RNF170 Antibody

What is RNF170 and what are its key cellular functions?

RNF170 is a Ring Finger Protein 170, an endoplasmic reticulum (ER) membrane-located E3 ubiquitin ligase with approximately 257 amino acids and a molecular weight of approximately 30 kDa (though it often migrates at approximately 21.5 kDa on SDS-PAGE) . The protein contains three transmembrane domains and a canonical RING-HC domain, with its N-terminus in the ER lumen and the RING domain and C-terminus in the cytosol .

RNF170 has several key cellular functions:

• IP3 Receptor Regulation: RNF170 mediates the ubiquitination and degradation of inositol 1,4,5-trisphosphate receptors (IP3Rs) via the ER-associated degradation (ERAD) pathway . It associates rapidly with activated IP3 receptors and facilitates their processing.

• Immune Response Modulation: RNF170 plays a novel role in selectively inhibiting TLR3-triggered innate immune responses by promoting TLR3 degradation . Studies show that RNF170 promotes K48-linked polyubiquitination of K766 in TLR3, leading to TLR3 degradation through the proteasome pathway.

• Calcium Signaling: RNF170 is involved in calcium homeostasis through its regulation of IP3 receptors, which are ER membrane calcium channels .

Importantly, mutations in RNF170 have been associated with neurological disorders including hereditary spastic paraplegia and autosomal dominant sensory ataxia (ADSA) .

How is RNF170 structured and what domains are important for antibody targeting?

RNF170 has a distinctive structure with several domains that are important for antibody targeting:

• Transmembrane Domains: RNF170 contains three predicted membrane-spanning helices that anchor it to the ER membrane . These transmembrane domains contain charged residues that form ionic interactions critical for protein stability .

• RING Domain: Contains a canonical RING-HC domain in the cytosolic portion of the protein, which is essential for its ubiquitin ligase activity . The RING domain encompasses residues around amino acids 99-140 and contains critical cysteine and histidine residues (e.g., Cys-101 and His-103) that are required for ubiquitin ligase function .

• N-terminus: Located in the ER lumen .

• C-terminus: Located in the cytosol and contains regions important for interaction with substrates .

For antibody targeting, several regions have proven effective:

• Amino acids 1-200 (N-terminal region)

• Amino acids 46-120 (containing part of the RING domain)

• Amino acids 110-159 (containing part of the RING domain)

• Amino acids 121-193 (containing the sequence LGAISCPICRQTVTLLLTVFGEDDQSQDVLRLHQDINDYNRRFSGQPRSIMERIMDLPTLLRHAFREMFSVGG)

• C-terminal region (containing the sequence FYLISPLDFVPEALFGILGFLDDFFVIFLLLIYISIMYREVITQRLTR)

These regions offer specific epitopes for antibody recognition while maintaining specificity to RNF170 .

What is the relationship between RNF170 mutations and neurological disorders?

RNF170 mutations have been strongly linked to several neurological disorders, providing important insights into its physiological functions:

• Hereditary Spastic Paraplegia (HSP): Bi-allelic variants in RNF170 are associated with HSP. For example, in one family, a homozygous missense variant c.304T>C, p.Cys102Arg in the RING domain of RNF170 segregated with the disease with a LOD-score of 2.4 . This mutation affects the RING domain, which is critical for the protein's ubiquitin ligase activity.

• Autosomal Dominant Sensory Ataxia (ADSA): A point mutation in RNF170 (arginine 199 to cysteine) causes ADSA, a rare neurodegenerative disease characterized by ataxic gait, reduced sensory perception, and neurodegeneration in the posterior columns of the spinal cord . This mutation inhibits RNF170 expression and disrupts signaling via IP3 receptors by:

  • Enhancing RNF170 autoubiquitination and proteasomal degradation

  • Disrupting ionic interactions between charged residues in the transmembrane domains

  • Impairing calcium mobilization in response to stimuli without altering calcium store content, IP3 receptor levels, or IP3 production

Research with ADSA lymphoblasts showed that the R199C mutation significantly impairs platelet-activating factor-induced Ca2+ mobilization, suggesting that disrupted calcium signaling may be a key mechanism underlying neurodegeneration in ADSA .

