Recombinant Xenopus laevis E3 ubiquitin-protein ligase RNF128 (rnf128)

What is RNF128 and what is its basic structure in Xenopus laevis?

RNF128 (Ring Finger Protein 128), also known as GRAIL (Gene Related to Anergy In Lymphocytes), is a membrane-localized E3 ubiquitin ligase. In Xenopus laevis, RNF128 contains a canonical sequence with a cytosolic zinc-binding RING finger domain that possesses catalytic activity and a luminal or extracellular PA domain that captures transmembrane protein targets for ubiquitination . The RING finger domain of RNF128 exhibits E3 ligase activity, and mutations in this domain disturb ubiquitin ligase activity . The protein structure includes conserved cysteine residues at positions 277 and 280 within the RING region that are necessary for E3 catalytic activity .

How does RNF128 function in the ubiquitination process in Xenopus laevis?

In Xenopus laevis, RNF128 functions as an E3 ubiquitin ligase that catalyzes the formation of polyubiquitin chains on target proteins. Research demonstrates that RNF128 can mediate different types of ubiquitin linkages depending on the substrate and cellular context:

• K63-linked polyubiquitination: RNF128 has been shown to catalyze K63-linked polyubiquitination on some substrates, which typically leads to altered protein trafficking or signaling rather than degradation .

• K27-linked polyubiquitination: In certain contexts, RNF128 promotes K27-linked polyubiquitination, facilitating lysosomal degradation of targets .

• Substrate specificity: RNF128 exhibits selective binding to specific transmembrane proteins through its PA domain, enabling targeted ubiquitination .

The RING domain is essential for the catalytic activity of RNF128, as demonstrated by experiments using RING domain deletion mutants (Flag-RNF128 ΔR) that fail to promote ubiquitination .

How is RNF128 expressed during Xenopus laevis development?

RNF128 expression in Xenopus laevis follows a regulated developmental pattern. While comprehensive stage-by-stage expression data is limited, research indicates that RNF128 expression can be detected at various developmental stages, including during late embryogenesis and in adult tissues. The normal table of Xenopus development (stages 1-66 from fertilization to metamorphosis) provides a framework for understanding when and where RNF128 might be expressed .

In adult Xenopus laevis, RNF128 expression has been detected in specific tissues and cell types, particularly in immune cells and adipose tissue macrophages (ATMs). Studies have shown that RNF128 expression in ATMs can be influenced by transcription factors and neuro-hormonal signals, indicating dynamic regulation based on physiological conditions .

What factors regulate RNF128 expression in Xenopus laevis tissues?

Several factors have been identified as regulators of RNF128 expression in Xenopus laevis:

• Neuroendocrine signaling: In studies of Xenopus adipose tissue macrophages, the G protein-coupled receptor GPR147 has been shown to influence RNF128 expression. Deficiency of GPR147 increased RNF128 transcription, while NPFF (neuropeptide FF) treatment had the opposite effect, decreasing RNF128 expression .

• Inflammation mediators: Treatment with inflammatory stimuli like pI:pC (polyinosinic:polycytidylic acid, a TLR ligand) or LPS (lipopolysaccharide) affects RNF128 expression levels in Xenopus macrophages .

• Developmental programming: The expression of RNF128 appears to be regulated during development, with specific patterns emerging during the transition from embryonic to adult stages .

• Tissue-specific factors: Different tissues in Xenopus exhibit varied levels of RNF128, suggesting tissue-specific regulatory mechanisms .

What are the most effective methods for producing recombinant Xenopus laevis RNF128 protein?

Based on research protocols, effective methods for producing recombinant Xenopus laevis RNF128 include:

• Bacterial expression systems: E. coli-based expression systems have been successfully used for producing recombinant RNF128, typically with N-terminal His-tags for purification purposes . The process involves:

  • Cloning the RNF128 coding sequence into an appropriate bacterial expression vector

  • Transforming E. coli cells and inducing expression

  • Lysing cells and purifying the protein using affinity chromatography

  • Further purification using size exclusion chromatography if needed

• Eukaryotic expression systems: For applications requiring post-translational modifications, RNF128 can be expressed in eukaryotic systems like:

  • Insect cells (Sf9 or Hi5) using baculovirus expression

  • Mammalian cell lines (HEK293T) for transient expression

• Storage considerations: Recombinant RNF128 is typically stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0. The protein should be aliquoted and stored at -20°C/-80°C to avoid repeated freeze-thaw cycles, which can compromise activity .

What techniques are most effective for studying RNF128 function in Xenopus laevis models?

