Recombinant Mouse E3 ubiquitin-protein ligase NRDP1 (Rnf41)

What is E3 ubiquitin-protein ligase NRDP1 (Rnf41) and what are its primary functions?

NRDP1 (Neuregulin receptor degradation protein-1), also known as RNF41 or FLRF, is a RING finger E3 ubiquitin ligase primarily expressed in the brain, heart, prostate, and skeletal muscle . The protein plays crucial roles in regulating various cellular processes including:

• Protein degradation through the ubiquitin-proteasome pathway

• Cell growth and proliferation regulation

• Apoptosis signaling

• Inflammatory responses

• Oxidative stress management

NRDP1 functions by promoting the ubiquitination of various substrate proteins, including USP8, ErbB3, ErbB4, BRUCE/Apollon, MyD88, and Parkin . This ubiquitination typically targets these proteins for degradation by the proteasome, effectively regulating their cellular levels and activities. NRDP1 also exhibits autoubiquitination activity, which regulates its own stability and cellular concentration.

What is the structural organization of NRDP1 and how does it relate to function?

NRDP1 contains several key structural domains that are essential for its functionality:

• A RING finger domain: Required for its E3 ubiquitin ligase activity

• A coiled-coil domain: Mediates protein oligomerization

• A C-terminal domain: Involved in substrate recognition and binding

The coiled-coil domain plays a particularly interesting role in NRDP1 function. Research has demonstrated that deletion of this domain abrogates NRDP1 oligomerization and suppresses NRDP1 autoubiquitination without affecting its ability to ubiquitinate substrates like ErbB3 . This suggests that oligomerization is specifically required for efficient NRDP1-mediated autoubiquitination but not for substrate ubiquitination, allowing for functional separation of these two activities .

How is NRDP1 expression regulated in different tissues and disease states?

NRDP1 expression varies significantly across tissues and is dynamically regulated under different physiological and pathological conditions:

• Tissue distribution: Highest expression in brain, heart, prostate, and skeletal muscle

• Cancer contexts: Present in normal mammary tissue but suppressed or lost in over half of human breast tumors and invariably lost in ErbB2 overexpression mouse models . NRDP1 is notably more labile when expressed in human breast tumor cell lines than in nontransformed lines, suggesting accelerated protein degradation may underlie its suppression in tumors .

• Ischemic conditions: Upregulated in cerebral cortical neurons following ischemia, with Nrdp1 mRNA expression increasing as early as 1 hour after oxygen-glucose deprivation (OGD) treatment and remaining elevated for at least 6 hours .

• Liver disease: RNF41 expression is down-regulated in CD11b+ macrophages recruited to mouse fibrotic liver and patient cirrhotic liver, regardless of cirrhosis etiology . Prolonged inflammation with TNF-α progressively reduces macrophage RNF41 expression .

The dysregulation of NRDP1 in various disease contexts highlights its potential significance as both a biomarker and therapeutic target.

How does NRDP1 oligomerization affect its function and substrate specificity?

NRDP1 oligomerization represents a fascinating regulatory mechanism that differentially affects its activities:

The coiled-coil domain of NRDP1 mediates its oligomerization, which is essential for efficient autoubiquitination. Studies have demonstrated that deletion of this domain abrogates NRDP1 oligomerization and suppresses NRDP1 autoubiquitination, but interestingly does not affect its ability to ubiquitinate substrates like ErbB3 .

This functional separation between autoubiquitination and substrate ubiquitination suggests a sophisticated regulatory mechanism:

• Oligomerization may increase NRDP1's capacity for autoubiquitination by bringing multiple RING domains into proximity

• Substrate ubiquitination appears to proceed through a different mechanism that doesn't require oligomerization

• This separation potentially allows for differential regulation of NRDP1 stability versus its enzymatic activity toward substrates

This dichotomy could provide opportunities for developing therapeutic strategies that selectively target NRDP1 autoubiquitination without affecting its substrate ubiquitination activity, which might be particularly relevant for breast cancer patients where NRDP1 stability is compromised .

What is the role of NRDP1 in ischemic neuronal injury and what mechanisms are involved?

