SH3GLB1 Human

What is the basic structure of the SH3GLB1 protein and how does it relate to its function?

SH3GLB1 (also known as Endophilin-B1 or Bax-interacting factor 1/Bif-1) belongs to the Bin/Amphiphysin/Rvs167 (BAR) family of proteins. It contains an N-terminal BAR domain responsible for membrane binding and bending, and a C-terminal SH3 (Src-homology 3) domain that facilitates protein-protein interactions . In the presence of biological membranes, SH3GLB1 dimers assemble into helical scaffolds around the membrane, driving tubulation . This membrane curvature function is critical for its roles in mitochondrial dynamics, autophagy, apoptosis, and endocytosis .

How does SH3GLB1 contribute to autophagosome formation?

SH3GLB1 plays a crucial role in autophagosome biogenesis through multiple mechanisms. It forms a complex with Beclin1 through UVRAG (ultraviolet irradiation resistant-associated gene) and promotes the activation of class III PI3 kinase (Vps34) . During nutrient starvation, SH3GLB1 accumulates in punctate foci where it co-localizes with autophagy markers LC3, Atg5, and Atg9 . SH3GLB1-positive, crescent-shaped small vesicles expand by recruiting and fusing with Atg9-positive small membranes to complete autophagosome formation . This process is indispensable for the initiation phase of autophagy .

What are the key protein interactions of SH3GLB1 and their significance?

SH3GLB1 interacts with multiple proteins crucial for cellular homeostasis:

• The pro-apoptotic factor Bcl-2-associated X protein (Bax) and SH3GLB2

• Beclin-1, which is essential for autophagosome formation

• Amphiphysin-1 and amphiphysin-2, involved in endocytosis

• Huntingtin protein, implicated in neuronal function

These interactions occur primarily through SH3GLB1's canonical SH3 domain that binds PxxP motif-containing proteins . The diversity of these binding partners highlights SH3GLB1's multifunctional role in cellular processes ranging from membrane dynamics to cell death pathways.

How can researchers effectively modulate SH3GLB1 expression in experimental models?

Several validated approaches for SH3GLB1 modulation in experimental settings include:

• RNA interference:

  • siRNA targeting SH3GLB1 (sequence: 5′-GGGAAUCAGCAGUACACAUTT-3′ and 3′-AUGUGUACUGCUGAUUCCCTT-5′) transfected using Lipofectamine® RNAiMAX reagent

  • Lentiviral-transfected shRNA for long-term modulation

• Overexpression systems:

  • Lentiviral vectors containing SH3GLB1 cDNA

  • Plasmid-based overexpression systems

• Pharmacological approaches:

  • HDAC inhibitors like SAHA, which can indirectly affect SH3GLB1 levels

For optimal results, researchers should validate knockdown or overexpression efficiency through western blotting, and consider cell type-specific transfection optimization protocols .

How does SH3GLB1 expression correlate with patient survival in different cancer types?

Analysis of clinical databases including the CGGA (Chinese Glioma Genome Atlas) and TCGA (The Cancer Genome Atlas) has revealed significant correlations between SH3GLB1 expression and patient outcomes. In glioblastoma specifically, survival curves of cases with higher SH3GLB1 expression showed worse prognosis . This counterintuitive finding (given SH3GLB1's role as a tumor suppressor in some contexts) suggests a complex and potentially context-dependent role in cancer progression.

The correlation between high SH3GLB1 expression and poor survival was particularly evident in recurrent tumors, as demonstrated by paired analysis of primary and recurrent tumor samples . High SH3GLB1 gene expression was also associated with higher disease grading in glioblastoma . These findings highlight the importance of comprehensive analysis of SH3GLB1 expression across different cancer stages and subtypes to fully understand its prognostic significance.

What is the role of SH3GLB1 in temozolomide resistance in glioblastoma?

