TGFBRAP1 Human

What is TGFBRAP1 and where is it located in the human genome?

TGFBRAP1 (transforming growth factor beta receptor associated protein 1) is a protein-coding gene located on chromosome 2 in the human genome. It is also known by synonyms TRAP-1, TRAP1, and VPS3, though these should not be confused with other proteins sharing similar nomenclature. The gene has multiple transcript variants, with multiple RefSeq entries including NM_004257 and NM_001142621 . TGFBRAP1 encodes a protein that specifically binds to TGF-beta receptors and plays a crucial role in TGF-beta signaling, acting as both a signaling mediator and receptor stabilizer.

What are the primary functions of TGFBRAP1 in cellular signaling?

TGFBRAP1 serves multiple functions in cellular signaling pathways:

• Acts as a chaperone in signaling downstream of TGF-beta

• Mediates signal-dependent association with SMAD4

• Stabilizes TGF-β receptor type 1 (TGFBR1) by protecting it from ubiquitination and degradation

• Competes with E3 ubiquitin ligases Smurf1/2 for binding to TGFBR1

• Participates in positive feedback regulation of the TGF-β signaling pathway

• Functions as a component of mammalian CORVET, a multisubunit tethering protein complex involved in fusion of early endosomes

The protein plays a pivotal role in maintaining and enhancing TGF-β signaling through its protective effect on TGFBR1, making it a significant factor in cellular processes regulated by this pathway.

How does TGFBRAP1 interact with the TGF-β signaling pathway?

TGFBRAP1 interacts with the TGF-β signaling pathway through several mechanisms:

• Receptor stabilization: TGFBRAP1 binds to and stabilizes TGFBR1 by preventing its ubiquitination and subsequent proteasomal degradation

• Competition with E3 ligases: It competes with E3 ubiquitin ligases Smurf1/2 for binding to TGFBR1, thereby inhibiting receptor degradation

• Promotion of SMAD signaling: By stabilizing TGFBR1, TGFBRAP1 enhances phosphorylation of SMAD2 and SMAD3, critical downstream effectors of TGF-β signaling

• Feedback regulation: TGFBRAP1 is itself transcriptionally upregulated by the SMAD2/3 complex, creating a positive feedback loop that further potentiates TGF-β signaling

This multilevel involvement makes TGFBRAP1 a key regulator of TGF-β signaling strength and duration in cells.

What are the recommended approaches for manipulating TGFBRAP1 expression in experimental settings?

For effective manipulation of TGFBRAP1 expression in experimental settings, researchers should consider the following methodologies:

• RNA interference:

  • shRNA lentiviral particles targeting TGFBRAP1 are available with verified sequences corresponding to the target gene with 100% identity

  • Multiple variants at the gene locus may require specific targeting; utilizing at least 4 unique 29mer target-specific shRNAs with a scramble control is recommended

  • Knockdown efficiency should be evaluated 72 hours post-transfection, preferably by Western blot rather than qPCR

• CRISPR activation/knockout:

  • CRISPR/dCas9 activation systems have been successfully used to upregulate TGFBRAP1

  • When designing CRISPR experiments, genome-wide screening approaches can identify TGFBRAP1 as a significant hit in phenotypic assays

• Ectopic expression:

  • Overexpression vectors can be used to study the effects of enhanced TGFBRAP1 activity

  • Rescue experiments involving TGFBRAP1 re-expression in knockout cells provide valuable insights into its function

When manipulating TGFBRAP1, it is critical to verify the specificity of the targeting approach and to assess effects on both receptor levels and downstream signaling events.

How can researchers effectively measure TGFBRAP1's impact on TGF-β signaling?

To quantify TGFBRAP1's impact on TGF-β signaling, researchers should employ multiple complementary approaches:

• Phosphorylation assessment:

  • Immunoblotting for phosphorylated SMAD2/3 levels with and without TGF-β stimulation

  • Monitor phosphorylated TGFBR1 levels as a direct indicator of receptor activation

• Protein stability assays:

  • Cycloheximide chase assays to measure TGFBR1 half-life in the presence or absence of TGFBRAP1

  • Proteasome inhibitor studies to confirm the role of proteasomal degradation

• Ubiquitination assays:

  • Immunoprecipitation followed by ubiquitin immunoblotting to assess TGFBR1 ubiquitination levels

  • Co-transfection with E3 ligases (Smurf1/2) to evaluate competitive binding

• Transcriptional readouts:

  • Reporter assays using SMAD-responsive elements

  • qRT-PCR for TGF-β target genes to assess pathway activation

• Protein-protein interaction studies:

  • Co-immunoprecipitation of TGFBRAP1 with TGFBR1 and other pathway components

  • Proximity ligation assays to visualize interactions in situ

These approaches provide a comprehensive assessment of how TGFBRAP1 modulates TGF-β signaling at multiple levels within the pathway.