NRDP1 plays a significant role in ischemic neuronal injury through several interconnected mechanisms:

Ischemia induces rapid and persistent upregulation of NRDP1 in cerebral cortical neurons, with Nrdp1 mRNA expression increasing at 1 hour after oxygen-glucose deprivation (OGD) treatment and remaining elevated through 6 hours of exposure . This upregulation has substantial functional consequences:

• Promotion of neuronal apoptosis: Knockdown of NRDP1 with siRNA reduces OGD-induced cell death/apoptosis, while overexpression of NRDP1 increases neuronal death

• Molecular pathway: NRDP1 targets USP8 (ubiquitin-specific protease 8) for degradation. Since USP8 normally protects hypoxia-inducible factor-1α (HIF-1α) from von Hippel-Lindau (pVHL)-mediated degradation, NRDP1 upregulation leads to decreased USP8 levels and consequently reduced HIF-1α levels

• Apoptotic signaling: NRDP1 activation affects apoptosis-associated proteins, including increased caspase-3 activation, PARP-1 cleavage, and elevated Bax/Bcl-2 ratio

Experimental evidence confirms that knockdown of NRDP1 partially reverses OGD-induced USP8 downregulation, while overexpression of NRDP1 exacerbates it . This NRDP1-USP8-HIF-1α pathway represents a potential therapeutic target for neuroprotection in ischemic stroke.

How does NRDP1 contribute to macrophage-driven fibrosis resolution and liver regeneration?

RNF41 (NRDP1) plays a crucial role in regulating macrophage function during liver injury, fibrosis, and regeneration:

Recent research has revealed that RNF41 expression is significantly down-regulated in CD11b+ macrophages recruited to fibrotic liver tissue in both mouse models and human cirrhotic liver samples, regardless of the underlying cirrhosis etiology . Prolonged inflammation with TNF-α progressively reduces macrophage RNF41 expression, linking inflammatory processes to RNF41 dysregulation .

The functional significance of this downregulation has been demonstrated through targeted manipulation of RNF41 expression:

• Therapeutic restoration: Inducing RNF41 expression specifically in CD11b+ macrophages using dendrimer-graphite nanoparticles (DGNPs) conjugated with plasmids ameliorates liver fibrosis, reduces liver injury, and stimulates hepatic regeneration in fibrotic mice with or without hepatectomy

• Mechanism of action: This therapeutic effect is primarily mediated through the induction of insulin-like growth factor 1 (IGF-1)

• Loss-of-function consequences: Conversely, depletion of macrophage RNF41 worsens inflammation, fibrosis, hepatic damage, and reduces survival rates

These findings establish RNF41 as a "master regulator" of macrophage function in chronic liver diseases and position it as a potential therapeutic target for liver fibrosis, cirrhosis, and possibly other diseases characterized by inflammation and fibrosis .

How does NRDP1-mediated degradation of BRUCE/apollon contribute to apoptosis regulation?

NRDP1 plays a significant role in apoptosis regulation through its interaction with BRUCE/apollon:

BRUCE (BIR repeat-containing ubiquitin-conjugating enzyme) is a 530 kDa membrane-associated inhibitor of apoptosis protein (IAP) that contains a ubiquitin-carrier protein (E2) domain and normally functions to suppress apoptosis . NRDP1 specifically targets BRUCE for ubiquitination and subsequent proteasomal degradation, which has important implications for cellular apoptosis:

• Direct interaction and degradation: NRDP1 physically associates with BRUCE and, in the presence of an E2 enzyme like UbcH5c, catalyzes BRUCE ubiquitination

• Response to apoptotic stimuli: In many cell types, apoptotic stimuli induce proteasomal degradation of BRUCE (but not other IAPs like XIAP or c-IAP1), and reducing NRDP1 levels by RNA interference significantly reduces this loss of BRUCE

• Functional consequences for apoptosis: Decreasing BRUCE content by RNA interference or overexpression of NRDP1 promotes apoptosis, confirming that BRUCE normally inhibits apoptosis and that NRDP1-mediated degradation of BRUCE contributes to apoptotic progression

• Stimulus-specific activation: Treatments like etoposide potentiate the NRDP1-dependent decrease in BRUCE levels, and expression of Nrdp1 siRNAs greatly reduces this etoposide-induced loss of BRUCE

This NRDP1-BRUCE regulatory axis represents an important mechanism in the initiation of apoptosis and could be targeted for therapeutic intervention in diseases characterized by dysregulated apoptosis.