SH3GLB1 plays a pivotal role in promoting temozolomide (TMZ) resistance in glioblastoma through multiple mechanisms:

• Enhanced autophagy: SH3GLB1 is indispensable for autophagy initiation, and TMZ-resistant cells show increased SH3GLB1-mediated autophagy

• Mitochondrial metabolism modulation: SH3GLB1 alters oxidative phosphorylation (OXPHOS) pathways, enhancing mitochondrial functions in resistant cells

• Key mitochondrial effects include:

  • Increased mitochondrial membrane potential

  • Enhanced mitochondrial respiration

  • Elevated ATP production

Experimental evidence demonstrates that SH3GLB1 knockdown in resistant cells resensitizes them to TMZ treatment, restoring drug efficacy by suppressing TMZ-induced autophagy and OXPHOS . This suggests SH3GLB1 as a potential therapeutic target for overcoming TMZ resistance in glioblastoma treatment.

How does SH3GLB1 regulate CD133 expression and tumor-initiating cell features in glioblastoma?

SH3GLB1 regulates CD133 (a marker for tumor-initiating cells) through epigenetic mechanisms:

• SH3GLB1 affects histone H4 lysine 5 (H4K5) acetylation at the CD133 promoter region

• Mechanistic pathway:

  • SH3GLB1 is distributed in nucleus, cytoplasm, and mitochondria

  • Nuclear SH3GLB1 influences H4K5 acetylation

  • Enhanced H4K5 acetylation increases CD133 transcription

• Functional consequences:

  • SH3GLB1 knockdown reduces spheroid formation frequency and size

  • CD133+ cells with high SH3GLB1 show increased resistance to cytotoxic treatments

  • SH3GLB1 levels correlate with tumor-initiating cell features in single-cell transcriptomic analyses

This regulatory mechanism links SH3GLB1 to the maintenance of tumor-initiating cell populations in glioblastoma, explaining its association with treatment resistance and tumor recurrence.

What molecular pathways connect SH3GLB1 to oxidative phosphorylation in glioblastoma cells?

Single-cell RNA transcriptomic analysis of glioblastoma tumors revealed distinct clusters with varying levels of SH3GLB1 expression and oxidative phosphorylation (OXPHOS) activity. The following molecular connections have been identified:

These associations suggest that SH3GLB1 may regulate specific components of the OXPHOS machinery, particularly in tumor-initiating cell (TIC) populations (cluster 4) . The exact mechanisms connecting SH3GLB1 to OXPHOS regulation may involve mitochondrial membrane remodeling through its BAR domain or indirect effects through autophagy-mediated mitochondrial quality control .

What are the most effective approaches for studying SH3GLB1 in patient-derived glioblastoma samples?

Based on recent research methodologies, the following approaches have proven effective for studying SH3GLB1 in patient-derived samples:

• Single-cell RNA transcriptomic analysis:

  • Enables identification of tumor cell subpopulations with varying SH3GLB1 expression

  • Allows correlation with tumor-initiating cell markers (CD133, Olig2, SOX2, Bmi1, Myc)

  • Reveals relationships between SH3GLB1 and metabolic pathways

• Protein expression analysis:

  • Western blotting of paired primary and recurrent tumor tissues

  • Immunohistochemistry to determine spatial distribution

• Bioinformatic approaches:

  • Correlation analysis using CGGA and TCGA databases

  • Ingenuity Pathway Analysis (IPA) to identify associated pathways

  • Survival analysis based on SH3GLB1 expression levels

• CD133+ cell isolation and characterization:

  • Cell sorting followed by functional assays

  • Spheroid formation assays

  • Resistance testing against TMZ or oxidative stress

These methods, particularly when combined, provide comprehensive insights into SH3GLB1's role in glioblastoma pathophysiology and treatment resistance.

How can researchers establish valid experimental models to study SH3GLB1-mediated autophagy?

