SH3GLB2 Human

What is the molecular structure and basic characterization of human SH3GLB2?

Human SH3GLB2 (Endophilin B2) is a 395 amino acid protein that belongs to the endophilin family. It shows approximately 65% identity to SH3GLB1 (Endophilin B1/Bif-1) . The protein contains an SH3 domain and a core coiled-coil region that is required for homo- and hetero-dimerization . In Western blot analyses, SH3GLB2 appears as a specific band at approximately 42-43 kDa .

Structurally, SH3GLB2 shares similarities with other endophilin family proteins throughout its sequence, suggesting a common fold and potentially a common mode of action . The protein primarily localizes to the cytoplasmic compartment and is excluded from the nucleus .

How does SH3GLB2 differ from its paralog SH3GLB1?

While SH3GLB2 shares 65% sequence identity with SH3GLB1, there are several key differences:

Both proteins contain SH3 domains and can form homo- and heterodimers through their coiled-coil regions . They colocalize in the cytoplasmic compartment with Bax protein, though SH3GLB2 does not significantly influence the onset or time course of Bax-mediated apoptosis in HeLa or 293T cells .

What are the known protein-protein interactions of SH3GLB2?

Based on experimental evidence, SH3GLB2 engages in several protein-protein interactions:

• SH3GLB2 forms homodimers with itself through its coiled-coil region .

• SH3GLB2 forms heterodimers with SH3GLB1 through the same coiled-coil domain region .

• SH3GLB2 colocalizes with Bax in the cytoplasmic compartment, though functional significance appears limited .

Importantly, the SH3 domain of SH3GLB2 is not involved in these dimerization interactions . The protein's involvement in endocytosis pathways suggests it likely interacts with other components of the endocytic machinery, though specific partners beyond SH3GLB1 and Bax are not clearly identified in the available research.

What antibodies and reagents are available for detecting human SH3GLB2?

Several validated antibodies and reagents are available for detecting human SH3GLB2 in experimental settings:

• Mouse Anti-Human Endophilin B1/B2 Monoclonal Antibody (Clone #807009, MAB7456):

  • Applications: Western blot (0.2 μg/mL), immunohistochemistry (15 μg/mL), Simple Western

  • Detects both Endophilin B1 and B2

  • Validated in Jurkat, Saos-2, and U-118-MG cell lines

• Recombinant Protein Control Fragments:

  • Human SH3GLB2 (aa 165-246) Control Fragment (RP-93012): For blocking experiments with antibody PA5-54407

  • Human SH3GLB2 Control Fragment (RP-93013): For blocking experiments with antibody PA5-55167

  • Recommended use: 100x molar excess of protein fragment, pre-incubated with antibody for 30 min at room temperature

When performing blocking experiments to validate antibody specificity, it's critical to use the matching control fragment with its corresponding antibody at the recommended concentrations to ensure reliable results.

What are the optimal conditions for detecting SH3GLB2 in Western blot experiments?

For optimal Western blot detection of human SH3GLB2, follow these methodological guidelines:

• Sample preparation:

  • Prepare cell lysates under reducing conditions

  • Use appropriate lysis buffers (e.g., Immunoblot Buffer Group 1 as referenced in protocols)

• Gel electrophoresis and transfer:

  • Separate proteins using standard SDS-PAGE techniques

  • Transfer proteins to PVDF membrane

• Primary antibody incubation:

  • For MAB7456: Use at 0.2 μg/mL concentration

  • For AF7456: Use at 1 μg/mL concentration

• Secondary antibody incubation:

  • For mouse primary (MAB7456): Use HRP-conjugated Anti-Mouse IgG (e.g., HAF018)

  • For sheep primary (AF7456): Use HRP-conjugated Anti-Sheep IgG (e.g., HAF016)

• Detection:

  • Look for a specific band at approximately 42-43 kDa

  • Include positive control cell lines such as Jurkat, Saos-2, or U-118-MG

This procedure has been validated in multiple cell lines, resulting in consistent and specific detection of SH3GLB2 protein.

How can researchers validate SH3GLB2 expression across different tissue and cell types?

