Recombinant Tetraspanning orphan receptor (TOR)

What is Tetraspanning orphan receptor (TOR) and what are its known biological functions?

Tetraspanning orphan receptors (TORs) comprise several distinct proteins found across different organisms. These proteins belong to the broader class of seven transmembrane-spanning proteins (7TM), also known as G protein-coupled receptors (GPCRs) . The term "orphan" indicates that the endogenous ligands for these receptors have not yet been definitively identified.

Several specific TOR proteins have been characterized:

• SmTOR: Found in the parasite Schistosoma mansoni, this tegument membrane protein is expressed most highly in cercariae. SmTOR contains an extracellular domain (SmTORed1) that binds complement component C2, interfering with its cleavage by C1s and limiting C3 convertase formation. This suggests SmTOR plays a role in immune evasion during early infection stages .

• Thymus Orphan Receptor: A nuclear receptor expressed predominantly in the thymus and T cell lines that binds to specific DNA sequences. This receptor can repress thyroid hormone receptor (TR) and retinoic acid receptor (RAR) activity, potentially modulating hormone signaling in the thymus .

• Trypanosoma cruzi TOR: Found in this parasitic organism, although specific functions are less extensively characterized in current literature .

What experimental approaches are commonly used to study TOR expression and function?

Researchers employ several methodological approaches to investigate TOR proteins:

Protein Expression and Characterization:

• Recombinant protein expression using bacterial systems with vectors like pET15b

• Protein purification via affinity chromatography (typically utilizing His-tags)

• Structural characterization through domain-specific cloning and expression

Functional Analysis:

• Complement binding assays for SmTOR to assess C2 interaction

• DNA binding studies for thymus orphan receptor using electrophoretic mobility shift assays with specific sequences (5′-AGGTCA-3′ direct repeats with defined nucleotide spacers)

• Transcriptional reporter assays to assess regulatory activity on target genes

Immunological and Vaccine Studies:

• Generation of specific antibodies using recombinant domains as immunogens

• Protection studies in animal models (e.g., mice immunized with rSmTORed1 showed 45-64% reduction in worm burden in schistosomiasis challenges)

• Analysis of antibody responses in infected human populations

How do tetraspanins like TOR organize membrane microdomains and regulate signaling?

Tetraspanins function as molecular scaffolds that organize proteins into highly structured microdomains containing adhesion, signaling, and adaptor proteins . These tetraspanin-enriched microdomains (TEMs) play crucial roles in cellular signaling through several mechanisms:

Membrane Organization:

• Tetraspanins create specialized membrane zones that concentrate signaling molecules

• They regulate the spatial distribution and clustering of receptors like EGFR, affecting their activation dynamics

• TEMs collaborate with glycosphingolipid-enriched microdomains to form functional signaling platforms

Signaling Regulation:

• Tetraspanins modulate various signaling pathways including:

  • EGFR signaling and endocytosis

  • RhoA activation and cytoskeletal organization

  • PKC membrane stabilization and downstream ERK1/2 activation

Structural Factors:

• Palmitoylation of tetraspanins is critical for their lateral associations within TEMs

• Loss of palmitoylation disrupts protein-protein interactions and reduces downstream signaling

• Ubiquitination at specific lysine residues (e.g., K8, K11, K17 in CD151) regulates tetraspanin surface expression and signaling capabilities

What challenges exist in producing recombinant TOR proteins for structural studies?

Production of recombinant tetraspanning proteins presents several technical challenges:

Expression System Selection:

• Bacterial systems (e.g., with pET15b vector) work well for soluble domains like SmTORed1

• Full-length membrane proteins often require eukaryotic expression systems

• Choice depends on specific requirements for post-translational modifications

Protein Solubility and Stability:

• Transmembrane domains create hydrophobicity challenges

• Strategies include:

  • Using only soluble domains (e.g., SmTORed1)

  • Fusion with solubility-enhancing tags

  • Optimizing detergent conditions during extraction and purification

Functional Validation:

• Critical to verify that recombinant protein retains native activity

• For SmTOR, this includes verifying C2 binding capability

• For thymus orphan receptor, DNA binding specificity must be preserved

Storage Considerations:

• Recombinant proteins typically require storage in optimized buffers

• For example, TOR proteins may be stored in Tris-based buffers with 50% glycerol at -20°C or -80°C

• Repeated freeze-thaw cycles should be avoided

How does the post-translational modification of tetraspanins affect TOR function?

