Recombinant Mouse RING finger protein 112 (Rnf112)

What is the molecular structure of mouse Rnf112?

Mouse Rnf112 is a 654 amino acid protein containing multiple functional domains. The protein includes a characteristic RING finger domain (located within amino acids 73-322) that confers E3 ubiquitin ligase activity. The RING domain contains conserved cysteine and histidine residues that coordinate zinc ions, which is essential for its ubiquitin ligase function. The full amino acid sequence includes recognizable motifs for GTPase activity and protein-protein interactions . The protein also contains regions that facilitate its regulatory functions in neuronal development and cellular signaling pathways.

What are the primary biological functions of Rnf112?

Rnf112 functions as both an E3 ubiquitin-protein ligase and a GTPase. As an E3 ligase, it facilitates the transfer of ubiquitin to target substrates, marking them for degradation via the proteasome pathway. The protein plays crucial roles in:

• Neuronal differentiation, including neurogenesis and gliogenesis during brain development

• Cell cycle regulation, particularly inducing G0/G1 phase arrest

• Maintenance of neural functions in the adult brain

• Protection of nervous tissue from oxidative stress-induced damage

• Regulation of dendritic spine density and synaptic neurotransmission

• Tumor suppression, particularly in gastric cancer through FOXM1 ubiquitination

What expression systems yield the highest functional activity for recombinant mouse Rnf112?

Mammalian expression systems, particularly HEK-293 cells, have proven most effective for producing functional recombinant mouse Rnf112. These systems provide appropriate post-translational modifications and protein folding environments. Expression in HEK-293 cells typically yields protein with >90% purity as determined by Bis-Tris PAGE, anti-tag ELISA, Western Blot, and analytical SEC (HPLC) . While E. coli systems can express truncated versions of the protein (specific domains), they may lack proper folding and post-translational modifications necessary for full activity. Cell-free protein synthesis (CFPS) systems have also been employed with 70-80% purity but may offer advantages for rapid production and modification screening .

What are the optimal storage conditions for maintaining recombinant mouse Rnf112 activity?

For maximum stability and retention of functional activity, recombinant mouse Rnf112 should be stored at -80°C. Repeated freeze-thaw cycles significantly decrease protein activity and should be strictly avoided . Working aliquots should be prepared during initial thawing to minimize degradation. The composition of storage buffer influences stability; buffers containing glycerol (10-20%), reducing agents like DTT (1-5 mM), and protease inhibitors help maintain protein integrity. Activity assessments should be performed immediately after thawing for optimal results, and long-term storage beyond 12 months is not recommended even at -80°C .

How can researchers effectively verify the E3 ubiquitin ligase activity of recombinant Rnf112?

Verification of E3 ubiquitin ligase activity can be accomplished through multiple complementary approaches:

• In vitro ubiquitination assays: Combine purified recombinant Rnf112 with E1 enzyme, E2 conjugating enzyme, ubiquitin, ATP, and potential substrate proteins. Analyze ubiquitination using western blotting with anti-ubiquitin antibodies.

• Functional RING domain verification: Create RING domain mutants (as control) by modifying key cysteine/histidine residues. Compare wild-type and mutant Rnf112 in ubiquitination assays. Research demonstrates that mutations in the RING domain abolish ubiquitination capacity while maintaining substrate binding .

• Substrate-specific assays: For known substrates like FOXM1, incubate recombinant Rnf112 with the substrate and detect ubiquitination using substrate-specific antibodies. Studies show that wild-type Rnf112 efficiently ubiquitinates FOXM1, leading to its degradation, while catalytically dead RNF112-Mut (with RING domain mutations) fails to ubiquitinate FOXM1 .

• Cellular ubiquitination assays: Express recombinant Rnf112 in cells alongside potential substrates and analyze changes in substrate protein levels and ubiquitination status.

What methods can be used to investigate Rnf112's role in neuronal protection mechanisms?

Several experimental approaches can be employed to study Rnf112's neuroprotective functions:

• Intracerebral hemorrhage (ICH) models: Utilize Rnf112 knockout or overexpression mouse models subjected to ICH procedures. Research has shown that Rnf112 deletion protects brain against ICH by inhibiting the TLR-4/NF-κB inflammatory pathway .

• Pathway analysis assays: Examine the effect of Rnf112 expression on TLR-4/NF-κB signaling components using western blotting, immunoprecipitation, and RT-qPCR to measure changes in protein levels, interactions, and gene expression.

• Oxidative stress resistance assays: Subject neuronal cells with varied Rnf112 expression to oxidative stress inducers (H₂O₂, glutamate) and measure cell viability, ROS levels, and apoptotic markers.

• Dendritic spine density analysis: Perform Golgi staining or GFP expression in neurons to visualize and quantify dendritic spine morphology and density in relation to Rnf112 expression levels. This approach helps understand how Rnf112's GTPase activity contributes to spine maintenance .

• Electrophysiological recordings: Conduct patch-clamp recordings to assess synaptic transmission in neurons with modified Rnf112 expression.

How does Rnf112 interact with the FOXM1 pathway in cancer models?

Rnf112 functions as a tumor suppressor in cancer models through direct regulation of FOXM1, a transcription factor that promotes cancer progression. Research demonstrates that:

• Direct interaction and ubiquitination: Rnf112 physically binds to FOXM1 and acts as an E3 ubiquitin ligase to ubiquitinate FOXM1, targeting it for proteasomal degradation. This interaction depends on the RING finger domain of Rnf112 .

• Transcriptional suppression: Rnf112 expression significantly downregulates FOXM1-dependent transcriptional activity, as measured using a luciferase reporter containing FKH binding sequences. This leads to reduced expression of FOXM1 downstream genes related to cell proliferation and invasion, including CKS1, CCNB1, SKP2, FN1, and ZEB1 .