To comprehensively validate SH3GLB2 expression across tissues and cell types, researchers should employ multiple complementary approaches:

• Immunohistochemistry (IHC):

  • Use validated antibodies (e.g., MAB7456 at 15 μg/mL)

  • For paraffin-embedded tissues, perform heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic

  • Counterstain with hematoxylin to visualize tissue architecture

  • Look for cytoplasmic staining, particularly in pancreatic islet cells

• Immunocytochemistry (ICC) for cell lines:

  • For adherent cells: Use immersion fixation

  • For non-adherent cells: Follow specialized protocols (e.g., Fluorescent ICC Staining of Non-adherent Cells)

  • Add nuclear counterstain (e.g., DAPI)

  • Look for cytoplasmic localization pattern

• Western blot analysis:

  • Compare expression across multiple cell lines and tissue lysates

  • Include positive controls (Jurkat, Saos-2, U-118-MG)

  • Quantify relative expression levels

• RNA expression analysis:

  • Consult The Human Protein Atlas for baseline expression data across 44 normal tissue types

  • Consider RNA-seq or RT-PCR to validate expression at the transcript level

Integrating data from these multiple approaches provides a more reliable assessment of expression patterns than any single method alone.

How does SH3GLB2 deficiency affect response to influenza infection?

A key study using SH3GLB2-deficient (B2-deficient) mice revealed significant effects on the response to influenza A virus (IAV) infection:

• Enhanced recovery parameters:

  • Swift body weight recovery after infection

  • Significantly better survival compared to wild-type mice

  • Improved restoration of alveolar homeostasis

• Molecular and cellular changes in B2-deficient lungs:

  • Induction of genes expressing surfactant proteins, ABCA3, GM-CSF, podoplanin, and caveolin mRNA after 7 days post-infection

  • Temporal induction of CCAAT/enhancer binding protein CEBPα, β, and δ mRNAs 3–14 days after infection

  • Improved alveolar extracellular matrix integrity

  • Enhanced respiratory mechanics

  • Robust recovery of alveolar macrophages

  • Increased recruitment of CD4+ lymphocytes

These findings suggest that targeting endophilin B2 could alleviate adverse effects of IAV infection on respiratory and immune cells, enabling more effective restoration of alveolar homeostasis. The study provides compelling evidence that SH3GLB2 may be a potential therapeutic target for severe influenza infections.

What experimental models are available for studying SH3GLB2 function?

Researchers can utilize several experimental models to investigate SH3GLB2 function:

• Transgenic mouse models:

  • B2-deficient mice have been developed and validated for studying the role of SH3GLB2 in influenza infection

  • These models allow for in vivo assessment of physiological functions in lung homeostasis and immune response

• Cell line models:

  • Human cell lines with documented SH3GLB2 expression:

    • Jurkat (acute T cell leukemia)

    • Saos-2 (osteosarcoma)

    • U-118-MG (glioblastoma/astrocytoma)

  • These cell lines provide platforms for in vitro analysis of protein function, localization, and interactions

• Protein interaction analysis:

  • Yeast two-hybrid system: Previously used to identify SH3GLB2 as an interacting partner of SH3GLB1

  • Co-immunoprecipitation assays: Can validate protein-protein interactions in mammalian cell contexts

• Recombinant protein systems:

  • E. coli-derived recombinant human protein fragments are available for structural and functional studies

When designing experiments, researchers should select the model most appropriate for their specific research question, considering the advantages and limitations of each system.

What is the relationship between SH3GLB2 and apoptotic pathways?

The relationship between SH3GLB2 and apoptotic pathways has been experimentally investigated:

• Cellular colocalization:

  • SH3GLB2 colocalizes with the apoptosis regulator Bax in the cytoplasmic compartment of cells

• Functional impact:

  • Despite this colocalization, experimental evidence indicates that SH3GLB2 does not significantly influence the onset or time course of Bax-mediated apoptosis in HeLa or 293T cells

  • This suggests that while there may be physical proximity between SH3GLB2 and Bax, this association might not translate into functional consequences for apoptotic signaling

• Comparison with SH3GLB1:

  • This contrasts with some findings regarding its paralog SH3GLB1/Bif-1, which has been implicated in certain apoptotic processes

  • The 65% sequence similarity between these proteins might suggest some shared and some distinct functions in cellular pathways

These findings highlight the importance of distinguishing between physical interaction and functional consequence when studying protein-protein relationships in apoptotic pathways.

How does tissue-specific expression of SH3GLB2 correlate with its function?