Post-translational modifications, particularly palmitoylation, significantly impact tetraspanin function and likely affect TOR proteins similarly:

Palmitoylation Effects:

• Critical for protein-protein interactions within TEMs

• Palmitoylation-deficient tetraspanins show weakened associations with partner proteins

• Example: Palmitoylation-deficient CD151 has diminished integration with integrins, reducing AKT phosphorylation

• CD82 palmitoylation affects PKC membrane stabilization and ERK1/2 activation

Ubiquitination Regulation:

• Tetraspanins are ubiquitinated at specific N-terminal lysine residues

• CD81 and CD151 interact with GRAIL (gene related to anergy in lymphocytes), promoting their ubiquitination

• Key sites include K8/K11 for CD81 and K8/K11/K17 for CD151

• TSPAN6 ubiquitination at K11, K16, and K43 regulates RLR signaling by inhibiting signalosome formation

Methodological Approaches:

• Site-directed mutagenesis to replace specific cysteine residues

• Comparing wild-type and modification-deficient mutants

• Mass spectrometry to identify modification sites

• Functional assays to assess impact on signaling and protein interactions

What strategies can identify potential ligands for orphan TORs?

Identifying ligands for orphan receptors remains challenging. Several complementary approaches can be employed:

Classical Deorphanization Strategies:

• Screening potential ligand libraries against receptor-expressing cells

• Monitoring changes in second messenger levels upon ligand binding

• Receptor binding assays with labeled candidate ligands

Alternative Approaches:

• Phenotypic characterization of organisms with silenced or overexpressed TOR proteins

• Assessment of constitutive receptor activity (ligand-independent signaling)

• Analysis of protein complexes containing TOR proteins

• Development of synthetic surrogate ligands

Functional Classification:

• Some orphan receptors may be "true orphans" for which no natural ligand exists

• Others may be "conditional orphans" that behave as orphans only in the absence of ligand

Specific Examples:

• For thymus orphan receptor TOR, transient transfection experiments have been used to test various nuclear receptor ligands, though no activating ligand has been identified

• TOR's relationship to ROR alpha/RZR alpha (90% similarity in DNA-binding domain, 53% in ligand-binding domain) may provide clues to potential ligands

How does SmTOR interact with complement system components and what are the implications for vaccine development?

SmTOR demonstrates important interactions with the complement system that make it a promising vaccine target:

Complement Regulation:

• SmTORed1 contains a C-terminal H17 motif that binds complement component C2

• This interaction interferes with C2 cleavage by C1s, limiting C3 convertase formation

• Highest expression in cercariae suggests a role in immune evasion during early infection

Vaccine Potential:

• Recombinant SmTORed1 immunization of BALB/c mice using CFA/IFA adjuvants generates high-titer antibodies

• Significant protection against parasite infection was observed:

  • 64% reduction in worm burden in first trial

  • 45% reduction in worm burden in second trial (compared to control groups)

Immunological Considerations:

• Natural infection rarely induces anti-SmTORed1 antibodies (only 10% of infected patients)

• This suggests immunization with isolated domains may be more effective than natural immunity

• The first extracellular domain appears particularly immunogenic when used as a recombinant protein

What is known about the role of thymus orphan receptor TOR in transcriptional regulation?