Mechanistically, analyses show that mutations in RNF170 lead to IP3R-1 accumulation, with an increase of IP3R-1 levels to approximately 1.8-fold compared to wild-type cells . This accumulation could be reversed by stable re-expression of wildtype RNF170, supporting the causality of RNF170 status for the observed IP3R-1 accumulation .

How can RNF170 antibodies be used to investigate IP3R degradation pathways?

RNF170 antibodies serve as valuable tools for investigating IP3R degradation pathways through several advanced approaches:

• Tracking RNF170-IP3R Interactions:

  • Immunoprecipitation using anti-RNF170 antibodies can pull down activated IP3R complexes to study temporal dynamics of their association

  • Immunofluorescence co-localization studies using RNF170 antibodies alongside IP3R antibodies reveal spatial association between these proteins at the ER membrane and during trafficking to early endosomes

• Monitoring IP3R Ubiquitination Patterns:

  • Western blotting with RNF170 and ubiquitin antibodies after stimulus-induced IP3R activation reveals the specific ubiquitin chains (K48-linked and K63-linked) conjugated to IP3Rs

  • Comparing wild-type cells with RNF170 knockout models shows that RNF170 mediates the addition of all known ubiquitin conjugates to activated IP3Rs (monoubiquitin and Lys48- and Lys63-linked ubiquitin chains)

• Quantifying IP3R Degradation Kinetics:

  • Western blot analysis using RNF170 antibodies alongside IP3R antibodies in pulse-chase experiments allows measurement of IP3R degradation rates

  • In RNF170 knockout models created via CRISPR/Cas9, IP3R1 accumulates to approximately 1.8-fold of wild-type levels, demonstrating RNF170's essential role in IP3R homeostasis

• Investigating Erlin1/2 Complex Interactions:

  • RNF170 antibodies can help study the association between RNF170 and the erlin1/2 complex, which has been shown to bind to IP3 receptors immediately after their activation

  • Co-immunoprecipitation using RNF170 antibodies reveals that a substantial proportion of RNF170 is constitutively associated with the erlin1/2 complex

Experimental data shows that depletion of endogenous RNF170 by RNA interference inhibits stimulus-induced IP3 receptor ubiquitination and degradation, while overexpression of a catalytically inactive RNF170 mutant (with Cys-101 and His-103 mutated to Ser and Ala) suppresses stimulus-induced IP3 receptor processing .

What methodological approaches can be used to study RNF170's role in innate immune signaling?

Investigating RNF170's role in innate immune signaling requires sophisticated methodological approaches that leverage RNF170 antibodies:

• RNF170-TLR3 Interaction Analysis:

  • Immunoprecipitation with RNF170 antibodies followed by detection of TLR3 can confirm their physical interaction in macrophages and other immune cells

  • GST pull-down assays combined with RNF170 antibody detection demonstrate direct binding between RNF170 and TLR3

  • Immunofluorescence colocalization studies show that RNF170 colocalizes with TLR3 both in resting conditions and after poly(I:C) stimulation

• Subcellular Trafficking Studies:

  • Multi-color immunofluorescence using RNF170 antibodies in combination with markers for cellular compartments (KDEL for ER, EEA1 for early endosomes) reveals that both RNF170 and TLR3 are transferred from the ER to early endosomes after poly(I:C) stimulation

• CRISPR/Cas9 Knockout Models:

  • Generation of Rnf170-deficient (Rnf170−/−) cell lines and mice via CRISPR/Cas9-mediated deletion of exon 4, with RNF170 antibodies confirming knockout efficiency