What are effective methods for studying NRDP1 expression and activity?

Several complementary approaches can be employed to comprehensively investigate NRDP1 expression and activity:

Expression Analysis:

• Real-time RT-PCR: Provides sensitive quantification of Nrdp1 mRNA expression levels as demonstrated in studies of primary rat cerebral cortical neurons and PC12 cells subjected to oxygen-glucose deprivation

• Western blotting: Enables detection of NRDP1 protein levels using specific antibodies, typically normalized to loading controls such as β-actin

• Immunohistochemistry/Immunofluorescence: Allows visualization of NRDP1 expression in tissue sections or cell cultures

Activity Assessment:

• Ubiquitination assays: In vitro assays using purified recombinant NRDP1, E1, E2 (typically UbcH5c), ubiquitin, ATP, and potential substrates can demonstrate NRDP1's E3 ligase activity

• Substrate degradation analysis: Monitoring the levels of known NRDP1 substrates (e.g., ErbB3, BRUCE, USP8) after NRDP1 manipulation provides functional evidence of its activity

• Co-immunoprecipitation experiments: Useful for detecting physical interactions between NRDP1 and its substrates or regulators

Manipulation Approaches:

• Overexpression systems: Transfection with NRDP1 cDNA or adenoviral vectors (e.g., Ad-Nrdp1) for gain-of-function studies

• RNA interference: siRNA or shRNA targeting NRDP1 for loss-of-function studies

• Domain mutations: Creating specific domain deletions or point mutations to study structure-function relationships

When designing these experiments, it's important to consider NRDP1's relatively short half-life due to autoubiquitination, which may necessitate the use of proteasome inhibitors like MG132 to stabilize NRDP1 protein levels for certain analyses.

How can researchers effectively investigate NRDP1-substrate interactions?

Investigating NRDP1-substrate interactions requires a multi-faceted approach combining biochemical, cellular, and functional techniques:

Physical Interaction Analysis:

• Co-immunoprecipitation (Co-IP): Pull down NRDP1 and probe for potential substrates, or vice versa. This technique has been successfully used to demonstrate interactions between NRDP1 and USP8, and between USP8 and HIF-1α in OGD-treated neurons

• GST pulldown assays: Using recombinant GST-tagged NRDP1 to pull down potential binding partners from cell lysates

• Proximity ligation assay (PLA): Enables visualization of protein-protein interactions in situ with subcellular resolution

Ubiquitination Assessment:

• In vitro ubiquitination assays: Reconstituting the ubiquitination reaction with purified components (E1, E2, NRDP1, ubiquitin, ATP, and potential substrate) to directly test NRDP1's ability to ubiquitinate specific targets

• Cell-based ubiquitination assays: Expressing NRDP1 along with tagged ubiquitin and potential substrates, followed by immunoprecipitation under denaturing conditions and western blotting to detect ubiquitinated species

• Mass spectrometry: To identify ubiquitination sites and ubiquitin chain types on substrates

Functional Validation:

• Correlation analysis: Examine whether NRDP1 overexpression decreases substrate levels and whether NRDP1 knockdown increases substrate stability

• Proteasome inhibition: Test whether proteasome inhibitors like MG132 prevent NRDP1-mediated substrate degradation, confirming the ubiquitin-proteasome pathway's involvement

• Mutational analysis: Create ubiquitination-resistant substrate mutants (e.g., by mutating key lysine residues) to confirm the functional significance of the modification

Domain Mapping:

• Deletion constructs: Generate truncation mutants of both NRDP1 and the substrate to map interaction domains

• Point mutations: Introduce specific mutations in key domains to disrupt binding or catalytic activity

These complementary approaches provide robust evidence for bona fide NRDP1-substrate relationships and help distinguish direct from indirect effects.

What animal models are most useful for investigating NRDP1 function in vivo?

