Recombinant Xenopus laevis E3 ubiquitin-protein ligase NRDP1 (rnf41)

What is NRDP1 and what is its role in Xenopus laevis?

NRDP1 (Neural precursor cell expressed developmentally down-regulated protein 1), also known as RNF41 (Ring Finger Protein 41), is an E3 ubiquitin ligase that plays crucial roles in various cellular processes. In Xenopus laevis, NRDP1 functions as both an adaptor protein and an E3 ubiquitin ligase that regulates multiple biological pathways including immune responses and developmental processes. Its primary function involves facilitating the addition of ubiquitin to target proteins, thereby marking them either for degradation or altering their cellular functions . The protein contains characteristic RING finger domains critical for its catalytic activity and substrate recognition regions that determine its binding specificity to target proteins .

Why is Xenopus laevis a suitable model for studying NRDP1 function?

Xenopus laevis represents an ideal experimental model for studying NRDP1 function for several reasons. First, as a phylogenetically intermediate species between aquatic vertebrates and land tetrapods, it exhibits an immune system fundamentally similar to mammals, including leukocytes involved in innate immunity as well as B and T lymphocytes . Second, Xenopus can easily be induced to breed in laboratory settings by injecting human gonadotrophin, making it accessible for developmental studies . Third, its evolutionary distance from mammals permits distinguishing species-specific adaptations from more conserved features of biochemical pathways involving NRDP1 . Additionally, the availability of genetically-defined inbred strains and clones, along with well-established techniques for genetic manipulation, makes it possible to study NRDP1 function in various physiological contexts .

What is the protein structure of Xenopus laevis NRDP1/RNF41?

Xenopus laevis NRDP1/RNF41 is a 317 amino acid protein with specific functional domains. The complete amino acid sequence is:

MGYDVSRFQGDVDEDLICPICSGVLEEPVQAPHCEHAFCNACITQWFSQQQTCPVDRSVVTVAHLRPVPRIMRNMLSKLQITCDNAVFGCTTIVRLDNLMSHLSDCEHNPKRPVTCEQGCGLEMPKDEVPNHNCIKHLRSVVQQQQIRIGELEKTAESKHQLSEQKRDIQLLKAYMRAIRSANPNLQNLEETIEYNEILEWVNSLQPARVTRWGGMISTPDAVLQAVIKRSLVESGCPASIVNEIIENAHERNWPQGLATLETRQMNRRYYEN YVAKRIPGKQAVVVMACENQHMGEDMVLEPGLVMIFAHGVEEI

The protein contains critical RING finger cysteine-rich domains (CICSGVLEEPVQ and CEHAFCNACITQ) which are essential for its E3 ubiquitin ligase activity. These domains coordinate zinc ions and facilitate the transfer of ubiquitin from E2 ubiquitin-conjugating enzymes to specific substrate proteins .

How does NRDP1 regulate TLR signaling pathways in Xenopus compared to mammalian systems?

NRDP1 exerts dual regulatory effects on Toll-like receptor (TLR) signaling pathways that distinguish it from many other E3 ubiquitin ligases. In Xenopus, as in mammalian systems, NRDP1 simultaneously inhibits the production of proinflammatory cytokines while increasing interferon-beta production in TLR-triggered macrophages . This differential regulation occurs through two distinct mechanisms: first, NRDP1 suppresses the MyD88-dependent activation of transcription factors NF-κB and AP-1 by directly binding to and polyubiquitinating MyD88, leading to its degradation . Second, it promotes activation of the kinase TBK1 and transcription factor IRF3 by polyubiquitinating TBK1, which enhances rather than degrades this substrate .

What role does NRDP1 play in reproduction and spermatogenesis based on knockout studies?

Recent knockout studies have revealed a critical role for NRDP1 in male reproduction. Global deletion of Nrdp1 leads to male infertility characterized by the formation of round-headed sperm (globozoospermia) . Mechanistically, Nrdp1 deficiency disrupts autophagy-associated acrosome biogenesis during spermatogenesis . The most prominent phenotypes observed in Nrdp1-knockout males include:

• Complete male infertility with preserved female fertility

• Normal testicular volume and weight

• No obvious defects in early spermatogenesis stages

• Round-headed sperm with multiple tails and swollen middle pieces

• Severely reduced sperm motility

• Disrupted mitochondrial arrangement with abnormal accumulation in sperm heads

• Abnormal mitochondrial structure

Proteomic analysis of Nrdp1-deficient spermatids and sperm showed significant alterations in lysosomal and mitochondrial proteins, with more proteins being upregulated than downregulated. This finding aligns with NRDP1's role in protein degradation through both proteasomal and lysosomal pathways .

How can contradictory data on NRDP1 function between in vitro and in vivo studies be reconciled?

Reconciling contradictory data between in vitro and in vivo studies of NRDP1 requires consideration of several factors:

• Substrate specificity context: In vitro studies often use purified recombinant NRDP1 protein with specific substrates, whereas in vivo, NRDP1 functions within complex protein networks that influence substrate availability and specificity. For example, NRDP1 functions both as an adaptor protein and an E3 ligase in TLR signaling pathways in vivo, but in vitro studies may only capture one of these functions .

