RASSF3 Antibody, HRP conjugated

What is RASSF3 and why is it significant in research?

RASSF3 (Ras Association Domain-containing protein 3) belongs to the RASSF family of proteins that primarily function as tumor suppressors. The significance of RASSF3 stems from its role as a direct effector of mitochondrial GTPases MIRO1 and MIRO2, which are crucial for mitochondrial homeostasis, trafficking, and PINK1/PARKIN-mediated mitophagy . Unlike some RASSF family members that interact with growth-promoting RAS GTPases, RASSF3 primarily interacts with growth inhibitory RAS GTPases including RASD2, RASL10B, and REM2 . This distinctive binding profile suggests RASSF3 may have unique functions in cellular signaling networks, making it an important target for cancer research, cell biology studies, and investigations into mitochondrial dysfunction.

What are the optimal applications for RASSF3 antibodies in research?

Based on available data, RASSF3 antibodies are primarily optimized for Western Blotting (WB) applications . Different RASSF3 antibodies may be suitable for additional techniques - for instance, some commercially available RASSF3 antibodies are reported to work in ELISA and immunohistochemistry (IHC) . For investigating RASSF3's interactions with GTPases, pull-down assays using GST-tagged RA domains have proven effective in research settings . When selecting a RASSF3 antibody, researchers should consider the specific experimental application, the species reactivity required (human, mouse, and rat reactivity is reported for some RASSF3 antibodies ), and whether direct detection (HRP-conjugated) or indirect detection (unconjugated) is more suitable for the experimental design.

What is the recommended protocol for using RASSF3 Antibody, HRP conjugated in Western Blotting?

For Western blotting with RASSF3 Antibody, HRP conjugated, a methodological approach based on standard protocols and available data would include:

• Sample Preparation: Prepare cell/tissue lysates in an appropriate buffer (e.g., buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2% glycerol, 1% Triton X-100, 1% NP-40, 1 mM DTT, and protease inhibitors) .

• Protein Separation: Separate proteins using SDS-PAGE. Given RASSF3's molecular weight of ~28 kDa , a 12-15% gel would provide optimal separation.

• Transfer: Transfer proteins to nitrocellulose or PVDF membrane using standard conditions (research protocols have used nitrocellulose successfully) .

• Blocking: Block the membrane with 5% skim milk in TBST (or 3% BSA for phospho-sensitive applications) for 1 hour at room temperature.

• Primary Antibody Incubation: Dilute the HRP-conjugated RASSF3 antibody (typically 1:1000 based on similar HRP-conjugated antibodies) in blocking buffer and incubate overnight at 4°C or 1-2 hours at room temperature.

• Washing: Wash the membrane 3-5 times with TBST to remove unbound antibody.

• Detection: Apply ECL substrate directly (no secondary antibody required) and detect signal using a digital imaging system .

Important methodological considerations include avoiding sodium azide in buffers (it inhibits HRP activity) and optimizing antibody concentration and incubation time for your specific experimental system.

How can researchers verify the specificity of RASSF3 Antibody in experimental systems?

Verifying antibody specificity is crucial for reliable experimental outcomes. A comprehensive methodological approach includes:

• Molecular Weight Verification: Confirm that the detected band appears at the expected molecular weight of ~28 kDa for RASSF3 .

• Knockdown/Knockout Validation: Compare antibody signal between wild-type samples and RASSF3 knockdown (siRNA/shRNA) or knockout (CRISPR-Cas9) samples. The specific band should be significantly reduced or absent in knockdown/knockout samples.

• Overexpression Studies: Compare signal between control samples and those overexpressing RASSF3, expecting enhanced signal in overexpression samples.

• Peptide Competition: Pre-incubate the antibody with the immunizing peptide (if available - some RASSF3 antibodies use "a synthetic peptide corresponding to residues in Human RASSF3" as immunogen ), which should abolish specific binding.

• Cross-Reactivity Assessment: Test the antibody against other RASSF family members to ensure specificity within this protein family.

• Positive Control Samples: Include samples known to express RASSF3 (based on literature or validated by other methods).

These validation steps are especially important when working with antibodies for proteins like RASSF3 that have multiple family members with structural similarities.

What are the expected results and potential pitfalls when detecting RASSF3 in different sample types?

When detecting RASSF3 across different experimental samples, researchers should anticipate:

Expected Results:

• A distinct band at approximately 28 kDa in Western blots

• Stronger signals in tissues/cells known to express RASSF3

• Potential co-localization with mitochondrial markers given RASSF3's interaction with mitochondrial GTPases MIRO1/2

Sample-Specific Considerations:

• Cell Lines: Expression levels may vary significantly between cell types. Cancer cell lines might show altered RASSF3 expression compared to normal cells given its potential tumor suppressor role.

• Tissue Samples: Require efficient extraction methods to preserve protein integrity. Fresh or properly stored frozen samples yield better results than formalin-fixed materials for Western blotting.

• Subcellular Fractions: Given RASSF3's interaction with mitochondrial GTPases , enrichment in mitochondrial fractions might be expected.

Potential Pitfalls:

• Low Endogenous Expression: RASSF3 may be expressed at low levels in some tissues, requiring sensitive detection methods.

• Cross-Reactivity: Possible cross-reactivity with other RASSF family members, necessitating careful antibody validation.

• Post-Translational Modifications: These may affect antibody recognition or cause band shifts from the expected molecular weight.

• Degradation Products: Improper sample handling may lead to degradation bands below the expected molecular weight.

To address these challenges, researchers should include appropriate positive controls and optimize protein extraction and detection protocols for their specific experimental system.

How does RASSF3 interact with GTPases and what implications does this have for experimental design?

RASSF3 exhibits distinct GTPase interaction patterns that differ from other RASSF family members, with significant implications for experimental design:

Key Interaction Characteristics:

• RASSF3 directly binds to mitochondrial GTPases MIRO1 and MIRO2 through its RA (Ras Association) domain

• Unlike some GTPase interactions (e.g., RASSF5-KRAS), RASSF3-MIRO binding is nucleotide-independent, occurring regardless of whether GTPases are loaded with GDP, GTP, or GTP analogs

• RASSF3 interacts with growth inhibitory RAS GTPases including RASD2, RASL10B, and REM2

• RASSF3 also shows interaction with selected RHO subfamily GTPases (RHOBTB1, RHOBTB2, RHOH, RHOF) and certain ARF subfamily members (ARL4C, ARL11, ARL5C)

Methodological Implications:

• Pull-down Assay Design: When designing pull-down experiments for RASSF3-GTPase interactions, nucleotide loading (GDP vs. GTP) may not be critical, unlike with other GTPase studies .

• Protein Domain Analysis: The RA domain of RASSF3 is sufficient for MIRO GTPase binding in pull-down assays, informing construct design for interaction studies .

• Subcellular Localization Studies: Given MIRO1/2 are mitochondrial GTPases, co-localization studies should examine RASSF3 in relation to mitochondrial markers.

• Functional Studies: Research designs should consider RASSF3's potential role in mitochondrial homeostasis, trafficking, and PINK1/PARKIN-mediated mitophagy pathways .

• Specificity Controls: When studying RASSF3-GTPase interactions, other RASSF family members (particularly RASSF5) serve as informative controls due to their different GTPase binding profiles .

These characteristics make RASSF3 a unique member of the RASSF family and suggest methodological approaches that differ from those used with other RASSF proteins.

What methodological approaches can be used to study RASSF3's role in mitochondrial function?

Given RASSF3's direct interaction with mitochondrial GTPases MIRO1 and MIRO2 , several methodological approaches can elucidate its role in mitochondrial function:

1. Mitochondrial Co-localization Studies:

• Immunofluorescence microscopy using validated RASSF3 antibodies alongside mitochondrial markers

• Live-cell imaging with fluorescently-tagged RASSF3 and mitochondrial markers

• Super-resolution microscopy to determine precise spatial relationships

2. Biochemical Fractionation:

• Isolate mitochondrial, cytosolic, and other subcellular fractions

• Western blotting with RASSF3 antibodies to determine enrichment in mitochondrial fractions

• Protease protection assays to determine topology of RASSF3 at mitochondria

3. Functional Mitochondrial Assays:

• Measure mitochondrial membrane potential using fluorescent dyes after RASSF3 manipulation

• Assess mitochondrial morphology through microscopy after RASSF3 knockdown/overexpression

• Oxygen consumption rate and extracellular acidification rate measurements using Seahorse analyzer

4. Mitophagy Assessment:

• Monitor mitophagy markers (PINK1, Parkin recruitment) in relation to RASSF3 levels

• Use mitophagy-inducing conditions to test RASSF3's involvement

• Track mitochondrial clearance using pH-sensitive mitochondrial markers

5. MIRO1/2 Interaction Studies:

• Investigate whether RASSF3-MIRO interaction affects MIRO's known functions in mitochondrial movement

• Perform mitochondrial trafficking assays in neurons or other relevant cell types

• Use RASSF3 mutants defective in MIRO binding to determine functional consequences

6. Proximity-Based Protein Labeling:

• BioID or APEX2 fusion proteins to identify proteins in proximity to RASSF3 at mitochondria

• Cross-linking mass spectrometry to capture direct interaction partners

These methodological approaches, used in combination, can provide comprehensive insights into RASSF3's role in mitochondrial biology and how this relates to its tumor suppressor functions.

How can researchers design experiments to investigate differential functions between RASSF3 and other RASSF family members?

Designing experiments to differentiate RASSF3 functions from other RASSF family members requires methodological approaches that leverage their known differences in interaction partners and regulation:

1. Comparative GTPase Interaction Profiling:

• Perform parallel pull-down assays using RA domains from different RASSF proteins

• Create a quantitative interaction map as demonstrated in the research literature

• Compare RASSF3's interaction with growth-inhibitory GTPases against RASSF5's binding to growth-promoting GTPases

2. Domain-Swap Experiments:

• Generate chimeric proteins containing RA domains from different RASSF proteins

• Test whether the RA domain alone determines GTPase binding specificity

• Assess functional consequences of domain swapping

3. Cellular Localization Studies:

• Compare subcellular localization patterns of different RASSF family members

• Test co-localization with mitochondria, plasma membrane, and other organelles

• Examine how localization changes upon various cellular stimuli

4. Signaling Pathway Analysis:

• Compare effects of different RASSF members on common downstream pathways (e.g., Hippo)

• Perform phosphoproteomic analysis after manipulation of specific RASSF proteins

• Use pathway inhibitors to determine which RASSF-mediated effects depend on specific signaling cascades

5. Cell-Type Specificity Investigation:

• Compare expression patterns of RASSF family members across tissues and cell types

• Test functional consequences of manipulating different RASSF members in multiple cell types

• Identify cell types where RASSF3 has distinctive roles compared to other family members

6. Cancer Context Differentiation:

• Compare effects of different RASSF proteins on hallmarks of cancer (proliferation, apoptosis, etc.)

• Analyze cancer genomic databases for distinctive patterns of alteration in different RASSF genes

• Test tumor suppressor capabilities in cellular and animal models

These methodological approaches provide a comprehensive framework for distinguishing RASSF3's functions from other family members, leveraging its unique GTPase interaction profile and potential mitochondrial roles.

What are common issues when using HRP-conjugated antibodies for RASSF3 detection and how can they be resolved?

When using HRP-conjugated antibodies for RASSF3 detection, researchers may encounter several technical challenges that require methodological solutions:

1. High Background Signal:

• Cause: Insufficient blocking, excess antibody concentration, or non-specific binding

• Solution: Optimize blocking conditions (try 5% milk, 3-5% BSA, or commercial blockers); increase antibody dilution; add 0.1-0.5% Tween-20 to washing buffers; extend washing time and frequency

2. Weak or No Signal:

• Cause: Low RASSF3 expression, antibody degradation, or inefficient transfer

• Solution: Increase protein loading (RASSF3 may be expressed at low levels); verify transfer efficiency with Ponceau S staining; reduce antibody dilution; use enhanced chemiluminescence substrates; ensure proper storage of HRP-conjugated antibody at -20°C in small aliquots to prevent repeated freeze-thaw cycles

3. Multiple Bands or Non-specific Signals:

• Cause: Cross-reactivity with other RASSF family members, degradation products, or post-translational modifications

• Solution: Validate antibody specificity using knockout/knockdown controls; include positive control samples with known RASSF3 expression; optimize protein extraction to minimize degradation by using fresh protease inhibitors

4. Inconsistent Results:

• Cause: Variable sample preparation, inconsistent transfer, or antibody degradation

• Solution: Standardize lysate preparation protocols; use internal loading controls; prepare fresh working solutions of antibody; consider using a densitometry system like ImageLab for quantification

5. HRP-Specific Issues:

• Cause: HRP inactivation by sodium azide or other inhibitors

• Solution: Ensure all buffers used with HRP-conjugated antibodies are free of sodium azide (note that some commercial antibodies contain sodium azide in storage buffer ); use freshly prepared detection reagents; optimize exposure time during imaging

These troubleshooting approaches should be systematically applied when optimizing RASSF3 detection with HRP-conjugated antibodies, with careful documentation of each modification to the protocol.

What methodological approaches can optimize detection of low-abundance RASSF3 in complex samples?

Detecting low-abundance proteins like RASSF3 in complex samples requires specialized methodological approaches:

1. Sample Enrichment Techniques:

• Immunoprecipitation: Concentrate RASSF3 from large sample volumes prior to Western blotting

• Subcellular Fractionation: Enrich for mitochondrial fractions where RASSF3 may be concentrated due to its MIRO1/2 interaction

• Protein Fractionation: Use techniques like ammonium sulfate precipitation or ion exchange chromatography to concentrate proteins in the relevant molecular weight range

2. Enhanced Western Blotting Methods:

• High-Sensitivity Substrates: Use femto-level ECL substrates specifically designed for detecting low-abundance proteins

• Signal Amplification: Apply tyramide signal amplification systems compatible with HRP

• Membrane Selection: Use low-fluorescence PVDF membranes for lower background and higher protein retention

• Optimized Transfer: Use wet transfer with optimized buffer systems for efficient transfer of ~28 kDa proteins

3. Alternative Detection Methods:

• Capillary Western: Systems like Wes (ProteinSimple) can detect proteins at lower levels than traditional Western blotting

• Mass Spectrometry: Use targeted MS approaches like selected reaction monitoring (SRM) for sensitive, specific detection

• Proximity Ligation Assay: For in situ detection of RASSF3 and its interaction partners with single-molecule sensitivity

4. Data Analysis Optimization:

• Multi-Exposure Analysis: Capture multiple exposure times to ensure detection within the linear range

• Image Enhancement: Use specialized software with background subtraction and signal normalization

• Replicate Analysis: Increase biological and technical replicates to improve statistical confidence in low-signal detection

5. Experimental Controls:

• Validation Standards: Include recombinant RASSF3 protein as a positive control at known concentrations

• Spike-in Controls: Add known quantities of recombinant RASSF3 to samples to verify detection limits

• Knockdown Controls: Include RASSF3 knockdown samples to confirm band specificity

By combining these methodological approaches, researchers can substantially improve detection of low-abundance RASSF3 protein in complex biological samples.

How can researchers effectively design experiments to study RASSF3's role in tumor suppression?

Designing experiments to investigate RASSF3's tumor suppressor function requires a multi-faceted methodological approach:

1. Expression Analysis in Normal vs. Cancer Tissues:

• Methodology: Compare RASSF3 expression levels across matched normal/tumor samples using RASSF3 antibodies in Western blotting and IHC

• Analysis: Quantify expression differences and correlate with clinical parameters

• Controls: Include multiple normal tissue controls and cancer specimens of various grades/stages

2. Functional Studies in Cell Models:

• Overexpression Experiments:

  • Transfect cancer cell lines with RASSF3 expression constructs

  • Measure changes in proliferation, apoptosis, migration, and invasion

  • Use empty vector controls and RASSF3 mutants (particularly in the RA domain that mediates GTPase interactions )

• Knockdown/Knockout Experiments:

  • Generate RASSF3 knockdown (siRNA/shRNA) or knockout (CRISPR-Cas9) in normal and cancer cell lines

  • Assess whether RASSF3 depletion promotes oncogenic phenotypes

  • Measure mitochondrial function given RASSF3's interaction with MIRO1/2

3. Mechanistic Investigations:

• GTPase Interaction Studies:

  • Determine how RASSF3 interactions with growth-inhibitory GTPases contribute to tumor suppression

  • Use GST-RA domain pull-downs to identify cancer-relevant binding partners

  • Create GTPase binding-deficient mutants to assess functional consequences

• Signaling Pathway Analysis:

  • Investigate RASSF3's impact on major oncogenic pathways (MAPK, PI3K/AKT, Hippo)

  • Perform phosphoproteomic analysis after RASSF3 manipulation

  • Conduct epistasis experiments with pathway activators/inhibitors

4. In Vivo Models:

• Xenograft Studies:

  • Compare tumor growth of control vs. RASSF3-overexpressing cancer cells in immunodeficient mice

  • Analyze tumors for proliferation, apoptosis, and vascularization markers

• Genetic Mouse Models:

  • Generate RASSF3 knockout mice and assess spontaneous tumor formation

  • Cross with established cancer models to determine if RASSF3 loss accelerates tumorigenesis

5. Clinical Correlation Studies:

• Survival Analysis:

  • Correlate RASSF3 expression/mutation status with patient outcomes

  • Stratify by cancer type, stage, and treatment regimen

• Biomarker Potential:

  • Assess whether RASSF3 expression or activity could serve as a diagnostic or prognostic marker

  • Develop methodologies for detecting RASSF3 status in clinical samples

These methodological approaches provide a comprehensive framework for investigating RASSF3's role in tumor suppression, leveraging its unique interaction with growth-inhibitory GTPases and mitochondrial proteins.

What are the optimal methods for studying RASSF3-MIRO interactions in mitochondrial dynamics?

Given RASSF3's direct interaction with mitochondrial GTPases MIRO1 and MIRO2 , several specialized methodologies can effectively examine this relationship in mitochondrial dynamics:

1. Biochemical Interaction Analysis:

• Direct Binding Assays: Use purified GST-RA domains from RASSF3 to pull down purified MIRO proteins as demonstrated in research literature

• Nucleotide Dependency Assessment: Test whether GTP, GDP, or GTP analogs affect binding, though existing research suggests RASSF3-MIRO binding is nucleotide-independent

• Domain Mapping: Create truncation mutants of both RASSF3 and MIRO1/2 to determine minimal binding regions

2. Live-Cell Mitochondrial Dynamics Imaging:

• Dual-Color Fluorescence Microscopy: Tag RASSF3 and MIRO1/2 with different fluorescent proteins

• Time-Lapse Microscopy: Track mitochondrial movement in neuronal processes or other relevant cell types

• FRAP Analysis: Measure recovery after photobleaching to assess dynamics of the RASSF3-MIRO complex

3. Mitochondrial Transport Assessment:

• Kymograph Analysis: Generate time-space plots of mitochondrial movement in cells expressing wild-type or mutant RASSF3

• Calcium Sensitivity Assays: Test whether RASSF3 affects MIRO's calcium-dependent regulation of mitochondrial transport

• Microtubule Co-tracking: Simultaneously visualize microtubules, mitochondria, and RASSF3 to assess transport mechanisms

4. Mitophagy Pathway Investigations:

• PINK1/Parkin Recruitment: Monitor PINK1 stabilization and Parkin recruitment to mitochondria in relation to RASSF3 levels

• Mitophagy Flux Assays: Quantify mitochondrial clearance using mitochondrially-targeted Keima or other pH-sensitive reporters

• Ubiquitination Studies: Assess whether RASSF3 affects MIRO1/2 ubiquitination during mitophagy

5. Structure-Function Analysis:

• Point Mutations: Create specific mutations in the RASSF3 RA domain based on structural predictions

• Chimeric Proteins: Swap domains between RASSF3 and other RASSF family members to determine GTPase binding specificity

• Inducible Dimerization Systems: Use chemically-induced proximity systems to trigger RASSF3-MIRO interaction and observe consequences

These methodological approaches, used in combination, can provide comprehensive insights into the functional significance of RASSF3-MIRO interactions in mitochondrial dynamics, transport, and quality control.

How can researchers integrate multi-omics approaches to study RASSF3 functions in cellular homeostasis?

Integrating multi-omics approaches provides comprehensive insights into RASSF3's biological functions and can reveal novel mechanisms in cellular homeostasis:

1. Transcriptomic Profiling:

• RNA-Seq Analysis: Compare gene expression profiles between RASSF3 knockout/knockdown and control cells

• Single-Cell Transcriptomics: Identify cell populations particularly sensitive to RASSF3 manipulation

• Alternative Splicing Analysis: Determine whether RASSF3 affects RNA processing events

• Integration Method: Identify transcription factors potentially mediating RASSF3-dependent gene expression changes

2. Proteomics Approaches:

• Whole Proteome Analysis: Quantify global protein changes upon RASSF3 modulation

• Phosphoproteomics: Map signaling networks affected by RASSF3 expression

• Proximity Labeling: Use BioID or APEX2 fused to RASSF3 to identify proximal proteins in living cells

• Integration Method: Compare protein vs. mRNA changes to identify post-transcriptional regulation

3. Metabolomics Investigation:

• Targeted Metabolite Analysis: Focus on mitochondrial metabolites given RASSF3's interaction with MIRO1/2

• Stable Isotope Tracing: Determine metabolic flux changes in pathways potentially affected by RASSF3

• Integration Method: Correlate metabolite changes with gene/protein expression alterations

4. Interactome Mapping:

• Affinity Purification-Mass Spectrometry: Identify RASSF3 protein interaction networks

• GTPase-Specific Interactions: Compare RASSF3 interactome in cells expressing constitutively active vs. dominant negative GTPases

• Integration Method: Map protein interactions to pathways identified in other omics datasets

5. Functional Genomics:

• CRISPR Screens: Identify genes that modify phenotypes caused by RASSF3 loss or overexpression

• Synthetic Lethality Assays: Discover gene dependencies created by RASSF3 status

• Integration Method: Overlay genetic dependencies with physical interaction networks

6. Multi-omics Data Integration:

These integrated approaches can reveal how RASSF3's interactions with growth-inhibitory GTPases and mitochondrial proteins coordinate cellular homeostasis through multiple molecular mechanisms.

What are the key considerations when planning RASSF3 research projects?

Planning comprehensive RASSF3 research requires attention to several critical methodological and biological considerations that will maximize meaningful outcomes:

Methodological Considerations:

• Antibody Validation: Given the complexity of the RASSF family, rigorous validation of RASSF3 antibodies (including HRP-conjugated versions) is essential. This should include verification of specificity across different RASSF family members, assessment of optimal working conditions, and confirmation of expected molecular weight detection (~28 kDa) .

• Expression Systems: Consider that endogenous RASSF3 levels may be low in many cell types, potentially requiring sensitive detection methods or enrichment strategies. When overexpressing RASSF3, account for potential artifacts from non-physiological expression levels.

• Interaction Studies: Design studies that carefully distinguish RASSF3's unique interaction profile (particularly with MIRO1/2 and growth-inhibitory GTPases) from those of other RASSF family members . Consider nucleotide independence of RASSF3-MIRO interactions when designing binding experiments .

• Model System Selection: Choose models relevant to RASSF3's biological functions, particularly those where mitochondrial dynamics and tumor suppression mechanisms can be effectively studied.

Biological Considerations:

• Dual Focus on GTPase Interactions: Address both RASSF3's interaction with growth-inhibitory RAS GTPases and its strong binding to mitochondrial MIRO1/2 GTPases , as these may represent distinct functional roles.

• Mitochondrial Connection: Investigate RASSF3's potential role in mitochondrial homeostasis, trafficking, and PINK1/PARKIN-mediated mitophagy based on its strong interaction with MIRO GTPases .

• RASSF Family Context: Position research within the broader context of RASSF family functions, noting RASSF3's distinctive binding profile that differs from better-studied members like RASSF5 .

• Tumor Suppressor Mechanism: Consider how RASSF3's interaction with growth-inhibitory GTPases might contribute to potential tumor suppressor functions distinct from other RASSF proteins.

By addressing these key considerations from the outset, researchers can design more focused and revealing studies that advance understanding of RASSF3's unique biological roles and molecular mechanisms.

What future research directions might yield the most significant insights about RASSF3 function?

Several promising research directions could significantly advance understanding of RASSF3's biological functions:

1. Mitochondrial Biology and Disease:

• Investigate RASSF3's role in mitochondrial quality control mechanisms based on its strong interaction with MIRO1/2

• Explore potential implications in neurodegenerative diseases where mitochondrial dysfunction is prominent

• Examine RASSF3's influence on mitochondrial dynamics during cellular stress responses

2. Tumor Suppression Mechanisms:

• Determine how RASSF3's interactions with growth-inhibitory RAS GTPases translate to tumor suppressor functions

• Investigate RASSF3 expression patterns across cancer types and correlation with clinical outcomes

• Compare RASSF3's tumor suppression mechanisms with other RASSF family members to identify unique contributions

3. Signaling Network Integration:

• Map how RASSF3 integrates signals from multiple GTPases (MIRO1/2, RASD2, RASL10B, REM2)

• Investigate potential crosstalk between RASSF3 and other signaling pathways implicated in cancer and mitochondrial function

• Develop mathematical models predicting cellular responses based on RASSF3's position within signaling networks

4. Structural Biology:

• Determine the crystal structure of RASSF3's RA domain in complex with its GTPase partners

• Compare structural features with other RASSF family members to understand differential binding preferences

• Design structure-based mutants with altered GTPase binding profiles for functional studies

5. Therapeutic Targeting:

• Explore whether RASSF3's tumor suppressor function could be restored or enhanced in cancers

• Investigate whether modulating RASSF3-MIRO interactions could influence mitochondrial dynamics in disease states

• Develop screening approaches to identify compounds that affect RASSF3's protein interactions or activity

6. Systems Biology Integration:

• Perform multi-omics studies to place RASSF3 within the broader cellular regulatory network

• Identify synthetic lethal interactions that could be exploited in contexts where RASSF3 is dysfunctional

• Develop computational models predicting cellular consequences of altered RASSF3 expression or function