  • These knockout models show increased expression of IFN-β and IL-6 in peritoneal macrophages specifically in response to poly(I:C) stimulation (TLR3 activator) but not to other TLR ligands

• TLR3 Ubiquitination Analysis:

  • Use RNF170 antibodies in conjunction with ubiquitin and TLR3 antibodies to demonstrate that RNF170 promotes K48-linked polyubiquitination of K766 in TLR3

  • Western blotting analysis reveals that this ubiquitination leads to TLR3 degradation through the proteasome pathway

• Cytokine Production Profiling:

  • Compare cytokine production (type I IFNs and proinflammatory cytokines) between wild-type and RNF170-deficient cells using ELISA and qPCR, with RNF170 antibodies confirming knockout status

  • Data from Rnf170−/− bone marrow-derived dendritic cells (BMDCs) shows increased expression of IFN-α, IFN-β, IL-6, and TNF-α specifically upon poly(I:C) stimulation

These approaches collectively demonstrate that RNF170 selectively inhibits TLR3-triggered innate immune responses by targeting TLR3 for degradation, but does not affect other innate immune signaling pathways like RIG-I-MAVS signaling .

How can mutations in RNF170 be characterized using antibody-based approaches?

Characterizing RNF170 mutations requires sophisticated antibody-based approaches that can reveal molecular mechanisms underlying associated disorders:

• Expression Level Analysis of Mutant RNF170:

  • Western blotting using RNF170 antibodies in cells expressing mutant variants (e.g., R199C causing ADSA) reveals reduced expression levels and altered migration patterns

  • Quantitative analysis shows that mutant RNF170 often migrates approximately 1 kDa more rapidly than wild-type counterparts on SDS-PAGE

  • Analysis of ADSA lymphoblasts demonstrates chronically reduced RNF170 levels compared to control samples

• Protein Stability and Degradation Assessment:

  • Pulse-chase experiments combined with immunoprecipitation using RNF170 antibodies can measure protein half-life differences between wild-type and mutant variants

  • Treatment with proteasome inhibitors followed by western blotting with RNF170 antibodies can determine if reduced mutant protein levels result from enhanced proteasomal degradation

  • Studies show that the R199C mutation enhances RNF170 autoubiquitination and proteasomal degradation

• Structural Impact Analysis Through Mutagenesis:

  • Generate additional point mutations (e.g., R198S) and analyze their effects with RNF170 antibodies to identify critical residues for protein stability

  • Use glycosylation-site introduction combined with endoglycosidase H (endo H) sensitivity testing to confirm protein topology and assess structural alterations in mutants

  • Research shows that ionic interactions between charged residues in the transmembrane domains of RNF170 are required for protein stability

• Functional Assays of Ubiquitin Ligase Activity:

  • Immunopurify wild-type and mutant RNF170 using antibodies, then perform in vitro ubiquitination assays to assess catalytic function

  • Compare ubiquitin ligase activities toward IP3 receptors between wild-type and mutant RNF170

  • Studies show that while the R199C mutation affects protein stability, the mutant protein retains apparently identical ubiquitin ligase activity toward IP3 receptors

• Calcium Signaling Assessment in Patient-Derived Cells:

  • Use RNF170 antibodies to confirm protein expression levels in patient-derived cells (e.g., ADSA lymphoblasts)

  • Combine with calcium imaging to reveal functional defects in calcium mobilization

  • Research demonstrates that ADSA lymphoblasts show impaired platelet-activating factor-induced Ca2+ mobilization despite normal Ca2+ store content, IP3 receptor levels, and IP3 production

These approaches have revealed that disease-causing mutations like R199C in RNF170 don't necessarily disrupt ubiquitin ligase activity toward IP3 receptors, but rather affect calcium signaling through other mechanisms or by altering interactions with other substrates .

What are the optimal conditions for using RNF170 antibodies in western blotting applications?

For optimal western blotting results with RNF170 antibodies, researchers should consider the following protocol parameters:

Sample Preparation:

• Lysis Buffer Composition: Use a buffer containing 150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 1% Triton X-100, with protease inhibitors (10 μM pepstatin, 0.2 mM PMSF, 0.2 μM soybean trypsin inhibitor) and 1 mM dithiothreitol, pH 8.0

• Alternative Detergents: For co-immunoprecipitation studies examining RNF170 interactions with complexes like erlin1/2, substitute 1% CHAPS for Triton X-100

• Cell Disruption Protocol: Disrupt cells for 30 minutes at 4°C with lysis buffer, followed by centrifugation at 16,000 × g for 10 minutes at 4°C

Electrophoresis Considerations:

• Gel Percentage: Use 10-12% SDS-PAGE gels for optimal resolution of RNF170, which typically migrates at ~21.5-27 kDa despite its predicted size of ~30 kDa

• Migration Pattern: Note that wild-type RNF170 consistently migrates ~1 kDa more slowly than certain mutants (e.g., R199C)

• Loading Controls: Include appropriate controls specific to the cellular compartment (e.g., calnexin for ER membrane proteins)

Antibody Dilutions and Detection:

• Primary Antibody Dilutions:

  • For rabbit polyclonal antibodies: 1:200-1:1000

  • For HRP-conjugated antibodies: 1:500-1:2000

• Incubation Conditions: Overnight at 4°C for primary antibodies

• Washing Steps: 3-5 washes with TBST (Tris-buffered saline with 0.1% Tween-20)

• Secondary Antibody Selection: Anti-rabbit or anti-mouse HRP-conjugated secondary antibodies depending on the host species of the primary antibody

• Signal Development: Enhanced chemiluminescence (ECL) detection systems are suitable for visualizing RNF170 bands

Validation and Controls:

• Positive Controls: Include human placenta tissue lysate, which has been validated for RNF170 detection

• Knockout Validation: CRISPR/Cas9 RNF170 knockout cell lines serve as excellent negative controls

• Overexpression Controls: Cells transfected with RNF170 expression constructs (wild-type or tagged versions) can serve as positive controls

Troubleshooting Notes:

• If RNF170 detection is weak, consider enriching membrane fractions before loading

• Multiple isoforms (30 kDa, 27 kDa, 22 kDa, 20 kDa, 19 kDa, and 13 kDa) may be detected due to alternative splicing

• RNF170 often appears as a doublet band in western blots, likely due to post-translational modifications or different splicing forms

How can researchers optimize RNF170 antibody performance in immunohistochemistry and immunofluorescence?

To achieve optimal results with RNF170 antibodies in immunohistochemistry (IHC) and immunofluorescence (IF) applications, researchers should follow these detailed recommendations:

Tissue/Cell Preparation:

• Fixation:

  • For tissues: 10% neutral buffered formalin fixation for 24-48 hours

  • For cells: 4% paraformaldehyde for 15-20 minutes at room temperature

• Antigen Retrieval:

  • Heat-induced epitope retrieval with TE buffer at pH 9.0 is recommended for optimal results

  • Alternative: citrate buffer at pH 6.0 can also be effective

  • Perform retrieval for 20 minutes at 95-98°C

Antibody Concentrations and Incubation Parameters:

• Primary Antibody Dilutions:

  • For IHC: 1:20-1:200 dilution range is suggested for most RNF170 antibodies

  • For IF: Start with higher concentrations (1:50) and titrate as needed

• Incubation Conditions:

  • IHC: Overnight at 4°C or 1-2 hours at room temperature

  • IF: 1-2 hours at room temperature in a humidified chamber

• Blocking:

  • 5-10% normal serum (matching the host species of secondary antibody) with 1% BSA in PBS

  • Include 0.1-0.3% Triton X-100 for permeabilization in IF applications

Detection Systems:

• IHC Detection:

  • HRP-polymer detection systems provide excellent signal-to-noise ratio

  • DAB (3,3'-diaminobenzidine) substrate is recommended for visualization

• IF Detection:

  • Alexa Fluor or similar fluorophore-conjugated secondary antibodies

  • DAPI counterstain for nuclear visualization

Controls and Validation:

• Positive Control Tissues:

  • Human breast cancer tissue and human kidney tissue have been validated for RNF170 antibody detection

• Negative Controls:

  • Primary antibody omission

  • Non-immune serum from the same species as the primary antibody

  • CRISPR/Cas9 knockout cells/tissues where available

Colocalization Studies:

• ER Localization:

  • Co-stain with ER markers such as KDEL to confirm RNF170's ER membrane localization

• Endosomal Trafficking:

  • Co-stain with EEA1 to track RNF170 movement to early endosomes after stimulation

• TLR3 Colocalization:

  • For immune studies, co-stain with TLR3 antibodies to visualize interaction and trafficking patterns

Troubleshooting Recommendations:

• High Background:

  • Increase blocking time or concentration

  • Additional washing steps with 0.1% Tween-20 in PBS

• Weak Signal:

  • Optimize antigen retrieval conditions

  • Increase primary antibody concentration or incubation time

  • Use signal amplification systems (e.g., tyramide signal amplification)

• Non-specific Staining:

  • Pre-absorb primary antibody with immunizing peptide where available

  • Increase dilution of primary antibody

For subcellular localization studies, research has shown that RNF170 colocalizes with ER markers in resting cells and with early endosome markers after stimulation, providing important validation checkpoints for IF applications .

What approaches should be used to validate RNF170 antibody specificity and functionality?

Comprehensive validation of RNF170 antibody specificity and functionality is essential for reliable research outcomes. Researchers should implement the following multi-layered validation approach:

1. Genetic Validation Methods:

2. Biochemical Validation Approaches:

3. Functional Validation Methods:

4. Specificity Controls for Immunohistochemistry/Immunofluorescence:

5. Mutation-Specific Validation:

For studies examining RNF170 mutations (e.g., R199C causing ADSA), additional validation involves comparing antibody detection between wild-type and mutant proteins. The R199C mutant typically shows reduced expression levels and migrates ~1 kDa more rapidly than wild-type in SDS-PAGE .

By systematically applying these validation approaches, researchers can ensure high confidence in antibody specificity and functionality, particularly important given RNF170's involvement in critical cellular processes and disease mechanisms.

How should researchers interpret conflicting RNF170 protein expression patterns across different tissues and experimental models?

When encountering conflicting RNF170 protein expression patterns across different experimental systems, researchers should consider several key factors for accurate interpretation:

1. Isoform Variation and Migration Patterns:

RNF170 exists in multiple isoforms due to alternative splicing, which can cause varying expression patterns:

• Six isoforms have been identified with molecular weights of 30 kDa, 27 kDa, 22 kDa, 20 kDa, 19 kDa, and 13 kDa

• Despite its predicted size of ~30 kDa, RNF170 typically migrates at ~21.5-27 kDa on SDS-PAGE

• RNF170 can appear as a doublet band in western blots, likely due to post-translational modifications or different splicing forms

When analyzing conflicting results, researchers should determine:

• Which isoforms are being detected by the specific antibody epitope

• Whether the antibody recognizes all or only subset of isoforms

• If tissue-specific isoform expression could explain the differences

2. Subcellular Localization and Extraction Methods:

RNF170's membrane localization affects its detection:

• As an ER membrane protein with three transmembrane domains, extraction efficiency varies with different lysis buffers

• Triton X-100 (1%) is effective for general extraction, while CHAPS (1%) better preserves protein-protein interactions

• Subcellular fractionation may show enrichment in membrane fractions versus whole cell lysates

Researchers should evaluate:

• Whether differences in sample preparation methods could explain conflicting results

• If membrane protein enrichment steps were included in some studies but not others

• The detergent compatibility with the specific antibody being used

3. Antibody Target Region Considerations:

Different antibodies target distinct RNF170 regions:

• N-terminal region (AA 1-200)

• RING domain region (AA 46-120, AA 110-159)

• Middle region (AA 121-193)

• C-terminal region

When interpreting conflicting data:

• Compare the epitope regions targeted by different antibodies

• Consider that post-translational modifications might mask certain epitopes

• Evaluate whether the antibody target region is conserved across species if comparing cross-species data

4. Mutation Effects on Detection:

RNF170 mutations can significantly impact antibody detection:

• The R199C mutation causing ADSA reduces protein expression and alters migration pattern (~1 kDa faster)

• Mutations may destabilize the protein, resulting in lower steady-state levels

• Point mutations can disrupt epitope recognition by specific antibodies

For studies involving disease models:

• Compare antibody epitope location relative to the mutation site

• Consider whether the mutation affects protein stability or expression

• Verify results with multiple antibodies targeting different regions

5. Data Integration Framework:

When faced with conflicting patterns, apply this systematic approach:

By systematically addressing these factors and implementing appropriate controls, researchers can resolve apparent conflicts in RNF170 expression patterns and develop a more accurate understanding of its biology across different experimental contexts.

What are the most common technical challenges when working with RNF170 antibodies and how can they be overcome?

Researchers working with RNF170 antibodies encounter several technical challenges that can impact experimental outcomes. Here's a comprehensive troubleshooting guide:

Protein Detection Issues in Western Blotting

Immunohistochemistry and Immunofluorescence Challenges

Co-immunoprecipitation Difficulties

Special Considerations for Mutant Protein Analysis

Antibody Validation Strategies

By implementing these targeted troubleshooting strategies, researchers can overcome technical challenges associated with RNF170 antibodies and generate more reliable and reproducible experimental results in both basic research and disease model applications.

How should researchers interpret RNF170 antibody data in the context of disease models and potential therapeutic applications?

Disease-Associated RNF170 Mutations: Pattern Recognition and Interpretation

When evaluating disease models:

• Compare antibody detection between patient-derived cells and controls

• Assess if protein level changes match mRNA expression patterns

• Determine if mutant proteins retain wild-type subcellular localization

• Evaluate potential compensatory mechanisms affecting expression

Pathway Analysis in Neurological Disease Models

When studying neurodegeneration mechanisms:

• IP3R-Ca2+ Signaling Axis:

  • Measure IP3R levels using western blotting alongside RNF170 detection

  • Quantify approximately 1.8-fold increase in IP3R levels in RNF170-deficient models

  • Assess calcium mobilization in response to stimuli in patient-derived cells

  • Determine if calcium store content, IP3 receptor levels, or IP3 production are altered

• Rescue Experiment Interpretation:

  • Evaluate if re-expression of wild-type RNF170 reverses IP3R accumulation

  • Assess whether calcium signaling defects are corrected by wild-type RNF170

  • Compare rescue efficiency between wild-type and functionally modified RNF170 constructs

Immune Pathway Analysis in RNF170-Deficient Models

For immunological research applications:

• TLR3-Mediated Innate Immune Responses:

  • Measure type I IFN and proinflammatory cytokine production in RNF170-deficient models

  • Assess specificity by comparing responses to different TLR ligands and viral stimuli

  • Determine if RNF170 deficiency selectively enhances TLR3-TRIF signaling without affecting RIG-I-MAVS signaling

• TLR3 Degradation Assessment:

  • Quantify TLR3 ubiquitination and degradation kinetics in presence/absence of RNF170

  • Verify if RNF170 promotes K48-linked polyubiquitination of K766 in TLR3

  • Monitor TLR3-RNF170 colocalization changes following stimulation

Therapeutic Implication Framework

Biomarker Development Considerations

RNF170 antibody data may inform biomarker development:

• Evaluate if RNF170 protein levels or modification patterns correlate with disease severity

• Determine if RNF170 substrate accumulation (e.g., IP3R) serves as surrogate marker

• Assess if disease-specific RNF170 protein conformations can be detected with conformation-specific antibodies

• Consider if RNF170-associated pathway components provide more robust biomarkers

By systematically applying these interpretation frameworks to RNF170 antibody data in disease contexts, researchers can generate insights that inform therapeutic development while avoiding oversimplification of complex disease mechanisms involving this multifunctional E3 ubiquitin ligase.