• Post-translational modifications: Endogenous NRDP1 undergoes various post-translational modifications in vivo that may not be replicated in in vitro systems, affecting its activity and substrate selection.

• Experimental design considerations: When designing experiments to reconcile contradictory data, researchers should:

  • Use both Nrdp1-knockdown and overexpression approaches in the same experimental system

  • Employ both purified protein interaction studies and cellular context experiments

  • Validate findings across multiple model systems, including Xenopus, mammalian cells, and in vivo models

• Tissue-specific effects: Knockout studies reveal that NRDP1 has tissue-specific functions, such as its critical role in spermatogenesis , which may not be apparent in more generalized in vitro studies focusing on immune function .

What are the optimal conditions for expressing and purifying recombinant Xenopus laevis NRDP1?

The optimal conditions for expressing and purifying recombinant Xenopus laevis NRDP1 involve several critical considerations:

Expression Systems:

• Yeast expression systems have proven effective for producing functional Xenopus NRDP1 with proper folding and post-translational modifications

• For structural studies requiring higher yields, E. coli systems may be used with optimization of induction temperature (16-18°C) and IPTG concentration (0.1-0.5 mM)

• Mammalian cell expression (particularly HEK293T cells) is recommended when studying interaction partners in a more native-like environment

Purification Protocol:

• Affinity chromatography using His-tag purification (as NRDP1 is often expressed with a His-tag)

• Size exclusion chromatography to separate monomeric from aggregated protein

• Ion exchange chromatography as a polishing step

Buffer Optimization:

• pH range: 7.2-7.8 (typically 7.4)

• Salt concentration: 150-300 mM NaCl

• Reducing agents: 1-5 mM DTT or 0.5-2 mM TCEP to maintain RING domain integrity

• Addition of 5-10% glycerol for stability during storage

Quality Control:

• Purity assessment via SDS-PAGE (>90% purity is standard for functional studies)

• Western blotting to confirm identity

• Activity assays using known substrates to verify functional integrity

How can researchers effectively study NRDP1's E3 ligase activity in Xenopus systems?

Studying NRDP1's E3 ligase activity in Xenopus systems requires specialized techniques to capture both substrate specificity and enzymatic activity:

In Vitro Ubiquitination Assays:

• Reconstitute ubiquitination reaction with purified components:

  • Recombinant Xenopus NRDP1 (E3)

  • Appropriate E1 (ubiquitin-activating enzyme)

  • E2 (ubiquitin-conjugating enzyme, typically UbcH5 family members)

  • Purified substrate protein (e.g., MyD88 or TBK1)

  • Ubiquitin (consider using tagged ubiquitin for easier detection)

  • ATP regeneration system

• Detect ubiquitination through:

  • Western blotting for substrate molecular weight shifts

  • Mass spectrometry to identify ubiquitination sites

Cell-Based Assays:

• Xenopus cell culture systems (such as XTC or XL177 cells)

• Transfection with wild-type or mutant NRDP1 constructs

• Co-immunoprecipitation followed by ubiquitination analysis

• Fluorescence-based reporters for tracking substrate degradation

In Vivo Approaches:

• Transgenic Xenopus approaches:

  • CRISPR/Cas9-mediated gene editing to create point mutations in catalytic domains

  • Overexpression of dominant-negative NRDP1 mutants

  • Tissue-specific knockout using conditional approaches

• Analysis methods:

  • Immunoprecipitation of endogenous substrates followed by ubiquitin detection

  • Proteomic profiling comparing wild-type and NRDP1-deficient tissues

  • Functional readouts relevant to known NRDP1 pathways (immune response, spermatogenesis)

What experimental design is most appropriate for investigating NRDP1's role in immune regulation in Xenopus?

To investigate NRDP1's role in immune regulation in Xenopus, the following experimental design is recommended:

Study Design Framework:

Specific Techniques:

• Primary Cell Isolation and Culture:

  • Isolation of Xenopus peritoneal macrophages or splenocytes

  • Culture conditions optimized for immune cell function

  • TLR stimulation using appropriate ligands (e.g., LPS, poly(I:C))

• In Vivo Immune Challenge Models:

  • Bacterial challenge (e.g., using heat-killed bacteria)

  • Viral challenge (e.g., Ranavirus infection)

  • Lipopolysaccharide-induced endotoxin shock model

• Molecular Analysis Techniques:

  • RT-qPCR for cytokine gene expression

  • ELISA for secreted cytokine quantification

  • Immunoblotting for signaling pathway activation

  • Immunoprecipitation for protein-protein interactions

  • ChIP assays for transcription factor binding

• Advanced Imaging:

  • Confocal microscopy to track NRDP1 localization during immune responses

  • Live cell imaging to monitor real-time signaling dynamics

• Systems Biology Approaches:

  • Transcriptomics to identify global gene expression changes

  • Proteomics to identify novel substrates and pathway components

  • Network analysis to integrate NRDP1 function into broader immune regulation networks

How does NRDP1 substrate specificity differ between its roles in immunity and reproduction?