To establish robust experimental models for studying SH3GLB1-mediated autophagy, researchers should consider:

• Cell line selection:

  • Use paired sensitive and resistant GBM cell lines (e.g., U87MG/U87MG-R, A172/A172-R)

  • Patient-derived primary cultures that maintain original tumor characteristics

• Autophagy assessment methods:

  • Monitor LC3B-II and p62 levels by western blotting

  • Fluorescent tracking of autophagosome formation

  • Electron microscopy for ultrastructural analysis of autophagosomes

• SH3GLB1 modulation approaches:

  • RNA interference (siRNA or shRNA) for knockdown studies

  • Overexpression systems using lentiviral vectors

  • CRISPR/Cas9-based gene editing for complete knockout

• Functional assays:

  • Membrane remodeling assays to assess BAR domain function

  • Co-immunoprecipitation to study interaction with Beclin1 and UVRAG

  • Analysis of autophagic flux using lysosomal inhibitors (chloroquine, bafilomycin A1)

• In vivo validation:

  • Xenograft models with SH3GLB1-modified cells

  • Treatment response studies (e.g., TMZ sensitivity)

These approaches provide complementary data on different aspects of SH3GLB1-mediated autophagy and its functional consequences.

What are the technical considerations for studying SH3GLB1 protein-protein interactions?

When investigating SH3GLB1 protein-protein interactions, researchers should consider several technical factors:

• Co-immunoprecipitation optimization:

  • Use mild lysis buffers to preserve membrane-associated complexes

  • Consider crosslinking approaches for transient interactions

  • Include appropriate controls (IgG, reverse IP)

• Domain-specific interaction studies:

  • Generate constructs with isolated domains (BAR domain, SH3 domain)

  • Create point mutations in key residues for functional validation

  • Use purified recombinant proteins for direct binding assays

• Subcellular localization considerations:

  • SH3GLB1 localizes to multiple compartments (cytoplasm, mitochondria, nucleus)

  • Compartment-specific isolation may be necessary

  • Use immunofluorescence with co-staining to verify interaction locations

• Advanced interaction techniques:

  • Proximity ligation assay for in situ detection of protein interactions

  • FRET/BRET for dynamic interaction studies

  • Biochemical assays to assess how interactions affect protein function (e.g., membrane tubulation assays)

• Specific interactions of interest:

  • SH3GLB1-Beclin1-UVRAG complex formation

  • SH3GLB1-Bax interaction during apoptosis

  • SH3GLB1 binding to PxxP motif-containing proteins

Careful attention to these technical considerations will ensure more reliable and physiologically relevant results when studying SH3GLB1 interactions.

How might SH3GLB1 be targeted therapeutically in glioblastoma?

Based on current understanding, several potential therapeutic strategies targeting SH3GLB1 in glioblastoma warrant investigation:

• Direct SH3GLB1 inhibition:

  • Small molecule inhibitors targeting the SH3 domain to disrupt protein-protein interactions

  • Peptide-based inhibitors mimicking key binding regions

  • Antisense oligonucleotides or siRNA delivery systems for in vivo knockdown

• Combination approaches:

  • SH3GLB1 inhibition + TMZ to overcome resistance

  • SH3GLB1 inhibition + autophagy inhibitors (chloroquine, hydroxychloroquine)

  • SH3GLB1 inhibition + OXPHOS inhibitors to target metabolic vulnerabilities

• Epigenetic modulation strategies:

  • HDAC inhibitors to modify SH3GLB1's effect on H4K5 acetylation

  • Targeting the regulatory elements controlling SH3GLB1 expression

• TIC-directed approaches:

  • Targeting SH3GLB1 specifically in CD133+ populations

  • Developing strategies to penetrate the blood-brain barrier effectively

Future clinical development should consider predictive biomarkers to identify patients most likely to benefit from SH3GLB1-targeted therapies, potentially based on SH3GLB1 expression levels or specific pathway activation signatures.

What are the contradictions in the literature regarding SH3GLB1's role in cancer progression?