What cell models are most appropriate for studying TGFBRAP1 functions?

Based on current research, the following cell models are particularly suitable for studying TGFBRAP1 functions:

• Hepatocellular carcinoma (HCC) cell lines:

  • Huh7 and PLC/PRF/5 cells have been effectively used to study TGFBRAP1's role in cancer stemness and drug resistance

  • These models show clear responses to TGFBRAP1 manipulation in terms of TGF-β signaling and stemness properties

• Patient-derived organoids:

  • Provide a more physiologically relevant context for studying TGFBRAP1's effects on cell growth and differentiation

  • Have demonstrated measurable responses to TGFBRAP1 overexpression

• Xenograft models:

  • In vivo models using TGFBRAP1-manipulated cells allow assessment of tumorigenic capacity and drug response

  • Enable evaluation of TGFBRAP1's role in tumor formation and treatment resistance

When selecting a model system, researchers should consider the baseline expression levels of TGFBRAP1 and TGF-β pathway components, as well as the specific cellular process being investigated (e.g., stemness, drug resistance, signaling dynamics).

How does TGFBRAP1 contribute to cancer stemness and drug resistance mechanisms?

TGFBRAP1 has been identified as a key regulator of cancer stemness and drug resistance, particularly in hepatocellular carcinoma (HCC), through several interconnected mechanisms:

• Enhancement of stemness properties:

  • TGFBRAP1 upregulates stem cell markers including CD24, CD133, CD44, and EpCAM

  • It promotes self-renewal capabilities as demonstrated by increased tumor sphere formation

  • Enables greater colony formation in soft agar assays

  • Enhances growth of patient-derived organoids

• Drug resistance development:

  • TGFBRAP1 overexpression confers resistance to tyrosine kinase inhibitors (TKIs) including Regorafenib, Sorafenib, and Lenvatinib

  • Xenograft models with TGFBRAP1-overexpressing cells show reduced sensitivity to Regorafenib treatment

  • Conversely, TGFBRAP1 silencing increases sensitivity to TKIs

• Signaling pathway modulation:

  • By strengthening TGF-β signaling through TGFBR1 stabilization, TGFBRAP1 creates a positive feedback loop

  • This enhanced signaling maintains a stem-like phenotype that is inherently more resistant to therapeutic agents

  • TGFBRAP1-regulated gene signatures correlate with TGF-β activity and stemness signatures in human HCC tissues

The identification of TGFBRAP1 as a TGF-β-inducible positive feedback regulator provides important insights into mechanisms of acquired drug resistance in cancer.

What is the molecular mechanism by which TGFBRAP1 stabilizes TGFBR1?

The molecular mechanism by which TGFBRAP1 stabilizes TGFBR1 involves a sophisticated interplay of protein-protein interactions and competition for binding sites:

• Competitive binding with E3 ubiquitin ligases:

  • TGFBRAP1 competes specifically with Smurf1 and Smurf2 E3 ubiquitin ligases for binding to TGFBR1

  • This competition occurs in a dose-dependent manner, with higher levels of TGFBRAP1 leading to greater inhibition of Smurf1/2-mediated ubiquitination

  • Notably, TGFBRAP1 does not affect TGFBR1 ubiquitination mediated by other E3 ligases such as WWP1 and NEDD4L

• Prevention of poly-ubiquitination:

  • By blocking Smurf1/2 binding, TGFBRAP1 prevents poly-ubiquitination of TGFBR1

  • This reduced ubiquitination protects TGFBR1 from proteasomal degradation

  • Consequently, TGFBRAP1 increases both total and phosphorylated TGFBR1 levels

• Selective receptor regulation:

  • TGFBRAP1 exhibits specificity for TGFBR1, with minimal effects on TGFBR2 levels

  • This selective stabilization enhances downstream signaling through the SMAD2/3 pathway

This mechanistic understanding provides critical insights into how TGFBRAP1 functions as a positive regulator of TGF-β signaling through receptor stabilization rather than as a traditional signaling molecule.

What role does TGFBRAP1 play in establishing feedback loops within the TGF-β signaling pathway?