The thymus orphan receptor variant of TOR functions as a nuclear receptor with specific roles in transcriptional regulation:

DNA Binding Properties:

• Binds specifically to direct repeats of the half-site sequence 5′-AGGTCA-3′ with four or five nucleotide spacers

• These sequences also serve as binding sites for thyroid hormone receptors (TR) and retinoic acid receptors (RAR)

Transcriptional Activity:

• Does not activate reporter genes in transient transfection experiments (with or without known nuclear receptor ligands)

• Functions primarily as a repressor, inhibiting TR and RAR activity on their respective response elements (DR-4-TREs or DR-5-RAREs)

Regulatory Mechanism:

• Similar to COUP-TF in its ability to negatively regulate retinoic acid and thyroid hormone signals

• Differs from COUP-TF in response element recognition and tissue expression pattern

Tissue Specificity:

• Expressed predominantly in the thymus and T cell lines

• Likely modulates retinoid and thyroid hormone signaling specifically in thymic tissues

How does TOR signaling influence cellular protein production, and what are the experimental systems to study this relationship?

TOR signaling affects protein production through several mechanisms, though some confusion exists due to shared acronyms:

Clarification:

• The Target of Rapamycin (TOR) pathway should not be confused with Tetraspanning orphan receptor (TOR)

• Both may influence protein production through different mechanisms

TOR Signaling Pathway:

• Central regulator of cell growth and protein synthesis

• Controls translation and ribosome biogenesis

• May influence recombinant protein production capacity in expression systems

Experimental Approaches:

• Comparing protein yields with TOR pathway modulators (e.g., rapamycin)

• Genetic manipulation of TOR pathway components

• Optimization of culture conditions affecting TOR signaling (nutrients, energy status)

Model Systems:

• Yeast expression systems have been valuable for studying TOR pathway effects

• Mammalian cell lines allow investigation of more complex regulatory networks

• Relationships between tetraspanning proteins and the TOR signaling pathway represent an area needing further research

What analytical techniques are most effective for studying TOR structure-function relationships?

Researchers employ multiple complementary techniques to elucidate TOR structure-function relationships:

Molecular Biology Approaches:

• Cloning strategies using sticky-end PCR and appropriate restriction sites (e.g., NdeI and BamHI)

• Site-directed mutagenesis to create specific domain variants

• Expression of isolated domains (e.g., SmTORed1) for functional studies

Structural Analysis:

• X-ray crystallography of soluble domains

• NMR spectroscopy for smaller domains and peptides

• Computational modeling based on related proteins with known structures

Functional Assays:

• Complement binding assays for SmTOR variants

• DNA binding studies for thymus orphan receptor

• Reporter gene assays to assess transcriptional regulation

• Protein-protein interaction studies (co-immunoprecipitation, FRET)

How can researchers effectively design experiments to study TOR in different cellular contexts?

Experimental design for TOR studies requires careful consideration of biological context:

Expression System Selection:

• Match expression system to research question:

  • Bacterial systems for protein production and structural studies

  • Mammalian systems for functional studies in relevant cellular contexts

  • Parasite models for host-pathogen interaction studies

Functional Analysis Strategy:

• For SmTOR: Focus on complement regulation and immune evasion

• For thymus orphan receptor: Examine transcriptional regulation in thymic cells

• For tetraspanin functions: Investigate membrane microdomain organization and signaling

Genetic Approaches:

• Gene silencing (RNAi, CRISPR) to assess loss-of-function phenotypes

• Overexpression studies to identify gain-of-function effects

• Domain-specific mutations to map functional regions

Contextual Considerations:

• Stage-specific expression (e.g., SmTOR in cercariae)

• Tissue-specific functions (e.g., thymus orphan receptor in T cells)

• Interactions with specific signaling pathways in different cell types

How should researchers interpret contradictory findings regarding TOR function across different experimental systems?

Researchers face several challenges when interpreting conflicting TOR data:

Source of Contradictions:

• Different TOR proteins across species (parasite vs. mammalian)

• Distinct cellular contexts (immune cells vs. parasites)

• Varied experimental approaches (in vitro vs. in vivo)

Resolution Strategies:

• Carefully define which TOR protein is under investigation

• Consider species-specific and context-dependent functions

• Directly compare experimental approaches in standardized systems

• Evaluate whether contradictions reflect true biological complexity or technical artifacts

Example Resolutions:

• For tetraspanin-related contradictions: CD151 loss showed increased RhoA activation in breast cancer cells (using FRET biosensors) but no change in other studies (using pull-down assays), possibly due to differences in detection sensitivity

• For thymus orphan receptor, contradictory findings might be reconciled by considering tissue-specific cofactors that modify its activity

What are the most promising research applications for recombinant TOR proteins?