The Human Protein Atlas data indicates that SH3GLB2 shows differential expression across tissues, which may correlate with tissue-specific functions:

• Normal tissue expression:

  • Cytoplasmic expression has been detected in pancreatic islet cells

  • The Human Protein Atlas contains expression data across 44 normal tissue types, though detailed comparative expression levels are not provided in the available search results

• Pathological tissue expression:

  • Expression has been detected in pancreatic cancer tissue

  • Expression in cancerous tissues may differ from normal counterparts, suggesting potential pathology-specific roles

• Methodological approach for researchers:

  • To investigate tissue-specific functions, researchers should first establish comprehensive expression profiles across tissues using RNA-seq, protein arrays, and immunohistochemistry

  • Follow with tissue-specific knockout or knockdown experiments to determine functional consequences

  • Compare phenotypes across different tissue types where SH3GLB2 is expressed

Further research is needed to fully elucidate the relationship between tissue-specific expression patterns and functional specialization of SH3GLB2 in different physiological contexts.

What are the evolutionary implications of SH3GLB2 conservation across species?

Analysis of SH3GLB2 conservation across species reveals important evolutionary insights:

• Sequence conservation:

  • Human SH3GLB2 control fragments show high sequence identity to mouse (92%) and rat (92%) orthologs

  • The human SH3GLB2 (aa 165-246) fragment shows even higher conservation, with 100% identity to both mouse and rat sequences

• Comparative analysis with SH3GLB1:

  • SH3GLB1 was identified as potentially homologous to a Caenorhabditis elegans SH3-containing protein (GenBank Accession No. U46675)

  • The conservation of both SH3GLB1 and SH3GLB2 across species suggests important functional roles maintained throughout evolution

• Research implications:

  • High conservation indicates functional importance and potential evolutionary pressure to maintain structure and function

  • Comparative studies across species may provide insights into conserved mechanisms

  • Model organisms like mice can likely provide relevant insights for human SH3GLB2 function due to high sequence identity

The significant conservation of SH3GLB2 across mammalian species suggests that findings from rodent models may be particularly relevant to understanding human SH3GLB2 function in health and disease contexts.

How might post-translational modifications regulate SH3GLB2 function?

While specific information about post-translational modifications (PTMs) of SH3GLB2 is limited in the available research, several methodological approaches can address this critical question:

• Potential regulatory PTMs to investigate:

  • Phosphorylation: May regulate protein-protein interactions, subcellular localization, or enzymatic activity

  • Ubiquitination: Could modulate protein stability, turnover, or targeting to specific cellular compartments

  • SUMOylation: Might affect protein localization or interaction capabilities

• Experimental approaches:

  • Mass spectrometry-based proteomic analysis of purified SH3GLB2 to identify and map modification sites

  • Site-directed mutagenesis of predicted modification sites followed by functional assays

  • Pharmacological manipulation of kinases, phosphatases, or other enzymes that regulate PTMs

  • Comparison of PTM patterns under different cellular conditions (e.g., stress, infection)

• Functional validation:

  • Generate phosphomimetic and phosphodeficient mutants to assess functional consequences

  • Examine how identified PTMs affect interactions with known partners like SH3GLB1

  • Investigate whether PTMs change in response to stimuli like viral infection

This research direction represents an important gap in our current understanding of SH3GLB2 regulation and could provide insights into how this protein's activity is modulated in different physiological and pathological contexts.

What approaches can be used to target SH3GLB2 for therapeutic development?

Given the findings that SH3GLB2 deficiency enhances recovery from influenza infection, developing therapeutic approaches targeting this protein may have clinical potential:

• Target validation strategies:

  • Confirm findings from mouse models in human cell and tissue systems

  • Validate that inhibition of SH3GLB2 provides therapeutic benefit without unacceptable side effects

  • Identify specific contexts (e.g., severe influenza) where modulation would be most beneficial

• Potential therapeutic modalities:

  • Small molecule inhibitors targeting the SH3 domain or coiled-coil region

  • Peptide-based disruptors of protein-protein interactions

  • RNA interference strategies (siRNA, antisense oligonucleotides) to reduce expression

  • CRISPR-based approaches for ex vivo cell therapeutic development

• Screening methodologies:

  • Structure-based virtual screening utilizing the SH3 domain structure

  • High-throughput functional assays measuring endocytosis or other SH3GLB2-dependent processes

  • Phenotypic screens in cellular models of influenza infection

• Considerations for therapeutic development:

  • Tissue-specific delivery to respiratory tissues for influenza applications

  • Assessment of effects on normal endocytic function

  • Evaluation of consequences of disrupting interactions with binding partners

The observation that B2-deficient mice demonstrate enhanced recovery from severe influenza infection provides a compelling rationale for developing SH3GLB2-targeted therapeutics for respiratory viral infections .