SH3BP5 Antibody

What is SH3BP5 and why is it an important research target?

SH3BP5 is a mitochondrial outer membrane scaffold protein characterized by an N-terminal SH3 domain binding site, a membrane spanning domain, and two D-motif (KIM) regions on its C-terminus . It holds significant research interest because it functions as a direct inhibitor of JNK (c-Jun N-terminal kinase) activity and serves as a recently identified effector of Humanin, a peptide with neuroprotective properties . SH3BP5 has emerged as a critical player in multiple pathological contexts:

• Neurodegeneration: SH3BP5 mediates Humanin's neuroprotective effects against Alzheimer's disease-related neuronal death

• Leukemia progression: Elevated SH3BP5 expression correlates with poor outcomes in acute myeloid leukemia (AML) patients

• Cardiovascular disease: Serum antibodies against SH3BP5 have been identified as potential biomarkers for atherosclerosis

The protein's strategic position at the intersection of multiple signaling pathways makes it an appealing target for researchers investigating cellular stress responses, apoptotic mechanisms, and disease biomarkers.

What are the available techniques for detecting SH3BP5 expression in experimental models?

Several methodological approaches have demonstrated efficacy for detecting SH3BP5 in research settings:

• Immunofluorescence microscopy: Cells can be fixed with 4% paraformaldehyde-PBS and sequentially immunostained with anti-SH3BP5 primary antibody followed by fluorescently-labeled secondary antibodies (Texas-red-conjugated or FITC-conjugated). Confocal microscopy (e.g., LSM710) provides optimal visualization of subcellular localization .

• Western blotting: Studies have successfully used western blot analysis to detect SH3BP5 protein expression in various cell lines, including AML cell lines like THP-1, U937, Kasumi-1, and MV4-11 .

• Quantitative RT-PCR: For mRNA expression analysis, qRT-PCR has been effectively employed to measure SH3BP5 transcript levels in leukemia cell lines and patient samples .

• Amplified luminescent proximity homogeneous assay (AlphaLISA): This highly sensitive method has been used to quantify serum antibody levels against SH3BP5 protein and peptides in patient samples .

These techniques provide complementary data regarding SH3BP5 expression patterns and can be selected based on specific research questions.

What epitope regions of SH3BP5 are most commonly targeted by commercial antibodies?

Based on the available research, antibodies targeting specific epitope regions of SH3BP5 have been identified through peptide array analysis. One significant epitope site recognized by serum antibodies is located within amino acids 161-174 of SH3BP5 . This region appears particularly immunogenic and has been used for developing detection systems.

For immunostaining applications, researchers have successfully used antibodies against SH3BP5 that can be preabsorbed with an excess amount of immunizing antigen (GST-SH3BP5) to deplete SH3BP5-recognizing immunoglobulin, providing an effective control for specificity testing .

Commercial antibodies may target various regions of the protein, but those recognizing functional domains (such as the SH3 binding domain or JNK interaction regions) are particularly valuable for mechanistic studies examining protein-protein interactions.

How should researchers design experiments to investigate SH3BP5's role in JNK signaling pathways?

When investigating SH3BP5's role in JNK signaling, researchers should consider the following experimental design elements:

• Manipulate SH3BP5 expression levels:

  • For overexpression studies, human or mouse SH3BP5 cDNAs can be PCR-amplified and cloned into appropriate expression vectors .

  • For knockdown approaches, shRNA-encoding lentivirus systems have proven effective in decreasing SH3BP5 expression in various cell lines .

• Assess effects on JNK activity: Following SH3BP5 manipulation, researchers should measure:

  • JNK phosphorylation status via western blotting

  • Downstream targets of JNK, such as phosphorylation of BAD

  • JNK-mediated cellular outcomes (apoptosis, cell viability)

• Evaluate subcellular localization: As SH3BP5 targets JNK to mitochondria, co-localization studies using fluorescently tagged proteins or subcellular fractionation followed by western blotting can reveal important mechanistic insights .

• Include appropriate controls:

  • Scramble shRNA controls for knockdown experiments

  • Empty vector controls for overexpression studies

  • JAK2 inhibitor (AG490) can be used when studying the Humanin-SH3BP5-JNK axis to block upstream signaling

This comprehensive approach allows researchers to establish causal relationships between SH3BP5 levels and JNK pathway activity in their experimental system.

What are the recommended methods for studying SH3BP5 antibody levels as biomarkers in clinical samples?

