitly mentioned in the search results, proximity ligation assays would be valuable for detecting and quantifying NDFIP1 protein interactions in situ with high sensitivity and specificity.

Functional Validation:
To establish the biological significance of NDFIP1's interactions and trafficking functions, researchers have employed functional assays such as proliferation measurements, examining how disrupting specific interactions affects downstream cellular processes . This connects molecular interactions to biological outcomes.

For comprehensive analysis, researchers should combine multiple approaches to establish both the physical nature of NDFIP1 interactions and their functional consequences in relevant biological contexts.

What are the key considerations for translating NDFIP1 research into clinical applications?

Translating NDFIP1 research into clinical applications involves several important considerations:

Diagnostic Biomarker Development:

• Research suggests that NDFIP1 tissue expression levels combined with exosomal TAZ levels could serve as diagnostic biomarkers, particularly for NSCLC .

• Standardization of detection methods is crucial for clinical application, including optimized IHC protocols for tissue samples and reliable methods for exosome isolation and analysis from patient serum.

• Validation in larger, diverse patient cohorts is essential, as existing studies acknowledge the need for expanded sample sizes .

• Multi-center studies would be needed to establish reproducibility across different clinical settings and patient populations.

Therapeutic Target Development:

• Given NDFIP1's roles in immune regulation and tumor suppression, therapeutic approaches could focus on either restoring NDFIP1 expression/function in cancers or modulating its activity in autoimmune conditions.

• For cancer applications, understanding whether NDFIP1 downregulation is a driver or consequence of malignant transformation is critical for determining therapeutic strategy.

• Drug discovery efforts might target the NDFIP1-TAZ interaction or the mechanisms controlling NDFIP1 expression.

• Potential therapeutic approaches could include small molecules that mimic NDFIP1's binding to target proteins or gene therapy approaches to restore NDFIP1 expression in tumors.

Immunotherapy Connections:

• The relationship between NDFIP1 and immune infiltrates, particularly in the context of immune checkpoint molecules, suggests potential relevance for immunotherapy response prediction .

• Research should investigate whether NDFIP1 status could help identify patients more likely to respond to immune checkpoint inhibitors.

• Combination approaches targeting both NDFIP1 pathways and immune checkpoints might enhance treatment efficacy.

For successful clinical translation, researchers must develop practical, cost-effective assays, validate findings across diverse populations, and carefully consider how NDFIP1-targeted interventions might affect its normal physiological functions to avoid unintended consequences.