The literature presents several apparent contradictions regarding SH3GLB1's role in cancer:

• Tumor suppressor vs. oncogenic functions:

  • Loss of SH3GLB1 is associated with carcinogenesis in some studies

  • Enhanced SH3GLB1 expression is linked to treatment resistance and worse prognosis in glioblastoma

• Autophagy role dichotomy:

  • SH3GLB1-mediated autophagy can promote cell death in some contexts

  • In GBM, SH3GLB1-mediated autophagy appears to promote survival and treatment resistance

• Context-dependent interactions:

  • SH3GLB1 interaction with Bax promotes apoptosis

  • Under stress conditions, SH3GLB1 may primarily engage with autophagic machinery rather than apoptotic pathways

These contradictions likely reflect:

• Cell type-specific functions

• Context-dependent signaling (stress conditions, genetic background)

• Different roles depending on disease stage and microenvironment

• Varying experimental models and conditions across studies

Resolving these contradictions will require comprehensive studies across multiple cancer types, using standardized methodologies and careful consideration of cellular context.

How does SH3GLB1 post-translational modification affect its function in different cellular contexts?

Post-translational modifications (PTMs) of SH3GLB1 represent an emerging area of research with implications for understanding its context-dependent functions:

• SUMOylation:

  • SUMO2-mediated SUMOylation of SH3GLB1 has been identified in cardiac cells

  • This modification promotes ionizing radiation-induced hypertrophic cardiomyopathy through mitophagy activation

  • The specific lysine residues targeted and the enzymes involved require further characterization

• Potential phosphorylation:

  • Computational prediction identifies several potential phosphorylation sites

  • Phosphorylation could affect membrane binding, protein interactions, or subcellular localization

  • Studies validating these sites and their functional consequences are needed

• Other potential modifications:

  • Ubiquitination may regulate SH3GLB1 protein levels

  • Acetylation could affect nuclear functions

  • Oxidative modifications might link SH3GLB1 function to cellular redox state

The study of SH3GLB1 PTMs would benefit from:

• Proteomics approaches to identify modification sites

• Generation of modification-specific antibodies

• Creation of modification-mimicking or modification-resistant mutants

• Investigation of how modifications change in response to cellular stress or disease states

Understanding these modifications may help explain the seemingly contradictory functions of SH3GLB1 in different contexts and potentially reveal new therapeutic opportunities.

What are the most significant unanswered questions about SH3GLB1 in human disease?

Despite significant advances, several critical questions remain unanswered:

• Mechanistic understanding:

  • How does SH3GLB1 specifically regulate mitochondrial fission/fusion events?

  • What determines whether SH3GLB1 promotes cell survival or cell death?

  • How does nuclear SH3GLB1 regulate gene expression?

• Clinical significance:

  • Can SH3GLB1 expression serve as a reliable prognostic or predictive biomarker?

  • How does SH3GLB1 function differ across cancer types and stages?

  • What is the role of SH3GLB1 in non-cancerous pathologies?

• Therapeutic potential:

  • Is SH3GLB1 a viable therapeutic target in treatment-resistant cancers?

  • What are the potential side effects of SH3GLB1 modulation given its multiple cellular roles?

  • Can SH3GLB1-targeted therapies be effectively delivered to the brain?

Addressing these questions will require interdisciplinary approaches combining structural biology, advanced imaging techniques, systems biology, and translational research using clinically relevant models.

How can researchers integrate multi-omics approaches to better understand SH3GLB1 biology?

A comprehensive multi-omics strategy for SH3GLB1 research could include:

• Genomic approaches:

  • Whole genome/exome sequencing to identify mutations

  • GWAS to link SH3GLB1 variants with disease susceptibility

  • eQTL analysis to identify regulatory variants

• Transcriptomic integration:

  • Single-cell RNA sequencing to identify cell populations with varying SH3GLB1 expression

  • Spatial transcriptomics to map SH3GLB1 expression in tissue context

  • Alternative splicing analysis to identify tissue-specific isoforms

• Proteomic strategies:

  • Proximity-dependent biotinylation (BioID) to identify the complete SH3GLB1 interactome

  • Phosphoproteomics to map signaling pathways

  • Structural proteomics to understand complex formation

• Metabolomic integration:

  • Analysis of metabolic changes associated with SH3GLB1 modulation

  • Stable isotope tracing to track metabolic flux

  • Lipidomics to study membrane composition changes

• Data integration frameworks:

  • Network analysis to identify functional modules

  • Machine learning approaches to predict SH3GLB1 function in different contexts

  • Clinical data integration to assess relevance to human disease