TGFBRAP1 establishes a critical positive feedback loop within the TGF-β signaling pathway that amplifies and sustains pathway activation:

• TGF-β-induced TGFBRAP1 expression:

  • TGF-β stimulation induces transcriptional upregulation of TGFBRAP1

  • This induction is mediated by the SMAD2/3 transcriptional complex

  • As a result, active TGF-β signaling leads to increased TGFBRAP1 protein levels

• TGFBRAP1-mediated receptor stabilization:

  • Elevated TGFBRAP1 levels protect TGFBR1 from degradation

  • Stabilized TGFBR1 promotes increased phosphorylation of SMAD2/3

  • This enhanced SMAD2/3 activation further increases TGFBRAP1 expression

• Amplification of signaling output:

  • The circular nature of this regulation creates a self-reinforcing loop

  • This positive feedback mechanism sustains and potentially amplifies TGF-β signaling

  • In cancer contexts, this can maintain a stem-like state and promote drug resistance

This feedback regulation represents a mechanism by which transient TGF-β signals can be converted into sustained pathway activation, with significant implications for normal development and disease states.

How is TGFBRAP1 expression correlated with clinical outcomes in cancer?

Based on the available research, TGFBRAP1 expression shows significant correlations with clinical outcomes in cancer, particularly in hepatocellular carcinoma (HCC):

• Expression patterns:

  • TGFBRAP1 expression is elevated in cancer stem cell (CSC) populations

  • High TGFBRAP1 expression correlates with TGF-β signaling activity in human HCC tissues

  • TGFBRAP1 levels are associated with stemness signatures in clinical HCC samples

• Prognostic implications:

  • High TGFBRAP1 expression is linked to higher tumorigenic capacity in xenograft models

  • TGFBRAP1-regulated gene signatures correlate with cancer progression markers

  • The connection to drug resistance suggests poorer response to standard therapies

• Therapeutic resistance:

  • TGFBRAP1 overexpression reduces sensitivity to tyrosine kinase inhibitors (TKIs) including Regorafenib, Sorafenib, and Lenvatinib

  • Depletion of TGFBRAP1 increases sensitivity to these therapies, suggesting its value as a predictive biomarker for treatment response

These findings indicate that TGFBRAP1 expression levels may serve as both a prognostic biomarker for cancer progression and a predictive marker for response to targeted therapies.

What are potential therapeutic strategies targeting TGFBRAP1 or its interactions?

Several potential therapeutic strategies targeting TGFBRAP1 or its interactions emerge from current research:

• Direct TGFBRAP1 inhibition:

  • RNA interference approaches using shRNA to downregulate TGFBRAP1 expression

  • Small molecule inhibitors that could disrupt TGFBRAP1 binding to TGFBR1

  • Peptide-based competitors that mimic TGFBRAP1 binding sites but lack functional activity

• Combination therapies:

  • Co-administration of TGFBRAP1 inhibitors with TKIs to overcome resistance

  • Dual targeting of both TGFBRAP1 and downstream TGF-β signaling components

  • Sequential therapy regimens that first disrupt TGFBRAP1 function before administering conventional treatments

• Targeting the feedback loop:

  • Disruption of the positive feedback between SMAD2/3 activation and TGFBRAP1 expression

  • Interventions that block transcriptional upregulation of TGFBRAP1

  • Agents that interfere with the competitive binding between TGFBRAP1 and Smurf1/2

Research suggests that blocking the feedback activation of TGF-β signaling by TGFBRAP1 could increase cancer cell sensitivity to TKIs by decreasing cancer stem cell stemness, highlighting a promising therapeutic approach for eliminating resistant cancer cells.

How might TGFBRAP1's dual role in endosomal function and TGF-β signaling be leveraged in research?

TGFBRAP1's dual functionality in both endosomal processes and TGF-β signaling presents unique research opportunities:

• Integrated signaling-trafficking studies:

  • Investigation of how endosomal trafficking affects TGF-β receptor stability and signaling

  • Exploration of whether TGFBRAP1's role in CORVET complex influences TGF-β receptor internalization and recycling

  • Assessment of whether differential subcellular localization of TGFBRAP1 affects its receptor-stabilizing functions

• Systems biology approaches:

  • Development of computational models that integrate trafficking dynamics with signaling outputs

  • Proteomics studies to identify TGFBRAP1 interaction partners in different cellular compartments

  • Spatiotemporal analysis of TGFBRAP1 functions in various cellular contexts

• Therapeutic targeting strategies:

  • Identification of compounds that selectively disrupt either the endosomal or signaling functions

  • Development of targeted approaches that exploit the dual functionality to achieve specific cellular effects

  • Exploration of whether the endosomal function contributes to drug resistance mechanisms

Understanding how these dual roles are coordinated could provide insights into fundamental cellular processes and reveal novel intervention points for diseases associated with dysregulated TGF-β signaling.

What are common challenges in detecting and measuring TGFBRAP1 protein levels?

Researchers often encounter several challenges when detecting and measuring TGFBRAP1 protein levels:

• Antibody specificity issues:

  • Cross-reactivity with related proteins, particularly other TRAP family members

  • Variable recognition of different TGFBRAP1 isoforms

  • Recommendation: Validate antibodies using positive and negative controls, including TGFBRAP1-overexpressing and TGFBRAP1-depleted cells

• Extraction and stability concerns:

  • Potential co-fractionation with membrane-associated proteins due to receptor interactions

  • Sensitivity to extraction conditions that might disrupt protein-protein interactions

  • Recommendation: Optimize lysis buffers and include appropriate protease inhibitors to preserve TGFBRAP1 integrity

• Expression level variations:

  • Cell type-specific expression patterns

  • Dynamic regulation in response to TGF-β signaling

  • Recommendation: Establish baseline expression levels for your specific experimental system

• Detection method sensitivity:

  • Western blot may be more reliable than qPCR for assessing knockdown efficiency

  • Immunofluorescence might require signal amplification techniques

  • Recommendation: Use multiple complementary detection methods for comprehensive analysis

These technical considerations should be carefully addressed when designing experiments involving TGFBRAP1 detection to ensure reliable and reproducible results.

How can researchers distinguish between TGFBRAP1's effects on receptor stability versus other signaling mechanisms?

To differentiate between TGFBRAP1's receptor stabilization effects and other potential signaling mechanisms, researchers should consider the following experimental approaches:

• Temporal analysis of signaling events:

  • Compare the kinetics of TGFBR1 stabilization with downstream SMAD2/3 phosphorylation

  • Use pulse-chase experiments to track receptor fate in the presence/absence of TGFBRAP1

  • Implement time-course analyses after TGF-β stimulation to identify the sequence of events

• Functional separation using mutants:

  • Create TGFBRAP1 mutants that retain binding to TGFBR1 but lack other functional domains

  • Utilize TGFBR1 mutants resistant to ubiquitination to bypass the stabilization effect

  • Express truncated versions of TGFBRAP1 to identify domains critical for different functions

• Specific pathway inhibition:

  • Use TGFBR1 kinase inhibitors (like SB431542) to block signaling while assessing receptor stability

  • Employ proteasome inhibitors to differentiate degradation-dependent from degradation-independent effects

  • Implement Smurf1/2 knockdown to mimic the competitive binding effect of TGFBRAP1

• Rescue experiments with hierarchy analysis:

  • Test whether TGFBR1 overexpression rescues phenotypes in TGFBRAP1-depleted cells

  • Assess whether constitutively active SMAD constructs bypass TGFBRAP1 requirements

  • Determine if proteasome inhibition rescues TGFBR1 levels in TGFBRAP1-knockdown cells

These strategies can help delineate the direct receptor stabilization function from potential additional signaling roles of TGFBRAP1.

What are key considerations when interpreting transcriptomic data related to TGFBRAP1 manipulation?

When analyzing transcriptomic data following TGFBRAP1 manipulation, several important considerations should guide interpretation:

• Pathway overlap assessment:

  • Determine overlap with established TGF-β response gene signatures

  • Distinguish direct TGF-β pathway effects from secondary transcriptional changes

  • Consider cross-talk with other signaling pathways that might be indirectly affected

• Temporal dynamics:

  • Account for early versus late response genes following TGFBRAP1 manipulation

  • Consider the kinetics of the positive feedback loop on transcriptional outputs

  • Evaluate sustained versus transient gene expression changes

• Cell context dependencies:

  • Recognize that TGFBRAP1 effects may vary by cell type and differentiation state

  • Consider baseline activity of the TGF-β pathway in your experimental system

  • Compare findings across multiple cell models when possible

• Functional enrichment analysis:

  • Beyond individual genes, analyze pathway enrichment and gene ontology categories

  • Focus on stemness-related gene sets when studying cancer contexts

  • Consider correlation with clinical datasets to validate biological relevance

• Technical validation:

  • Confirm key transcriptional changes using qRT-PCR

  • Validate protein-level changes for selected targets

  • Use multiple statistical approaches to identify robust gene expression patterns

These considerations help ensure accurate interpretation of the complex transcriptional changes that occur in response to TGFBRAP1 manipulation.

What unexplored aspects of TGFBRAP1 biology warrant further investigation?

Several promising but underexplored aspects of TGFBRAP1 biology deserve further research attention:

• Isoform-specific functions:

  • Multiple transcript variants of TGFBRAP1 exist (NM_001142621, NM_004257, NM_001328646), but their potential differential functions remain largely uninvestigated

  • Research is needed to determine whether specific isoforms preferentially interact with different pathway components

  • Tissue-specific expression patterns of these variants could reveal specialized roles

• Non-canonical TGF-β pathway interactions:

  • While SMAD-dependent functions are established, TGFBRAP1's potential involvement in non-canonical TGF-β signaling through MAP kinases, Rho-like GTPases, or PI3K/AKT pathways remains poorly understood

  • These alternative pathways could contribute to phenotypes not fully explained by canonical signaling

• Broader role in endosomal biology:

  • As a component of the mammalian CORVET complex involved in early endosome fusion, TGFBRAP1 may influence trafficking of multiple receptor types

  • The interplay between its endosomal function and specific receptor stabilization deserves systematic investigation

• Regulation by post-translational modifications:

  • The potential regulation of TGFBRAP1 itself through phosphorylation, ubiquitination, or other modifications represents a significant knowledge gap

  • Such modifications could provide additional layers of control over its activity and interactions

These research areas could yield important insights into both fundamental cell biology and disease mechanisms involving TGFBRAP1.

How might advanced technologies enhance our understanding of TGFBRAP1 functions?

Cutting-edge technologies offer promising approaches to deepen our understanding of TGFBRAP1 functions:

• Proximity labeling proteomics:

  • BioID or APEX2 fusion proteins could identify the complete interactome of TGFBRAP1 in living cells

  • Compartment-specific proximity labeling could reveal differential binding partners in distinct subcellular locations

  • Temporal analysis following TGF-β stimulation would map dynamic interaction changes

• Cryo-electron microscopy:

  • Structural studies of TGFBRAP1 in complex with TGFBR1 and/or Smurf1/2

  • Visualization of how TGFBRAP1 integrates into the CORVET complex

  • Identification of critical binding interfaces for potential therapeutic targeting

• Live-cell imaging techniques:

  • FRET/FLIM approaches to monitor TGFBRAP1-receptor interactions in real-time

  • Optogenetic control of TGFBRAP1 function to assess temporal aspects of signaling

  • Super-resolution microscopy to visualize endosomal dynamics influenced by TGFBRAP1

• Single-cell multi-omics:

  • Correlation of TGFBRAP1 expression with transcriptomic and proteomic profiles at single-cell resolution

  • Identification of cell state transitions influenced by TGFBRAP1 levels

  • Characterization of heterogeneous responses within populations

These technological approaches could resolve current knowledge gaps and provide unprecedented insights into TGFBRAP1's multifaceted cellular functions.

What are potential therapeutic applications of TGFBRAP1 research beyond cancer?

While current research focuses predominantly on TGFBRAP1 in cancer, several other therapeutic applications warrant exploration:

• Fibrotic disorders:

  • Given TGF-β's central role in fibrosis, TGFBRAP1 inhibition might attenuate excessive scarring

  • Potential applications in liver fibrosis, pulmonary fibrosis, and renal fibrosis

  • Targeted delivery to fibroblasts could provide specificity in anti-fibrotic approaches

• Immune regulation:

  • TGF-β signaling shapes immune responses; TGFBRAP1 modulation might influence immunosuppression

  • Potential applications in autoimmune disorders where TGF-β signaling is dysregulated

  • Combination with immunotherapies could enhance cancer treatment outcomes

• Regenerative medicine:

  • Controlled manipulation of TGFBRAP1 might influence stem cell differentiation pathways

  • Temporal modulation could promote tissue repair while limiting fibrotic responses

  • Applications in wound healing and tissue engineering contexts

• Developmental disorders:

  • Given TGF-β's role in embryogenesis, TGFBRAP1 variants might contribute to developmental abnormalities

  • Understanding these connections could lead to prenatal interventions or gene therapies

  • Model organisms could help elucidate TGFBRAP1's role in development