Based on published research methodologies, the following approach is recommended for studying SH3BP5 antibody levels as potential biomarkers:

• Sample collection and processing:

  • Collect serum samples from patients with the condition of interest and from healthy donors

  • Process samples consistently to minimize pre-analytical variability

• Detection method selection:

  • The amplified luminescent proximity homogeneous assay (AlphaLISA) has demonstrated high sensitivity and stability for detecting anti-SH3BP5 antibodies in serum samples

  • This method incorporates glutathione- or streptavidin-donor beads and anti-human-IgG-acceptor beads

• Antigen preparation:

  • Express and purify GST-fused SH3BP5 proteins in E. coli using affinity chromatography

  • For epitope-specific detection, use synthetic peptides corresponding to identified epitope regions (e.g., amino acids 161-174)

• Data analysis and interpretation:

  • Employ receiver operating characteristic (ROC) curve analysis to assess predictive values

  • Set cutoff values that maximize the combined sensitivity and specificity

  • Use two-tailed statistical tests with P values < 0.05 considered significant

• Validation cohorts:

  • Test findings in independent patient populations

  • Stratify results based on clinical parameters (age, disease stage, etc.)

This methodological framework provides a robust approach for evaluating SH3BP5 antibodies as potential biomarkers in various disease contexts.

What cell lines and model systems are most appropriate for studying SH3BP5 function?

The choice of experimental models should align with the specific aspect of SH3BP5 biology under investigation:

Cell Line Models:

• Neuronal models:

  • Human neuroblastoma SH-SY5Y cells have been successfully used for studying SH3BP5's role in neuroprotection

  • Neurohybrid F11 cells provide another viable model system for neuronal studies

• Leukemia models:

  • AML cell lines (THP-1, U937, Kasumi-1, MV4-11) show high expression of SH3BP5 and are appropriate for studying its role in leukemia

  • ALL cell lines (Kasumi-2, Rch-Acv, NALM-6) display variable SH3BP5 expression and can serve as comparative models

Expression systems:

• For recombinant protein production, E. coli expression systems using vectors like pGEX-2T (for GST-fusion proteins) and pQE30 (for His-tagged proteins) have proven effective

In vivo models:

• While not explicitly described in the provided search results, transgenic or knockout mouse models with altered SH3BP5 expression would provide valuable insights into its in vivo functions

The selection of appropriate model systems should be guided by the specific research question, with consideration for endogenous SH3BP5 expression levels and the relevant signaling pathway components present in each model.

How can researchers distinguish between direct and indirect effects when studying SH3BP5's impact on cellular pathways?

Distinguishing direct from indirect effects requires a multi-faceted experimental approach:

• Protein-protein interaction studies:

  • Direct binding assays using purified recombinant proteins

  • Co-immunoprecipitation of endogenous proteins

  • Proximity ligation assays to visualize protein interactions in situ

• Domain mapping experiments:

  • Generate SH3BP5 deletion mutants to identify critical interaction domains

  • Use the pQE30 vector system to express 6×His-SH3BP5 deletion mutants for binding specificity studies

• Kinase activity assays:

  • In vitro kinase assays using purified components can determine if SH3BP5 directly inhibits JNK enzymatic activity

  • Compare phosphorylation of known JNK substrates in the presence/absence of SH3BP5

• Temporal analyses:

  • Time-course experiments to establish the sequence of events following SH3BP5 manipulation

  • Use of specific pathway inhibitors (e.g., JAK2 inhibitor AG490) to block upstream signaling events

• Rescue experiments:

  • After SH3BP5 knockdown, reintroduce wild-type or mutant SH3BP5 to determine which domains are essential for restoring function

These approaches help establish causality rather than mere correlation, allowing researchers to map the direct molecular targets of SH3BP5 and distinguish these from downstream pathway effects.

How do post-translational modifications affect SH3BP5 function and antibody recognition?

While the provided search results don't specifically address post-translational modifications (PTMs) of SH3BP5, this represents an important area for advanced research investigation. Researchers investigating this aspect should consider:

• Identification of PTM sites:

  • Mass spectrometry-based proteomics approaches to identify phosphorylation, ubiquitination, or other modifications

  • Bioinformatic prediction of potential modification sites based on consensus sequences

• Functional impact assessment:

  • Generation of phosphomimetic or phosphodeficient mutants at identified or predicted PTM sites

  • Compare activity and binding properties of modified versus unmodified SH3BP5

• Antibody selection considerations:

  • Determine whether commercial antibodies recognize modified or unmodified forms of SH3BP5

  • For phospho-specific studies, consider developing antibodies that specifically recognize modified forms

  • Validate antibody specificity using modified and unmodified recombinant proteins

• Context-dependent regulation:

  • Investigate how cellular stress, growth factors, or disease conditions might alter the PTM status of SH3BP5

  • Examine how modifications might affect subcellular localization, particularly mitochondrial targeting

Understanding the impact of PTMs on SH3BP5 function and antibody recognition will provide deeper insights into its regulation and could reveal novel therapeutic intervention points.

What are the challenges in interpreting contradictory findings regarding SH3BP5's role in different disease contexts?

Researchers face several challenges when reconciling seemingly contradictory findings about SH3BP5 across different disease contexts:

• Context-dependent protein functions:

  • SH3BP5 appears to have protective effects in neurodegenerative conditions by mediating Humanin's neuroprotection

  • Conversely, elevated SH3BP5 correlates with poor outcomes in AML, suggesting a potential oncogenic role

  • These opposing functions may reflect tissue-specific signaling networks or different binding partners

• Methodological differences:

  • Variation in experimental approaches (overexpression vs. knockdown)

  • Different cell types and model systems

  • Varying methods for measuring outcomes

• Integration of antibody-based findings:

  • Serum antibodies against SH3BP5 serve as biomarkers for atherosclerosis

  • Determining whether these antibodies are causative, protective, or merely correlative requires careful interpretation

• Research design recommendations:

  • Employ multiple complementary techniques within the same study

  • Include appropriate disease and tissue-specific controls

  • Consider dose-dependent and temporal aspects of SH3BP5 function

  • Validate findings across multiple model systems

• Data integration strategies:

  • Pathway analysis to identify context-specific interacting partners

  • Meta-analysis of published findings with attention to methodological differences

  • Development of computational models that incorporate tissue-specific factors

By carefully considering these factors, researchers can develop more nuanced hypotheses about SH3BP5's role in health and disease, potentially revealing how a single protein can exert distinct effects in different cellular contexts.

What are the optimal conditions for using SH3BP5 antibodies in various experimental techniques?

Based on published methodologies, the following technical parameters are recommended for different applications:

Immunofluorescence microscopy:

• Fixation: 4% paraformaldehyde in PBS

• Primary antibody: Anti-SH3BP5 antibody (dilution should be optimized for each specific antibody)

• Secondary antibody: Texas-red-conjugated or FITC-conjugated goat anti-rabbit polyclonal antibody

• Nuclear counterstain: DAPI can be used when nuclear visualization is needed

• Imaging: Confocal microscopy (e.g., LSM710) for optimal resolution

Western blotting:

• Sample preparation: Standard cell lysis buffers appear adequate for SH3BP5 extraction

• Controls: Include positive control samples with known SH3BP5 expression

• Specificity validation: Pre-absorption of the SH3BP5 antibody with immunizing antigen (GST-SH3BP5) can serve as a negative control

AlphaLISA for serum antibody detection:

• Bead preparation: Glutathione- or streptavidin-donor beads and anti-human-IgG-acceptor beads

• Antigen: Recombinant GST-SH3BP5 or synthetic peptides corresponding to epitope regions

• Sample dilution: Optimize serum dilutions to ensure measurements within the linear range of detection

Researchers should always validate antibody specificity in their specific experimental system using appropriate positive and negative controls.

How can researchers validate the specificity of SH3BP5 antibodies?

Ensuring antibody specificity is critical for obtaining reliable results. The following validation strategies are recommended:

• Genetic approaches:

  • Use cells with SH3BP5 knockdown (via shRNA or CRISPR) as negative controls

  • Compare staining/detection patterns in cells with varying endogenous SH3BP5 expression levels

• Biochemical validation:

  • Pre-absorption control: Incubate the antibody with excess purified antigen (GST-SH3BP5) to deplete specific antibodies

  • Include a negative control using GST alone to control for potential reactivity with the tag

• Epitope competition assays:

  • Use synthetic peptides corresponding to the epitope region (e.g., amino acids 161-174) to compete for antibody binding

  • Declining signal with increasing peptide concentration confirms specificity

• Multiple antibody concordance:

  • Compare results using antibodies targeting different epitopes of SH3BP5

  • Consistent patterns across different antibodies increase confidence in specificity

• Recombinant protein controls:

  • Include purified recombinant SH3BP5 as a positive control in western blots

  • Use dilution series to establish detection limits and linear range

These validation steps should be documented and included in research publications to enhance reproducibility and confidence in the reported findings.

How can SH3BP5 antibody levels be effectively utilized as biomarkers in patient stratification?

The application of SH3BP5 antibody measurements for patient stratification requires methodological rigor and clinical correlation:

• Established clinical associations:

  • Elevated serum antibodies against SH3BP5 have been associated with various conditions, including diabetes mellitus, acute-phase cerebral infarction, transient ischemic attack, cardiovascular disease, and chronic kidney disease

• Standardized measurement protocols:

  • AlphaLISA methodology has demonstrated effectiveness for quantifying anti-SH3BP5 antibodies in patient sera

  • Consistent sample collection, processing, and testing procedures are essential for reliable results

• Patient stratification approach:

  • Set appropriate cutoff values using ROC curve analysis to maximize sensitivity and specificity

  • Consider combining SH3BP5 antibody measurements with established clinical risk factors for improved predictive power

• Subgroup analyses:

  • Stratify patients by age (e.g., <60 vs. ≥60 years)

  • Consider cytogenetic risk categories and other clinical parameters that might influence interpretation

• Longitudinal monitoring:

  • Serial measurements may provide prognostic information beyond single time point assessment

  • Evaluate changes in antibody levels in response to treatment or disease progression

The integration of SH3BP5 antibody measurements into clinical algorithms could enhance risk assessment and personalized treatment approaches across multiple disease contexts.

What methodological considerations are important when developing SH3BP5-targeted therapeutic approaches?

While the search results don't directly address SH3BP5-targeted therapeutics, the biological insights provided suggest several methodological considerations for therapeutic development:

• Target validation strategies:

  • Confirm SH3BP5's causative role in disease pathogenesis through genetic manipulation models

  • Identify tissue-specific functions to anticipate potential on-target adverse effects

  • Determine whether inhibition or enhancement of SH3BP5 function would be beneficial in specific disease contexts

• Therapeutic modality selection:

  • Small molecule inhibitors targeting SH3BP5-JNK interaction

  • Peptide-based approaches mimicking functional domains

  • RNA interference strategies for reducing SH3BP5 expression in diseases where it contributes to pathology

• Delivery considerations:

  • As a mitochondrial outer membrane protein, therapeutic agents must reach this subcellular compartment

  • Cell type-specific delivery systems may be needed for targeted therapeutic effects

• Efficacy assessment:

  • In vitro: Cell viability, apoptosis measurements, JNK phosphorylation status

  • In vivo: Disease-relevant phenotypic outcomes in appropriate animal models

• Biomarker integration:

  • Utilize anti-SH3BP5 antibody levels as potential predictive biomarkers for treatment response

  • Monitor changes in pathway activity (e.g., JNK-BAD signaling) as pharmacodynamic markers

These methodological considerations provide a framework for developing SH3BP5-targeted therapeutic approaches, particularly in conditions like AML where SH3BP5 appears to promote disease progression.

What emerging technologies could enhance our understanding of SH3BP5 function and antibody-based applications?

Several cutting-edge methodologies could significantly advance SH3BP5 research:

• Single-cell analyses:

  • Single-cell RNA sequencing to identify cell populations with differential SH3BP5 expression

  • Single-cell proteomics for protein-level characterization in heterogeneous tissues

• Advanced imaging techniques:

  • Super-resolution microscopy for detailed subcellular localization

  • Live-cell imaging with fluorescently tagged SH3BP5 to track dynamic cellular processes

  • Correlative light and electron microscopy to visualize SH3BP5 at the mitochondrial membrane

• Structural biology approaches:

  • Cryo-electron microscopy to determine SH3BP5's structure in complex with binding partners like JNK

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

• High-throughput screening platforms:

  • CRISPR activation/inhibition screens to identify genetic modifiers of SH3BP5 function

  • Small molecule libraries to discover compounds that modulate SH3BP5-protein interactions

• Computational methods:

  • Molecular dynamics simulations to understand SH3BP5's conformational changes

  • Machine learning approaches to predict patient outcomes based on SH3BP5 expression and antibody profiles

These technological advances would provide deeper mechanistic insights into SH3BP5's functions and potentially reveal novel therapeutic opportunities across various disease contexts.

What are the key unresolved questions regarding SH3BP5 biology that warrant further investigation?

Despite significant progress, several fundamental questions about SH3BP5 remain unanswered:

• Regulatory mechanisms:

  • How is SH3BP5 expression regulated in different tissues and disease states?

  • What post-translational modifications affect SH3BP5 function?

  • Are there tissue-specific isoforms with distinct functions?

• Signaling pathway integration:

  • How does SH3BP5 coordinate its roles in both Humanin-mediated neuroprotection and JNK pathway regulation?

  • What determines whether SH3BP5 exerts protective (neuronal) versus pathological (leukemia) effects?

  • Are there additional, undiscovered interaction partners beyond JNK and Btk?

• Clinical significance:

  • Do anti-SH3BP5 antibodies play a causal role in disease pathogenesis or are they merely biomarkers?

  • Can therapeutic targeting of SH3BP5 provide clinical benefit in conditions like AML?

  • What is the predictive value of SH3BP5 expression or antibody levels for treatment response?

• Evolutionary biology:

  • How conserved is SH3BP5 function across species?

  • What can comparative biology teach us about SH3BP5's fundamental roles?

• Technical challenges:

  • Development of more specific antibodies targeting distinct functional domains

  • Creation of conditional knockout models to study tissue-specific functions

Addressing these questions through rigorous scientific investigation will advance our understanding of SH3BP5 biology and potentially reveal novel therapeutic approaches for conditions ranging from neurodegeneration to cancer.