NRDP1 exhibits distinct substrate specificity profiles in its roles in immunity versus reproduction, which explains its diverse physiological functions:

Immune System Substrates:

• MyD88: NRDP1 directly binds and polyubiquitinates MyD88, leading to its degradation and subsequent inhibition of NF-κB and AP-1 activation

• TBK1: NRDP1 polyubiquitinates TBK1, but in this case, ubiquitination leads to activation rather than degradation, promoting IRF3 phosphorylation and interferon-beta production

Reproductive System Substrates:

• Analysis of Nrdp1-deficient testes and epididymis samples revealed significant alterations in protein expression patterns:

  • 440 upregulated and 201 downregulated proteins in testicular haploid cells

  • 374 upregulated and 113 downregulated proteins in epididymal sperm

  • 18 commonly upregulated transmembrane proteins between both tissues

  • Only 1 commonly downregulated protein (Ints12)

The differential substrate targeting appears to be regulated by:

• Tissue-specific expression of co-factors that influence NRDP1 substrate selection

• Compartment-specific localization of NRDP1 within cells

• Post-translational modifications of NRDP1 itself that alter its substrate preference

• Different ubiquitin chain topologies (K48 versus K63-linked) depending on cellular context

These differences highlight NRDP1's versatility as a regulator of protein homeostasis across different biological systems and suggest that therapeutic targeting of NRDP1 would need to account for these tissue-specific functions .

What are the implications of NRDP1 research in Xenopus for understanding human disease mechanisms?

Research on NRDP1 in Xenopus laevis has significant implications for understanding several human disease mechanisms:

Immunological Disorders:
The role of NRDP1 in balancing proinflammatory and interferon responses suggests potential involvement in autoimmune and inflammatory diseases. Nrdp1-transgenic mice show resistance to lipopolysaccharide-induced endotoxin shock and viral infections, indicating that modulating NRDP1 activity could be a therapeutic strategy for sepsis and certain viral diseases . The conservation of immune signaling pathways between Xenopus and humans makes these findings particularly relevant for translational research.

Male Infertility:
The finding that Nrdp1 deficiency leads to globozoospermia (round-headed sperm) and male infertility in mouse models has direct relevance to human male infertility cases with similar phenotypes . Approximately 1% of human male infertility cases present with globozoospermia, and NRDP1 mutations could potentially contribute to this condition. Understanding the molecular mechanisms through which NRDP1 regulates acrosome biogenesis and mitochondrial arrangement in sperm could lead to new diagnostic and therapeutic approaches for specific forms of male infertility.

Neurodegenerative Disorders:
While not directly addressed in the provided search results, NRDP1's function as an E3 ubiquitin ligase suggests potential roles in protein quality control pathways relevant to neurodegenerative diseases characterized by protein aggregation. The ability to study NRDP1 in the relatively simple nervous system of Xenopus tadpoles provides opportunities to investigate its neuronal functions in a controlled setting .

Cancer Biology:
Dysregulation of ubiquitin-proteasome pathways is a hallmark of many cancers. The identification of NRDP1's role in autophagy and protein degradation pathways suggests potential involvement in cancer development or progression. Xenopus models provide a unique system for investigating these connections through their well-characterized developmental pathways and the availability of transgenic approaches.

What methodological advancements are needed to further NRDP1 research in Xenopus systems?

Several methodological advancements would significantly enhance NRDP1 research in Xenopus systems:

Advanced Genetic Tools:

• Development of tissue-specific and inducible CRISPR/Cas9 systems for Xenopus to enable precise temporal and spatial control of NRDP1 expression

• Creation of knock-in models with tagged endogenous NRDP1 to facilitate tracking of the protein in its native context

• Establishment of Xenopus cell lines with stable NRDP1 modifications for high-throughput screening applications

Improved Protein Analysis Techniques:

• Development of Xenopus-specific antibodies with higher specificity and sensitivity for NRDP1 and its modified forms

• Adaptation of proximity labeling techniques (BioID, APEX) for Xenopus systems to identify transient NRDP1 interactions

• Implementation of advanced mass spectrometry approaches to map ubiquitination sites and chain topologies on NRDP1 substrates

Novel Imaging Methods:

• Live imaging protocols for visualizing NRDP1 activity in developing Xenopus embryos

• Super-resolution microscopy adaptations for Xenopus tissues to resolve subcellular localization of NRDP1 and its substrates

• Intravital imaging techniques to monitor NRDP1 function in immune cells responding to pathogens in vivo

Physiological Models:

• Development of Xenopus models for human diseases where NRDP1 dysregulation is implicated

• Standardized immune challenge protocols to evaluate NRDP1's role in various pathogen responses

• Creation of reproductive biology models to further investigate NRDP1's role in gametogenesis

Data Integration Platforms:

• Xenopus-specific databases integrating transcriptomic, proteomic, and phenotypic data related to NRDP1 function

• Computational models predicting NRDP1 substrate interactions across different tissues and developmental stages

• Cross-species analysis tools to facilitate translation of Xenopus findings to mammalian and human systems

These methodological advancements would address current technical limitations in studying the complex functions of NRDP1 in different biological contexts and would accelerate the translation of findings from Xenopus models to human health applications .