Several techniques have proven effective for studying RNF128 function in Xenopus laevis:

• CRISPR/Cas9-mediated gene editing:

  • Injection of Cas9 mRNA (2 ng/embryo) and sgRNA (400 pg/embryo) into the animal pole of one-cell stage embryos

  • Verification of targeting efficiency using T7 endonuclease I assay on genomic DNA extracted from stage 32 embryos

• Morpholino-mediated knockdown:

  • Injection of antisense morpholino oligonucleotides targeting RNF128 mRNA

  • Verification of knockdown efficiency by RT-qPCR and Western blot

• In vitro ubiquitination assays:

  • Reconstitution of the ubiquitination reaction using purified recombinant components

  • Analysis of ubiquitin chain formation by Western blotting

• Cell-based assays:

  • Transfection of Xenopus-derived cells with RNF128 constructs

  • Analysis of ubiquitination patterns using co-immunoprecipitation

  • Functional readouts such as protein stability and trafficking

• Transgenic reporter lines:

  • Generation of transgenic Xenopus expressing fluorescent RNF128 fusion proteins

  • Live imaging of protein localization and dynamics

What are the known substrates of RNF128 in Xenopus laevis, and how does substrate recognition occur?

Research has identified several substrates of RNF128 in Xenopus laevis and related experimental systems:

• Scavenger Receptor B1 (SRB1): RNF128 has been shown to directly bind to SRB1 and catalyze its K63-linked polyubiquitination at lysine 478 on the cytoplasmic C-terminus. This ubiquitination prevents SRB1 degradation through the lysosomal system .

• IL-3 Receptor α (IL-3Rα): RNF128 selectively binds to IL-3Rα (but not the common beta chain IL-3Rβ) and promotes its K27-linked polyubiquitination, facilitating lysosomal degradation .

• Transmembrane proteins: The PA domain of RNF128 is involved in recognizing transmembrane protein targets. Studies have shown that RNF128 interacts with the extracellular region of target proteins through this domain .

• Potential interaction with ribonucleoprotein complexes: In Xenopus egg extracts, RNF128 has been found to be part of RNA-binding complexes, suggesting it may have roles in RNA metabolism or regulation that are distinct from its E3 ligase activity .

Substrate recognition involves:

• Direct binding through the PA domain to extracellular/luminal regions of target proteins

• Positioning of the RING domain near cytoplasmic lysine residues for ubiquitination

• Substrate specificity determined by protein-protein interaction domains

How does RNF128 interact with other components of the ubiquitin-proteasome and endosomal/lysosomal systems?

RNF128 interacts with several components of cellular trafficking and degradation systems:

• Endosomal trafficking components:

  • RNF128 promotes the association between ubiquitinated SRB1 and Rab11, facilitating recycling of SRB1 to the cell membrane rather than lysosomal degradation

  • The interaction between SRB1 and Rab11 is reduced in RNF128-knockout cells, indicating RNF128's role in regulating this trafficking pathway

• Lysosomal pathway interactions:

  • RNF128 can direct proteins to either recycling pathways or lysosomal degradation depending on the substrate and ubiquitin linkage type

  • K27-linked ubiquitination by RNF128 promotes lysosomal degradation of IL-3Rα

  • K63-linked ubiquitination of SRB1 by RNF128 prevents lysosomal degradation

• Membrane trafficking:

  • RNF128 localizes to endosomes and influences receptor internalization and sorting during endocytosis

  • It regulates the balance between recycling and degradation for membrane proteins

How can RNF128 mutations be designed to study structure-function relationships in Xenopus laevis models?

Several strategic approaches to RNF128 mutagenesis have proven valuable for structure-function studies:

• RING domain mutations:

  • Deletion of the RING domain (Flag-RNF128 ΔR) abolishes E3 ligase activity while preserving protein-protein interactions

  • Point mutations in conserved cysteine residues (C277A and C280A) disrupt zinc coordination and catalytic activity

  • These mutations allow researchers to distinguish between ubiquitin ligase-dependent and independent functions of RNF128

• PA domain modifications:

  • Mutations in the PA domain affect substrate recognition without altering catalytic activity

  • This approach helps identify key residues involved in specific substrate interactions

• Glycosylation site mutations:

  • RNF128 contains N-glycosylation sites that can be mutated (H297/300N) to study the role of glycosylation in protein function and localization

  • These mutations help determine how post-translational modifications affect RNF128 activity

• Lysine mutants of substrate proteins:

  • Generating lysine-to-arginine mutations in potential ubiquitination sites of substrates (e.g., K478R in SRB1) is an effective strategy to identify specific ubiquitination sites

  • This approach has revealed that lysine 478 in SRB1 is the primary site for RNF128-mediated K63-linked ubiquitination

What are the challenges in maintaining activity of recombinant Xenopus laevis RNF128 for in vitro studies?