• In vivo tumor suppression: Xenograft tumor models show that Rnf112 overexpression decreases tumor growth and weight, coupled with decreased FOXM1 and its downstream target expression. Conversely, Rnf112 depletion increases tumor growth and FOXM1 expression .

• Clinical relevance: Gene expression analysis of cancer patients revealed negative correlation between RNF112 and FOXM1 expression levels. High RNF112 expression was associated with inhibition of cell cycle, proliferation, migration, and invasion pathways .

• Dependency on ubiquitin ligase activity: Catalytically dead RNF112 (RNF112-Mut) with mutations in the RING domain maintains binding to FOXM1 but fails to ubiquitinate and degrade it, resulting in loss of tumor suppressor function .

How can Rnf112 be utilized to study neuronal differentiation mechanisms?

Rnf112 provides a valuable tool for investigating neuronal differentiation mechanisms through several experimental approaches:

• Temporal expression analysis: Track Rnf112 expression patterns during different stages of neuronal development to correlate with specific differentiation processes.

• Cell cycle regulation studies: Investigate how Rnf112 induces cell cycle arrest at G0/G1 phase through upregulation of cell-cycle regulatory proteins, which is essential for initiating neuronal differentiation .

• Neurogenesis vs. gliogenesis fate determination: Manipulate Rnf112 expression in neural progenitor cells to observe shifts in differentiation towards neurons or glial cells. This helps elucidate the role of Rnf112 in cell fate decisions.

• Target identification: Perform proteomic analysis following Rnf112 immunoprecipitation to identify novel substrates that mediate its effects on neuronal differentiation.

• Domain-specific function analysis: Generate recombinant Rnf112 variants with mutations in specific domains (RING domain, GTPase domain) to dissect their individual contributions to neuronal differentiation. Comparative studies using wild-type and mutant forms can identify which functions (E3 ligase vs. GTPase) are critical for specific aspects of differentiation .

What experimental systems best represent the physiological functions of Rnf112 in vivo?

The most relevant experimental systems for studying physiological Rnf112 functions include:

What are common challenges in obtaining functionally active recombinant mouse Rnf112?

Researchers frequently encounter several challenges when producing active recombinant mouse Rnf112:

• Protein solubility issues: The full-length 654 amino acid protein can form inclusion bodies or aggregate during expression, particularly in bacterial systems. Solution: Express in mammalian HEK-293 cells, which provide appropriate chaperones and folding machinery .

• Maintaining RING domain integrity: The cysteine-rich RING domain is susceptible to oxidation, which compromises E3 ligase activity. Solution: Include reducing agents (DTT, β-mercaptoethanol) in all buffers and handle proteins under nitrogen atmosphere when possible.

• Post-translational modifications: Rnf112 may require specific post-translational modifications for full activity. Solution: Compare activity of protein expressed in different systems (bacterial, insect, mammalian) to identify the optimal expression system .

• Dual function preservation: Preserving both E3 ligase and GTPase activities simultaneously can be challenging. Solution: Carefully optimize purification conditions and verify both activities separately using specific assays.

• Tag interference: Purification tags may interfere with protein folding or activity. Solution: Compare different tag positions (N-terminal vs. C-terminal) and types (His, Strep, GST) to identify constructs with minimal functional impact .

How can researchers resolve conflicting data on Rnf112 function across different experimental models?

When facing contradictory results regarding Rnf112 function, consider these methodological approaches:

What critical controls should be included when studying Rnf112-mediated protein ubiquitination?

Rigorous ubiquitination studies require several essential controls:

• Catalytically inactive Rnf112: Include RING domain mutants (RNF112-Mut) that maintain substrate binding but lack E3 ligase activity as negative controls. Research shows these mutants fail to ubiquitinate targets like FOXM1 despite maintaining physical interaction .

• Substrate binding controls: Perform co-immunoprecipitation with wild-type and mutant Rnf112 to verify that differences in ubiquitination are not due to altered substrate binding.

• Ubiquitination reaction components: Include control reactions lacking individual components (E1, E2, ATP) to verify specificity of the ubiquitination signal.

• Proteasome inhibition: To visualize ubiquitinated species that would normally be rapidly degraded, include conditions with proteasome inhibitors (MG132, bortezomib).

• Linkage-specific ubiquitin antibodies: Use antibodies that recognize specific ubiquitin linkages (K48, K63) to distinguish between degradative and non-degradative ubiquitination.

• In vivo validation: Confirm in vitro findings by examining endogenous substrate levels in cells with normal or altered Rnf112 expression.

What are promising areas for future Rnf112 research in neurodegenerative disorders?

Several unexplored aspects of Rnf112 function offer potential for neurodegenerative disease research:

• TLR pathway modulation: Given that Rnf112 deletion protects against intracerebral hemorrhage by inhibiting the TLR-4/NF-κB pathway, investigating Rnf112 as a therapeutic target in other neuroinflammatory conditions is warranted .

• Oxidative stress resistance: Further explore how Rnf112 protects nervous tissue cells from oxidative stress-induced damage, which is a common feature in neurodegenerative diseases .

• Dendritic spine maintenance: Investigate the GTPase activity of Rnf112 in maintaining dendritic spine density in models of synaptopathies like Alzheimer's disease .

• Substrate identification: Conduct comprehensive proteomic screens to identify additional Rnf112 substrates in neuronal contexts that may be relevant to neurodegeneration.

• Therapeutic modulation strategies: Develop approaches to selectively enhance or inhibit specific Rnf112 functions (E3 ligase vs. GTPase) depending on the pathological context.

How can gene expression and protein correlation data enhance Rnf112 research?

Advanced genomic and proteomic analyses can significantly enhance Rnf112 research: