Recombinant Xenopus tropicalis Probable E3 ubiquitin-protein ligase RNF144A (rnf144a)

What is RNF144A and what is its primary function in biological systems?

RNF144A is an E3 ubiquitin ligase belonging to the RING-between-RING (RBR) family of ubiquitin ligases. These proteins function as RING-HECT hybrid E3 ligases, which play crucial roles in protein ubiquitination and subsequent degradation. RNF144A primarily functions to catalyze the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins, targeting them for proteasomal degradation. The enzyme catalyzes ubiquitin linkages specifically at the K6-, K11-, and K48-positions of ubiquitin in vitro .

The biological significance of RNF144A lies in its role in DNA damage response pathways. Research has demonstrated that RNF144A is induced in a p53-dependent manner during DNA damage and targets cytosolic DNA-dependent protein kinase catalytic subunit (DNA-PKcs) for ubiquitination and degradation. This regulation is critical for proper apoptotic response during DNA damage, suggesting RNF144A may function as a tumor suppressor .

How is RNF144A evolutionarily conserved across species?

The RNF144A protein, particularly its transmembrane domain, shows significant evolutionary conservation across species. This high degree of conservation suggests fundamental importance to its biological function. The TM domain of RNF144A contains the GXXXG motif, which is preserved in five TM-containing RBR E3 ligases, including RNF144A, RNF144B, RNF19A/Dorfin, RNF19B, and RNF217 .

In terms of subfamily relationships, RNF144A is most closely related to RNF144B at the protein level. Together, these two proteins comprise a distinct subdomain within the larger RBR family of proteins. This conservation extends to their functional mechanisms, as studies have shown that RNF144B also self-associates, suggesting a common regulatory principle within this subfamily .

The Xenopus tropicalis RNF144A shares key structural and functional domains with human RNF144A, making the amphibian protein a valuable model for studying the fundamental properties of this E3 ligase. The conservation of critical residues involved in zinc coordination, E2 binding, and self-association suggests mechanistic similarities in their enzymatic functions across vertebrate species.

What are the known substrates and interacting partners of RNF144A?

RNF144A has been identified to target several key proteins for ubiquitination and subsequent degradation, most of which are involved in DNA repair, heat shock/chaperone function, and cellular signaling pathways. The current known substrates include:

The interaction between RNF144A and its substrates is typically mediated by its RING finger domain, which recognizes specific structural features on target proteins. For efficient ubiquitination activity, RNF144A requires both proper membrane localization (via its TM domain) and self-association capability .

What role does the transmembrane domain play in RNF144A function?

The transmembrane (TM) domain of RNF144A serves dual critical functions that significantly impact its enzymatic activity and cellular localization:

• Membrane Localization: The TM domain anchors RNF144A to cellular membranes, particularly to endosomal structures. Deletion of the TM domain abolishes membrane localization .

• E3 Ligase Activation: The TM domain is required for optimal ubiquitin ligase activity. Experimental evidence shows that deletion of this domain significantly reduces RNF144A's enzymatic function .

• Self-Association Mediation: The TM domain facilitates RNF144A self-association through a classic GXXXG interaction motif (specifically G252XXXG256 in human RNF144A). This self-association is critical for its ubiquitin ligase activity .

Mutations affecting the GXXXG motif demonstrate the dual but independent roles of this domain:

• RNF144A-G252L/G256L mutant: Preserves membrane localization but is defective in self-association and ubiquitin ligase activity

• RNF144A-G252D mutant (found in human cancers): Retains self-association and ligase activity but loses membrane localization and undergoes rapid turnover

These findings underscore the importance of both proper membrane localization and self-association for optimal RNF144A function, suggesting a sophisticated regulatory mechanism that may apply broadly to other RBR family E3 ligases.

What experimental approaches are most effective for studying RNF144A enzymatic activity?

For comprehensive investigation of RNF144A enzymatic activity, researchers should consider the following experimental approaches:

In vitro ubiquitination assays:

• Purified recombinant RNF144A can be used in a reconstituted system with E1, E2 enzymes, ubiquitin, ATP, and substrate proteins

• Analysis should include immunoblotting for ubiquitinated products and mass spectrometry to identify ubiquitination sites and chain types (K6, K11, K48 linkages)

Structural characterization:

• NMR spectroscopy has proven effective for determining the solution structure of the RING finger domain of RNF144A

• Zinc binding stoichiometry can be determined using metallochromic indicators to confirm proper folding and activity

• Thermal unfolding curves (measuring tryptophan fluorescence emission) can assess protein stability of wild-type versus mutant proteins

Transmembrane domain analysis:

• Deletion and point mutation studies of the TM domain (particularly the GXXXG motif) provide insights into the dual roles in membrane localization and catalytic activation

• Subcellular fractionation and confocal microscopy to track membrane localization

• Co-immunoprecipitation assays to analyze self-association properties

Substrate identification and validation:

• Affinity purification coupled with mass spectrometry (AP-MS)

• Proximity-dependent biotin identification (BioID) methods

• Validation through direct ubiquitination assays with recombinant substrates

• Cell-based degradation assays comparing wild-type RNF144A with catalytically inactive mutants

When working with recombinant Xenopus tropicalis RNF144A specifically, researchers should ensure proper expression systems (typically E. coli for full-length protein with N-terminal His-tag) and appropriate storage conditions (avoiding repeated freeze-thaw cycles). Reconstitution in deionized sterile water to 0.1-1.0 mg/mL with 5-50% glycerol is recommended for long-term storage at -20°C/-80°C .

How does the GXXXG motif in RNF144A contribute to its self-association and functional regulation?

The GXXXG motif in RNF144A's transmembrane domain represents a classical helix-helix interaction motif that plays a crucial role in protein self-association and functional regulation. Research has revealed several key insights:

Molecular mechanism of GXXXG-mediated self-association:

• The GXXXG motif (specifically G252XXXG256 in human RNF144A) creates a smooth surface on transmembrane helices that facilitates close packing of adjacent helices

• This close packing enables van der Waals interactions and potential hydrogen bonding between transmembrane helices, promoting dimerization or higher-order oligomerization

• Mutation studies demonstrate that substituting these glycine residues with bulkier amino acids (G252L/G256L) disrupts self-association while preserving membrane localization

Functional consequences of self-association:

• Self-association through the GXXXG motif is required for optimal E3 ligase activity

• This requirement appears to be independent of membrane localization, as membrane localization-deficient mutants can retain self-association and E3 activity

• When both properties are compromised (through combined mutations), enzyme activity is severely impaired

Cancer-associated mutations:

• Mutations of GXXXG motifs in RNF144A have been found in human cancers, including a G252D mutation

• The G252D mutation preserves self-association and ubiquitin ligase activity but causes loss of membrane localization

• This mutant is turned over rapidly, suggesting that proper membrane localization is important for RNF144A stability in cells

This regulatory mechanism may represent a common feature across the RBR E3 ligase family, as all five TM-containing RBR E3 ligases (RNF144A, RNF144B, RNF19A/Dorfin, RNF19B, and RNF217) contain the RBR-TM(GXXXG) superstructure . These findings highlight a sophisticated layer of regulation where both proper subcellular localization and quaternary structure are required for optimal enzymatic function.

What methods are most effective for expression and purification of functional recombinant RNF144A?

Successful expression and purification of functional recombinant RNF144A requires careful consideration of several experimental parameters:

Expression systems and conditions:

• E. coli expression: The full-length Xenopus tropicalis RNF144A has been successfully expressed in E. coli with an N-terminal His-tag

• Tagged constructs: N-terminal His-tagging appears to preserve enzymatic function while facilitating purification

• Domain-specific considerations: For structural studies of the RING finger domain alone, bacterial expression systems with optimized conditions for zinc incorporation are recommended

Purification strategy:

• Affinity chromatography using Ni-NTA resins for His-tagged proteins

• Size exclusion chromatography to ensure monomeric/dimeric state and remove aggregates

• Ion exchange chromatography for further purification if needed

• Quality control by SDS-PAGE (>90% purity recommended)

Buffer optimization and protein stability:

• Final storage in Tris/PBS-based buffer with 6% Trehalose, pH 8.0

• Addition of 5-50% glycerol for long-term storage (50% being optimal)

• Lyophilization for extended stability

• Avoid repeated freeze-thaw cycles by preparing working aliquots stored at 4°C for up to one week

Reconstitution protocol:

• Brief centrifugation of vial prior to opening

• Reconstitution in deionized sterile water to 0.1-1.0 mg/mL

• Addition of glycerol to 50% final concentration

• Aliquoting for long-term storage at -20°C/-80°C

Functional validation assays:

• In vitro ubiquitination assays to confirm catalytic activity

• Thermal stability assessments via fluorescence spectroscopy

• Zinc content verification using metallochromic indicators

• Structural integrity confirmation via circular dichroism or NMR spectroscopy

For researchers working specifically with transmembrane domain mutants, special attention should be paid to detergent selection during purification to maintain proper folding and self-association properties of the protein.

How does RNF144A contribute to DNA damage response and apoptotic pathways?

RNF144A plays a sophisticated role in balancing DNA repair and apoptotic responses following genotoxic stress:

Induction mechanism:

• RNF144A expression is induced in a p53-dependent manner following DNA damage

• This induction represents part of the cellular DNA damage response (DDR) program

• The temporal regulation of RNF144A suggests it functions as a molecular switch between DNA repair and apoptotic pathways

DNA-PKcs regulation:

• DNA-PKcs (DNA-dependent protein kinase catalytic subunit) is a critical component of the non-homologous end joining (NHEJ) DNA repair pathway

• Beyond its nuclear functions, cytosolic DNA-PKcs exhibits pro-survival activity

• RNF144A specifically targets cytosolic DNA-PKcs for ubiquitination and degradation

• This degradation helps shift cellular responses from survival to apoptosis when DNA damage is extensive

Apoptotic promotion mechanism:

• By degrading pro-survival factors like DNA-PKcs, RNF144A promotes apoptotic responses

• This function is particularly important for eliminating cells with extensive DNA damage

• The process helps maintain genomic integrity at the organismal level by preventing propagation of cells with damaged DNA

Subcellular localization significance:

• RNF144A localizes to endosomal membranes via its transmembrane domain

• This localization appears critical for its function in DNA damage response

• Cancer-associated mutations that disrupt membrane localization (e.g., G252D) impair RNF144A stability and presumably its tumor suppressor function

Tumor suppressor implications:

• The role of RNF144A in promoting appropriate apoptotic responses to DNA damage suggests tumor suppressor functions

• Somatic mutations of RNF144A have been cataloged in cancer genetic databases across multiple tumor types, including breast, stomach, lymphoma, glioblastoma, uterine, and lung cancers

• These observations position RNF144A as a potentially important regulator of genomic integrity whose dysfunction may contribute to tumorigenesis

How does Xenopus tropicalis RNF144A compare with human RNF144A in structure and function?

Comparing Xenopus tropicalis RNF144A with its human counterpart reveals important insights about evolutionary conservation and species-specific adaptations:

Structural comparison:

• Domain architecture: Both Xenopus and human RNF144A maintain the characteristic RBR (RING1-IBR-RING2) domain arrangement and transmembrane domain

• Transmembrane region: The TM domain is highly conserved across species, highlighting its fundamental importance to function

• GXXXG motif: This self-association motif within the TM domain is preserved, suggesting conservation of self-association mechanisms

• Zinc-binding: Both proteins coordinate two zinc atoms within their RING domains in a cross-braced arrangement

Sequence homology:
The Xenopus tropicalis RNF144A protein (292 amino acids) shares significant sequence homology with human RNF144A, particularly in functional domains. Key residues involved in zinc coordination, E2 binding, and substrate recognition are conserved, suggesting mechanistic similarities in their enzymatic functions.

Functional conservation:

Experimental considerations for cross-species studies:

• Xenopus tropicalis RNF144A serves as a valuable model for studying fundamental properties of this E3 ligase

• When using recombinant Xenopus RNF144A for studies on human substrates, researchers should consider potential species-specific interaction differences

• Complementation studies in human cell lines using Xenopus RNF144A can help determine functional equivalence

• Structural studies of Xenopus RNF144A may provide insights applicable to the human protein due to high conservation of key domains

This cross-species conservation makes Xenopus tropicalis RNF144A a valuable research tool, particularly for structural and biochemical studies that require recombinant protein expression. The successful expression of full-length Xenopus tropicalis RNF144A in E. coli systems may offer advantages over human RNF144A for certain experimental applications.

What are the optimal storage and handling conditions for recombinant RNF144A?

Proper storage and handling of recombinant RNF144A is critical for maintaining protein stability and enzymatic activity. Based on empirical data, the following guidelines should be observed:

Storage conditions:

• Store lyophilized powder at -20°C/-80°C upon receipt

• For reconstituted protein, store at -20°C/-80°C with 5-50% glycerol (50% being optimal)

• Working aliquots can be stored at 4°C for up to one week

• Repeated freeze-thaw cycles should be strictly avoided as they significantly impact protein stability and activity

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% recommended)

• Prepare multiple small aliquots to minimize freeze-thaw cycles

Buffer composition:

• Optimal storage buffer: Tris/PBS-based buffer, 6% Trehalose, pH 8.0

• For activity assays, buffers containing divalent cations (particularly zinc) may be necessary to maintain proper folding of the RING domain

Quality control considerations:

• Verify protein integrity by SDS-PAGE before experimental use (>90% purity)

• Activity can be assessed through in vitro ubiquitination assays

• Thermal stability can be monitored through fluorescence-based thermal shift assays

• For structural integrity, consider limited trypsin digestion to confirm proper folding

These handling guidelines are particularly important when working with full-length RNF144A containing the transmembrane domain, as this hydrophobic region can contribute to aggregation if improperly handled.

What experimental controls are essential when studying RNF144A ubiquitin ligase activity?

Rigorous experimental design for studying RNF144A activity requires several key controls:

Positive and negative enzyme controls:

• Catalytically inactive mutant: RING domain mutations that disrupt E2 binding or zinc coordination serve as negative enzymatic controls

• Transmembrane domain mutants: G252L/G256L mutations (disrupting self-association) provide partial activity controls

• Domain deletion constructs: TM domain deletion variants show reduced but not eliminated activity

• Known active E3 ligase: Include a well-characterized E3 ligase (e.g., CHIP or Parkin) as positive control

Substrate specificity controls:

• Non-substrate proteins: Include structurally similar proteins not targeted by RNF144A

• Binding-deficient substrate mutants: Modify substrate recognition regions to confirm specificity

• Competition assays: Use excess untagged substrate to compete with tagged substrate

Ubiquitination reaction controls:

• ATP dependence: Reactions without ATP demonstrate energy requirement

• E1/E2 dependence: Omitting either E1 or E2 enzymes confirms the complete ubiquitination cascade

• Ubiquitin mutants: Lysine-to-arginine ubiquitin mutants help determine chain type specificity (K6R, K11R, K48R)

• Deubiquitinating enzyme: Addition of a DUB confirms reversibility and specificity of ubiquitin linkages

Cellular localization controls:

• Subcellular fractionation quality controls: Marker proteins for membrane vs. cytosolic fractions

• Localization mutants: TM domain mutants that alter membrane association

• Membrane disruption: Detergent treatments to disrupt membrane associations

Experimental setup table:

Implementation of these controls ensures reliable data interpretation and minimizes experimental artifacts when studying this complex E3 ubiquitin ligase.

How can researchers address the challenges of working with transmembrane-containing E3 ligases like RNF144A?

Working with transmembrane-containing E3 ligases like RNF144A presents unique challenges that require specific experimental strategies:

Expression and purification challenges:

• Solubilization approaches: Use mild detergents (CHAPS, DDM, or digitonin) that preserve native membrane protein structure

• Fusion tag strategies: N-terminal solubility-enhancing tags (MBP, SUMO) coupled with C-terminal purification tags can improve expression

• Cell-free expression systems: Consider membrane-mimetic environments for direct expression into liposomes or nanodiscs

• Insect cell expression: For mammalian proteins, baculovirus expression systems may provide better folding environments than bacterial systems

Functional analysis in membrane contexts:

• Nanodisc reconstitution: Incorporate purified protein into defined lipid nanodiscs for activity studies

• Liposome-based assays: Assess activity in artificial membrane systems with defined composition

• Native membrane isolation: Study the protein in its natural membrane environment through careful subcellular fractionation

• Detergent screening: Systematically evaluate detergent effects on self-association and activity

Self-association assessment techniques:

• Chemical crosslinking: Membrane-permeable crosslinkers can capture transient interactions

• Fluorescence resonance energy transfer (FRET): Tag variants with fluorescent proteins to measure association in live cells

• Co-immunoprecipitation from solubilized membranes: Preserve interactions through careful solubilization

• Analytical ultracentrifugation: Determine oligomeric states in detergent solutions

Mutational analysis strategies:

• Transmembrane domain chimeras: Replace TM domain with alternative TM domains to assess specificity

• Targeted GXXXG motif mutations: Distinguish self-association from membrane localization effects

• Domain-swapping experiments: Exchange domains between RNF144A and RNF144B to identify family-specific functions

Technical troubleshooting approaches:

These strategies enable researchers to overcome the inherent difficulties of working with membrane-associated E3 ligases, facilitating more comprehensive functional and mechanistic studies of RNF144A.

What are the emerging research areas for RNF144A in disease models and therapeutic development?

The current understanding of RNF144A suggests several promising research directions with potential therapeutic implications:

Cancer biology and therapeutic targeting:

• Tumor suppressor validation: Further investigation into RNF144A's proposed tumor suppressor function across different cancer types

• Mutation profiling: Comprehensive analysis of RNF144A mutations in cancer genomic databases to identify potential driver mutations

• Synthetic lethality approaches: Identification of genes showing synthetic lethality with RNF144A deficiency in cancer cells

• Small molecule modulators: Development of compounds that could stabilize or enhance RNF144A activity as potential cancer therapeutics

DNA damage response pathway integration:

• Pathway mapping: Detailed characterization of how RNF144A integrates with other DNA damage response factors

• Resistance mechanisms: Investigation of RNF144A's role in resistance to DNA-damaging chemotherapeutics

• Cell fate determination: Further elucidation of how RNF144A influences the balance between DNA repair and apoptosis

Comparative biology using Xenopus tropicalis as a model organism:

• Developmental roles: Investigation of RNF144A function during embryonic development in Xenopus

• Tissue-specific functions: Analysis of tissue-specific expression patterns and functions in the amphibian model

• Evolutionary insights: Comparative studies across vertebrate species to understand conserved and divergent functions

Structural biology and drug design opportunities:

• Complete structure determination: Resolution of full-length RNF144A structure including the transmembrane domain

• E2-RNF144A interface: Detailed mapping of the interaction interface between RNF144A and its cognate E2 enzymes

• Allosteric regulation sites: Identification of potential druggable sites that could modulate RNF144A activity

• Structure-based design: Development of peptides or small molecules targeting specific RNF144A functional domains

Expanded substrate identification:

• Proteomics approaches: Global ubiquitinome analysis in cells with RNF144A overexpression or knockout

• Substrate recognition motifs: Determination of consensus sequences or structural elements recognized by RNF144A

• Substrate network analysis: Systems biology approaches to understand the broader impact of RNF144A activity on cellular proteostasis

These research directions highlight the multifaceted potential of RNF144A as both a fundamental biological regulator and a possible therapeutic target, with Xenopus tropicalis RNF144A serving as a valuable research tool in advancing our understanding of this important E3 ligase.

How might cross-species conservation studies of RNF144A contribute to understanding its fundamental biological roles?

Cross-species comparative analysis of RNF144A offers powerful insights into its core biological functions and evolutionary significance:

Evolutionary conservation analysis:

• Phylogenetic profiling: Systematic comparison of RNF144A across vertebrate species can reveal selection pressures on specific domains

• Domain conservation mapping: Identification of highly conserved vs. variable regions provides clues to functional importance

• Species-specific adaptations: Analysis of species-specific sequence variations may reveal specialized functions in different organisms

Functional conservation testing:

• Cross-species complementation: Testing whether Xenopus tropicalis RNF144A can rescue phenotypes in human cell lines with RNF144A deficiency

• Substrate conservation: Determining whether substrates identified in one species (e.g., human DNA-PKcs) are also targeted by RNF144A orthologs from other species

• Regulatory mechanism comparison: Analysis of whether the GXXXG-mediated self-association mechanism is conserved across species

Developmental biology insights:

• Xenopus embryonic expression patterns: Characterization of spatial and temporal expression during development

• Functional studies in developing embryos: CRISPR-mediated knockout or morpholino knockdown in Xenopus embryos

• Cellular differentiation roles: Investigation of potential roles in cell fate decisions during development

Structural comparison approaches:

• Solution structure comparison: NMR studies comparing the RING domain structures across species

• Transmembrane domain analysis: Comparative studies of TM domain properties from different species

• Species-specific post-translational modifications: Identification of conserved and variable modification sites

Translational research applications:

• Model system selection guidance: Information on which species most closely resembles human RNF144A function

• Conserved regulatory networks: Identification of evolutionarily conserved pathways regulated by RNF144A

• Drug target validation: Cross-species conservation of binding sites can inform therapeutic development efforts

This evolutionary perspective not only enhances our fundamental understanding of RNF144A biology but also helps distinguish essential functions from species-specific adaptations, ultimately improving translational research efforts and therapeutic development strategies.

What are the key considerations for researchers beginning work with recombinant RNF144A?

Researchers initiating studies with recombinant RNF144A should consider the following key points to ensure successful experimental outcomes:

Protein expression and purification considerations:

• Select appropriate expression systems: E. coli works well for Xenopus tropicalis RNF144A with N-terminal His-tagging

• Pay careful attention to buffer composition and storage conditions: Tris/PBS-based buffer with 6% Trehalose, pH 8.0, with 50% glycerol for long-term storage

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Verify protein quality by SDS-PAGE (>90% purity recommended) before experimental use

Functional activity assessments:

• Include appropriate controls in all ubiquitination assays

• Consider both membrane localization and self-association in experimental design

• For structure-function studies, incorporate both wild-type and mutant proteins (particularly GXXXG motif mutants)

• Validate activity in both in vitro reconstituted systems and cellular contexts

Technical challenges awareness:

• Recognize the challenges associated with the transmembrane domain

• Consider detergent effects on protein activity and self-association

• Monitor zinc content and protein folding for optimal enzymatic activity

• Design experiments that account for RNF144A's dual functions in membrane localization and E3 ligase activity

Biological context integration:

• Consider RNF144A's role in DNA damage response pathways

• Explore potential tumor suppressor functions in appropriate experimental systems

• Investigate interactions with known substrates (DNA-PKcs, PARP1, HSPA2, BMI1, RAF1)

• Design experiments that address both basic mechanisms and disease relevance

By addressing these considerations, researchers can establish robust experimental systems for investigating this fascinating E3 ubiquitin ligase and contribute to our understanding of its biological functions and potential therapeutic applications.

How does current research on RNF144A inform our understanding of the broader RBR E3 ligase family?

Research on RNF144A provides significant insights into the broader RBR E3 ligase family, illuminating common mechanistic principles and regulatory features:

Shared structural and functional mechanisms:

• RNF144A exemplifies the RING-HECT hybrid mechanism characteristic of RBR ligases

• Studies on RNF144A's zinc coordination in its RING domain inform structural understanding of other family members

• The self-association regulatory mechanism identified in RNF144A may apply to other RBR ligases

Transmembrane domain significance:

• Five RBR E3 ligases contain transmembrane domains with GXXXG motifs (RNF144A, RNF144B, RNF19A/Dorfin, RNF19B, and RNF217)

• The dual role of this domain in membrane localization and E3 ligase activation likely represents a conserved regulatory principle

• This suggests a common evolutionary adaptation for membrane-associated ubiquitination functions

Disease relevance patterns:

• Like RNF144A, other RBR family members have been implicated in human diseases

• Parkin (another RBR ligase) is associated with Parkinson's disease

• HOIL-1L and HOIP(RNF31) are linked to immunological disorders

• This suggests a broader pattern of RBR ligases as critical regulators of cellular homeostasis

Substrate recognition principles:

• Insights from RNF144A substrate targeting may inform understanding of how other RBR ligases select their targets

• The combination of RING domain substrate recognition with HECT-like catalytic mechanism represents a unique feature of this enzyme family

• Cross-comparison of substrates across the family may reveal shared recognition principles

Therapeutic development implications:

• Strategies developed for targeting or modulating RNF144A may be applicable to other RBR family members

• Understanding the regulatory mechanisms of RNF144A provides a conceptual framework for approaching other RBR ligases

• The dual membrane localization/self-association paradigm suggests multiple potential intervention points for therapeutic development