Researchers face several challenges when working with recombinant Xenopus laevis RNF128:

• Protein stability issues:

  • The RING domain contains zinc-coordinating cysteine residues susceptible to oxidation

  • Maintaining a reducing environment with DTT or β-mercaptoethanol is essential

  • Repeated freeze-thaw cycles significantly reduce enzymatic activity

• Buffer composition considerations:

  • Optimal activity requires specific buffer conditions (Tris/PBS-based buffer with 6% trehalose at pH 8.0)

  • Presence of zinc ions (10-50 μM ZnCl₂) helps maintain RING domain structure

  • Glycerol (5-50%, with 50% being optimal for long-term storage) provides stability during freezing

• Co-factor requirements:

  • In vitro ubiquitination assays require the presence of E1 and E2 enzymes

  • Selection of appropriate E2 enzymes is critical as RNF128 works preferentially with specific E2s

  • ATP and ubiquitin must be fresh and functional for successful assays

• Reconstitution procedures:

  • Proper reconstitution from lyophilized powder in deionized sterile water to a concentration of 0.1-1.0 mg/mL is recommended

  • Centrifugation prior to opening vials helps retrieve all protein content

How does Xenopus laevis RNF128 compare structurally and functionally to its homologs in other vertebrates?

Comparative analysis reveals important similarities and differences between Xenopus laevis RNF128 and its homologs in other vertebrates:

What insights can be gained from studying RNF128 in Xenopus laevis that are not possible in mammalian models?

Xenopus laevis offers unique advantages for studying RNF128 biology:

• Developmental insights:

  • Xenopus embryos develop externally and are transparent, allowing direct observation of developmental processes influenced by RNF128

  • The well-characterized developmental stages (from fertilization to metamorphosis) provide a framework for studying RNF128's role throughout development

  • Metamorphosis, a unique feature of amphibian development, offers insights into RNF128's potential role in major tissue remodeling events

• Experimental advantages:

  • Xenopus eggs and embryos are larger than mammalian counterparts, facilitating microinjection and manipulation

  • Ease of generating gene knockouts using CRISPR/Cas9 by injecting into one-cell stage embryos

  • The ability to perform experiments in egg extracts provides a biochemically manipulable system for studying RNF128 function

• Evolutionary perspective:

  • As an amphibian, Xenopus occupies an important evolutionary position between fish and mammals

  • Comparing RNF128 function across these evolutionary transitions provides insights into conserved mechanisms

  • Studies in Xenopus have revealed that adipose tissue macrophages develop before bone marrow in evolution, with RNF128 playing a role in their activation state

How is RNF128 involved in pathological processes, and how can Xenopus laevis models contribute to understanding these mechanisms?

Research has implicated RNF128 in several pathological processes, with Xenopus laevis models providing valuable insights:

What advanced techniques can be applied to study RNF128-dependent ubiquitination in Xenopus laevis systems?

Several cutting-edge techniques can be applied to study RNF128-dependent ubiquitination in Xenopus laevis:

• Proximity-based labeling approaches:

  • BioID fusion with RNF128 to identify proximal interacting proteins in living cells

  • RNF128 has been successfully inserted into expression vectors encoding C-terminally FLAG-tagged BirA for this purpose

  • This technique can identify transient interactions that might be missed by traditional co-immunoprecipitation

• Quantitative ubiquitinomics:

  • Mass spectrometry-based approaches to identify and quantify ubiquitination sites on RNF128 substrates

  • SILAC or TMT labeling can be used to compare ubiquitination patterns in wild-type versus RNF128-deficient samples

  • This approach can reveal the complete spectrum of RNF128 substrates and ubiquitination sites

• Live imaging of ubiquitination dynamics:

  • Fluorescent ubiquitin sensors to visualize RNF128-mediated ubiquitination in real-time

  • FRET-based reporters that detect changes in protein conformation upon ubiquitination

  • This allows monitoring of the spatial and temporal dynamics of RNF128 activity during development or in response to stimuli

• In vitro reconstitution systems:

  • Xenopus egg extracts provide a biochemically tractable system for studying RNF128 function

  • Addition of recombinant proteins, inhibitors, or antibodies to these extracts allows manipulation of RNF128 activity

  • This approach has been used to study other E3 ligases in Xenopus and can be adapted for RNF128