Several animal models have proven valuable for studying NRDP1 function in different physiological and pathological contexts:

Neurological Models:

• Middle cerebral artery occlusion (MCAO) in rats: This stroke model has demonstrated that cerebral ischemia induces NRDP1 mRNA expression in the ischemic cerebral cortex, making it valuable for studying NRDP1's role in ischemic neuronal injury

• Oxygen-glucose deprivation (OGD) in primary rat cerebral cortical neurons: While not an in vivo model, this ex vivo approach has been instrumental in studying the molecular mechanisms of NRDP1 in neuronal ischemia

Liver Disease Models:

• Liver fibrosis models: These have been crucial for studying RNF41's role in hepatic fibrosis and regeneration. Various approaches including carbon tetrachloride (CCl₄) administration, bile duct ligation, or high-fat diets can be used to induce liver fibrosis in mice

• Partial hepatectomy models: Combined with fibrosis induction, these models allow study of RNF41's role in liver regeneration

• Macrophage-specific targeting: Dendrimer-graphite nanoparticles (DGNPs) conjugated with RNF41-expressing plasmids provide a sophisticated approach for cell type-specific manipulation in these models

Cancer Models:

• ErbB2 overexpression mouse model of breast cancer: This model has revealed that NRDP1 is invariably lost during mammary tumorigenesis, suggesting its importance in breast cancer development

• Xenograft models: Human cancer cell lines with manipulated NRDP1 expression can be implanted in immunodeficient mice to study tumor growth and progression

Genetic Models:

• Conditional knockout mice: While not specifically mentioned in the search results, tissue-specific or inducible NRDP1/RNF41 knockout models would provide valuable tools for studying its function in specific contexts

• Transgenic overexpression models: Similarly, conditional overexpression of NRDP1 could help elucidate its function in various tissues

The choice of animal model should be guided by the specific research question, the particular disease context of interest, and the molecular pathway being investigated.

What are effective strategies for manipulating NRDP1 expression or activity for experimental purposes?

Researchers can employ several strategies to modulate NRDP1 expression or activity, each with specific advantages for different experimental objectives:

Gain-of-Function Approaches:

• Plasmid transfection: Transient or stable transfection with NRDP1-expressing plasmids in cell culture systems

• Viral vectors: Adenoviral vectors (e.g., Ad-Nrdp1) provide efficient delivery of NRDP1 cDNA into various cell types, including primary neurons

• Nanoparticle-mediated delivery: For in vivo studies, dendrimer-graphite nanoparticles (DGNPs) conjugated with NRDP1-expressing plasmids enable targeted delivery to specific cell populations, such as CD11b+ macrophages

• Recombinant protein: Purified recombinant NRDP1 protein can be used for in vitro biochemical assays or potentially for protein transduction approaches

Loss-of-Function Approaches:

• RNA interference: siRNA or shRNA targeting NRDP1 mRNA for transient or stable knockdown

• DNA vector-based RNAi: Systems like BS/U6/Nrdp1 that express 21-nt siRNAs targeting the coding region of NRDP1

• CRISPR-Cas9 gene editing: For generating knockout cell lines or animal models

• Dominant negative constructs: Expressing catalytically inactive NRDP1 mutants that can interfere with endogenous NRDP1 function

Structure-Function Analysis:

• Domain deletion mutants: Removing specific domains (e.g., the coiled-coil domain) to study their role in NRDP1 function

• Point mutations: Introducing specific amino acid changes in catalytic residues or substrate binding regions

• Chimeric proteins: Creating fusion proteins to study subcellular localization or to develop regulated forms of NRDP1

Pharmacological Approaches:

• Proteasome inhibitors: MG132 or bortezomib can be used to stabilize NRDP1 protein levels by preventing its autoubiquitination-mediated degradation

• E1/E2 inhibitors: Compounds that inhibit ubiquitin-activating or conjugating enzymes can be used to block NRDP1-mediated ubiquitination

The choice of manipulation strategy should be guided by the specific research question, the cell type or animal model being used, and the desired temporal and spatial control over NRDP1 activity.

What are emerging roles of NRDP1 in disease pathogenesis?