What emerging applications of RNF170 antibodies might advance our understanding of neurodegenerative mechanisms?

RNF170 antibodies are poised to drive advances in understanding neurodegenerative mechanisms through several innovative applications:

Spatio-temporal Mapping of RNF170 in Neurodegeneration Models

Advanced imaging techniques combined with RNF170 antibodies can provide unprecedented insights:

• Super-resolution microscopy with RNF170 antibodies can reveal nanoscale distribution changes in disease states

• CLARITY and expansion microscopy enable 3D visualization of RNF170 distribution in intact neural tissues

• Multiplexed immunofluorescence can simultaneously track RNF170, IP3R, and other interacting proteins in disease progression

These approaches could reveal how RNF170 distribution changes during disease progression in models of hereditary spastic paraplegia and sensory ataxia . Research shows that mutations in RNF170 lead to IP3R-1 accumulation and disrupted calcium signaling, but the spatial dynamics of these changes remain poorly understood .

Single-cell Analysis of RNF170-dependent Pathways

Emerging single-cell technologies with RNF170 antibodies can reveal cellular heterogeneity:

• Single-cell mass cytometry (CyTOF) with RNF170 antibodies can identify differential expression across neural cell populations

• Imaging mass cytometry can map RNF170 expression in tissue context with subcellular resolution

• Proximity ligation assays can detect RNF170-substrate interactions in situ with single-molecule sensitivity

These techniques could address whether specific neuronal populations are particularly vulnerable to RNF170 dysfunction, potentially explaining the selective neurodegeneration observed in the posterior columns of the spinal cord in ADSA patients .

Dynamic Monitoring of RNF170-mediated Protein Quality Control

Live-cell imaging approaches using RNF170 antibody-based sensors can track degradation dynamics:

• Split-fluorescent protein complementation systems using RNF170 antibody fragments to monitor substrate interactions

• Fluorescence resonance energy transfer (FRET) sensors to detect RNF170-substrate proximity

• Fluorescent timer fusion proteins to measure substrate half-life changes in RNF170 mutant backgrounds

This could provide real-time information about how disease-causing mutations affect the kinetics of substrate ubiquitination and degradation, particularly for IP3 receptors which accumulate to approximately 1.8-fold of normal levels in RNF170-deficient cells .

Investigating Non-canonical Functions of RNF170 in Neuronal Homeostasis

RNF170 antibodies can help identify previously uncharacterized functions:

• Proximity-based biotinylation (BioID or TurboID) combined with RNF170 antibodies to capture transient interactors

• Immunoprecipitation-mass spectrometry to identify novel neuronal substrates beyond IP3R

• Spatial proteomics to map RNF170 to specific neuronal compartments (axons, dendrites, synapses)

These approaches might reveal why mutations in an ubiquitously expressed protein like RNF170 lead to highly specific neurodegeneration patterns, potentially through tissue-specific substrates or functions.

Therapeutic Monitoring Applications

RNF170 antibodies can facilitate development and monitoring of therapeutic interventions:

• Pharmacodynamic biomarker development using RNF170 antibodies to track target engagement

• Nanobody-based intrabodies derived from RNF170 antibodies to stabilize mutant proteins in vivo

• In vivo optical imaging using labeled RNF170 antibodies to monitor brain-region specific changes

This could help develop therapies targeting the disrupted ionic interactions between charged residues in RNF170's transmembrane domains that are required for protein stability but are disrupted in disease-causing mutations .