Recombinant TOR proteins offer several valuable research and therapeutic applications:

Vaccine Development:

• SmTOR as a vaccine candidate against schistosomiasis

• Recombinant SmTORed1 demonstrated significant protection in mouse models:

  • 64% reduction in worm burden in first trial

  • 45% reduction in second trial

• Focus on parasite-specific TORs to minimize cross-reactivity with host proteins

Drug Discovery:

• Screening platforms for compounds that modulate TOR function

• Development of synthetic ligands for orphan receptors

• Target-based drug design using structural information

Diagnostic Applications:

• Detection of anti-TOR antibodies in infection (though natural infection rarely induces them)

• TOR-based immunoassays for parasitic infections

Basic Research Tools:

• Probes for studying membrane organization and signaling

• Models for understanding orphan receptor biology and evolution

• Systems for investigating host-parasite interactions

How can researchers effectively integrate TOR studies with broader research on membrane organization and signaling?

TOR research can be integrated with broader membrane biology through several approaches:

Tetraspanin Web Investigation:

• Study TOR within the context of tetraspanin-enriched microdomains (TEMs)

• Examine co-localization with other tetraspanins and signaling molecules

• Investigate how TORs contribute to the "tetraspanin web" organization

Signaling Pathway Integration:

• Connect TOR function to established signaling networks:

  • For tetraspanin TORs: EGFR, RhoA, and PKC pathways

  • For thymus orphan receptor: Retinoid and thyroid hormone signaling pathways

  • For parasite TORs: Complement regulation pathways

Multidisciplinary Approaches:

• Combine structural biology, cell biology, and systems biology

• Use computational modeling to predict TOR functions in complex networks

• Develop integrated experimental systems that capture physiological complexity

Translational Applications:

• Connect basic TOR research to applications in:

  • Infectious disease (parasite TORs)

  • Immunology (thymus orphan receptor)

  • Cancer biology (tetraspanin signaling)

What are the most significant gaps in our understanding of TOR biology?

Despite progress, several critical knowledge gaps remain:

Ligand Identification:

• Endogenous ligands for many TOR proteins remain unknown

• Distinguishing between "true orphans" and "conditional orphans"

• Developing more effective deorphanization strategies

Structural Information:

• Limited high-resolution structures for full-length TOR proteins

• Incomplete understanding of conformational changes during activation

• Need for structural studies in membrane environments

Physiological Roles:

• Incomplete understanding of TOR functions in normal physiology

• Limited knowledge of developmental and tissue-specific roles

• Unclear evolutionary relationships between different TOR proteins

Integration with Other Systems:

• Connections between TOR and other membrane organization mechanisms

• Interactions with cytoskeletal elements and trafficking machinery

• Relationships between different TOR types (parasite, thymus, etc.)

What emerging technologies and approaches will advance TOR research?

Several cutting-edge approaches show promise for TOR research:

Advanced Imaging:

• Super-resolution microscopy to visualize TEMs and protein organization

• Single-molecule tracking to monitor TOR dynamics in living cells

• Correlative light and electron microscopy for structural-functional studies

Genetic Engineering:

• CRISPR-based approaches for precise manipulation of TOR genes

• Conditional knockout models to study tissue-specific functions

• Domain-specific mutations to map structure-function relationships

Systems Biology:

• Multi-omics approaches to understand TOR in complex networks

• Computational modeling of TOR-mediated signaling

• Network analysis to identify key interaction partners

Translational Applications:

• High-throughput screening for TOR-targeting compounds

• Advanced vaccine development strategies using recombinant TOR domains

• Biomarker development based on TOR expression or modification patterns