NRDP1 is increasingly recognized as a significant contributor to various disease processes, with emerging roles in multiple pathological contexts:

Cancer Biology:

• NRDP1 is suppressed or completely lost in over half of human breast tumors and is invariably absent in the ErbB2 overexpression mouse model of mammary cancer

• This loss appears to occur post-transcriptionally through accelerated protein degradation, particularly in breast tumor cell lines compared to non-transformed lines

• The consequent stabilization of ErbB3 likely enhances growth factor signaling, potentially contributing to tumor progression and therapeutic resistance

Ischemic Stroke and Neuronal Injury:

• NRDP1 is rapidly upregulated in cerebral cortical neurons following ischemia, with expression increasing within 1 hour of oxygen-glucose deprivation

• This upregulation promotes neuronal apoptosis through a pathway involving USP8 degradation and subsequent HIF-1α destabilization

• NRDP1 activation affects multiple apoptotic proteins, including caspase-3, PARP-1, and the Bax/Bcl-2 ratio, amplifying cell death signals

Liver Fibrosis and Regeneration:

• RNF41 (NRDP1) expression is significantly downregulated in macrophages during liver fibrogenesis in both mouse models and human cirrhotic liver samples

• This downregulation occurs regardless of cirrhosis etiology, suggesting it represents a common pathway in chronic liver disease progression

• The loss of macrophage RNF41 impairs liver regeneration and promotes fibrosis, identifying a previously unrecognized role in tissue repair mechanisms

Apoptosis Regulation:

• NRDP1 specifically targets BRUCE/apollon, an inhibitor of apoptosis protein, for ubiquitination and degradation

• This mechanism appears to be important in the initiation of apoptosis in response to various stimuli, including chemotherapeutic agents like etoposide

• The NRDP1-BRUCE axis represents a unique regulatory node in apoptotic signaling distinct from other IAP-regulatory pathways

These diverse roles highlight NRDP1's significance as both a potential biomarker and therapeutic target across multiple disease contexts, and suggest that its dysregulation may represent a common mechanism in pathogenesis.

How is NRDP1 being explored as a therapeutic target?

NRDP1/RNF41 is emerging as a promising therapeutic target in several disease contexts, with different strategies being developed based on its specific role in each condition:

Breast Cancer Approaches:

• The finding that oligomerization is required for NRDP1 autoubiquitination but not for substrate ubiquitination reveals a potential therapeutic strategy

• Compounds that stabilize NRDP1 by preventing its autoubiquitination while preserving its ability to ubiquitinate ErbB3 could potentially restore NRDP1 function in breast tumors

• This approach could provide a novel treatment strategy for breast cancer patients, particularly those with ErbB2/ErbB3-dependent tumors

Liver Disease Therapeutics:

• Macrophage-selective gene therapy using dendrimer-graphite nanoparticles (DGNPs) conjugated with RNF41-expressing plasmids has shown promising results in experimental models

• This approach ameliorates liver fibrosis, reduces liver injury, and stimulates hepatic regeneration in fibrotic mice with or without hepatectomy

• The therapeutic effect is primarily mediated through the induction of insulin-like growth factor 1 (IGF-1)

• This strategy represents a potential treatment for cirrhosis and chronic liver inflammation

Neurological Applications:

• Since NRDP1 upregulation contributes to ischemic neuronal injury, inhibiting NRDP1 expression or activity could provide neuroprotection in stroke

• Targeting the NRDP1-USP8-HIF-1α pathway may preserve neuronal viability under ischemic conditions

• RNA interference approaches targeting NRDP1 have demonstrated protective effects in experimental models of oxygen-glucose deprivation

Apoptosis Modulation:

• The role of NRDP1 in regulating BRUCE/apollon levels provides opportunities for modulating apoptotic sensitivity in various disease contexts

• Inhibiting NRDP1 could potentially protect cells from apoptosis in conditions where excessive cell death is problematic

• Conversely, enhancing NRDP1 activity could sensitize cells to apoptosis in conditions where resistance to cell death is a concern, such as cancer

These therapeutic strategies are still in early developmental stages, but the emerging understanding of NRDP1's role in disease pathogenesis provides a strong rationale for their continued exploration and refinement.

What methodological challenges remain in NRDP1 research?

Despite significant advances, researchers studying NRDP1/RNF41 continue to face several important methodological challenges:

Protein Stability and Detection Issues:

• NRDP1's autoubiquitination activity results in a relatively short half-life, making it difficult to detect endogenous protein in many cellular contexts

• Proteasome inhibitors may be required to stabilize NRDP1 for detection, but these compounds can have widespread effects on cellular physiology

• Development of more sensitive and specific antibodies for various experimental applications (western blotting, immunoprecipitation, immunohistochemistry) remains an ongoing challenge

In Vivo Manipulation Difficulties:

• Cell type-specific targeting of NRDP1 in vivo requires sophisticated delivery systems, as demonstrated by the use of dendrimer-graphite nanoparticles for macrophage-specific delivery

• The development of conditional knockout or transgenic mouse models would benefit the field but has not yet been widely reported

• Tissue-specific effects of NRDP1 modulation may vary significantly, requiring careful consideration of targeting strategies

Substrate Specificity Determination:

• The mechanisms governing NRDP1's substrate specificity remain incompletely understood

• Distinguishing direct NRDP1 substrates from proteins affected indirectly through downstream pathways requires rigorous biochemical validation

• The functional significance of different ubiquitin chain types (K48, K63, etc.) on NRDP1 substrates needs further investigation

Temporal Dynamics:

• NRDP1 expression and activity can change rapidly in response to various stimuli, necessitating careful time-course analyses

• The relationship between NRDP1 and its regulators (e.g., USP8) involves complex feedback loops that can be difficult to dissect experimentally

• Capturing these dynamic changes in vivo presents additional challenges

Translational Barriers:

• Moving from preclinical models to clinical applications requires overcoming drug delivery challenges, especially for nucleic acid-based therapies

• The development of small molecule modulators of NRDP1 activity with suitable pharmacokinetic properties remains an important goal

• Biomarkers for patient selection and treatment response monitoring need to be identified and validated

Addressing these methodological challenges will require continued technological innovation and interdisciplinary collaboration, but will ultimately advance our understanding of NRDP1 biology and its therapeutic potential.

What are the key unanswered questions in NRDP1 biology?

Despite significant progress in understanding NRDP1/RNF41, several fundamental questions remain unanswered:

Structural Biology Questions:

• What is the complete three-dimensional structure of NRDP1, particularly in its oligomeric state?

• How does NRDP1 recognize different substrates with high specificity?

• What structural changes occur during the catalytic cycle of ubiquitin transfer?

Regulatory Mechanisms:

• What are the upstream signals that regulate NRDP1 expression in different cellular contexts?

• How is NRDP1 activity modulated post-translationally beyond autoubiquitination?

• What determines the balance between NRDP1 autoubiquitination and substrate ubiquitination?

Substrate Repertoire:

• What is the complete set of physiological NRDP1 substrates in different cell types and tissues?

• How does substrate availability change under different cellular conditions?

• Are there non-degradative roles for NRDP1-mediated ubiquitination (e.g., signaling functions)?

Physiological Function:

• What is the role of NRDP1 in normal development and tissue homeostasis?

• How does NRDP1 function differ across various cell types and tissues?

• What are the consequences of NRDP1 dysregulation for whole-organism physiology?

Disease Mechanisms:

• How exactly does NRDP1 loss contribute to breast cancer progression beyond ErbB3 stabilization?

• What mechanisms lead to NRDP1 downregulation in liver macrophages during fibrogenesis?

• Are there other disease contexts where NRDP1 dysregulation plays a significant role?

Therapeutic Development:

• Can NRDP1 be selectively targeted with small molecules for therapeutic purposes?

• What biomarkers might predict response to NRDP1-targeted therapies?

• How can NRDP1-based therapies be effectively delivered to specific cell populations in vivo?

Addressing these questions will require integrated approaches combining structural biology, biochemistry, cell biology, animal models, and clinical studies. The answers will not only advance our fundamental understanding of NRDP1 biology but also inform the development of novel therapeutic strategies for diseases characterized by NRDP1 dysregulation.