Recombinant Bovine Transmembrane protein 100 (TMEM100)

What is the functional significance of TMEM100 in cellular signaling?

TMEM100 functions as a regulatory protein in multiple signaling pathways. Research has identified TMEM100 as a tumor suppressor gene that modulates the TGF-β signaling pathway in cancer cells. Specifically, TMEM100 overexpression inhibits the activation of the TGF-β signaling pathway by reducing TGF-β expression and the phosphorylation of downstream mediators Smad2 and Smad3 . In neuronal tissue, TMEM100 serves as an adaptor protein that regulates the physical and functional interaction between TRPA1 and TRPV1 ion channels, which are critical for pain signal transduction .

How is TMEM100 expression regulated in normal and pathological conditions?

In normal tissues, TMEM100 maintains baseline expression, but its levels are significantly altered in pathological states. In colorectal cancer (CRC), TMEM100 is markedly downregulated compared to normal colorectal tissue . Conversely, in neuroinflammatory conditions such as temporomandibular joint (TMJ) inflammation or masseter muscle injury, TMEM100 expression increases in trigeminal ganglion (TG) neurons . This differential regulation suggests tissue-specific transcriptional control mechanisms that respond to pathological stimuli.

What protein interactions does TMEM100 participate in?

TMEM100 has been identified as a critical regulator of protein complexes:

How does TMEM100 function as a tumor suppressor in colorectal cancer?

TMEM100 suppresses colorectal cancer progression through multiple mechanisms:

• Inhibition of cell proliferation: Overexpression of TMEM100 in NCI-H498 colorectal cancer cells significantly reduces cell proliferation as demonstrated by MTT assays and colony formation experiments .

• Suppression of migration and invasion: TMEM100 overexpression markedly inhibits cancer cell migration in scratch healing assays and reduces invasion capacity in Transwell assays .

• Regulation of EMT: TMEM100 overexpression elevates epithelial marker E-cadherin while downregulating mesenchymal markers N-cadherin and vimentin, thereby inhibiting the EMT process that facilitates metastasis .

• Modulation of TGF-β signaling: TMEM100 reduces TGF-β expression and Smad2/3 phosphorylation, effectively blocking this pathway which typically promotes tumor progression in advanced cancer stages .

What experimental approaches are most effective for studying TMEM100's role in cancer?

Researchers investigating TMEM100 in cancer contexts should consider these methodological approaches:

• Gene expression manipulation: Utilize siRNA for knockdown (si-TMEM100) and overexpression vectors (oe-TMEM100) to modulate TMEM100 levels in cancer cell lines .

• Functional assays:

  • MTT assay for proliferation assessment

  • Colony formation assay for clonogenic potential

  • Scratch healing assay for migration

  • Transwell assay for invasion capacity

• Molecular pathway analysis:

  • Western blot to detect changes in TGF-β pathway components (TGF-β, phospho-Smad2, phospho-Smad3)

  • Analysis of EMT markers (E-cadherin, N-cadherin, vimentin)

• Transcriptomic analysis: Gene Set Enrichment Analysis (GSEA) to identify signaling pathways associated with TMEM100 expression .

How does TMEM100 regulate pain signaling through TRPA1-TRPV1 interaction?

TMEM100 serves as a critical adaptor protein that modulates the interaction between TRPA1 and TRPV1 in sensory neurons:

• Molecular mechanism: TMEM100 weakens the physical association between TRPA1 and TRPV1, resulting in disinhibition of TRPA1 activity. In the absence of TMEM100, TRPV1 forms a tight complex with TRPA1 that suppresses TRPA1 function .

• Pain modulation: Higher TMEM100 expression leads to increased TRPA1 activity in sensory neurons, which enhances nociceptive signaling. This has been demonstrated in trigeminal ganglion neurons, where TMEM100 expression increases following TMJ inflammation or masseter muscle injury .

• Experimental evidence: Ca²⁺-imaging in GCaMP3-expressing TG neurons shows that the percentage of neurons responding to TRPA1 agonist JT010 increases after TMJ inflammation or masseter muscle injury. This enhanced TRPA1 activity can be suppressed by the TMEM100 inhibitor T100-Mut .

What are effective models for studying TMEM100 in pain research?

Researchers studying TMEM100's role in pain signaling can employ these experimental models:

• Animal models of pain:

  • TMJ inflammation model: CFA injection into the TMJ

  • Masseter muscle injury model (TASM)

• Genetic models:

  • Conditional knockout mice: Advillin-creER::Tmem100 fl/fl allows tamoxifen-inducible deletion of TMEM100 specifically in ~98% of TG/DRG neurons

  • Pirt-GCaMP3 mice: Express Ca²⁺ indicator GCaMP3 in >96% of sensory neurons for functional imaging

• Behavioral assessment:

  • Bite force test to measure TMD masticatory pain

  • Normalized bite force changes as quantitative pain indicators

• Pharmacological interventions:

  • TMEM100 inhibitor: T100-Mut (a TMEM100-mutant-derived peptide) administered locally (i.a. or i.m.)

  • TRPA1 inhibitor: HC030031

  • TRPV1 inhibitor: SB366791

What are optimal protocols for detecting TMEM100 co-expression with other proteins?

To effectively study TMEM100's co-localization with interacting proteins:

• Immunohistochemistry techniques:

  • Triple immunofluorescence staining for TMEM100, TRPA1, and TRPV1

  • Retrograde labeling of target tissue-innervating neurons combined with immunostaining

• Quantification methods:

  • Count percentage of TMEM100-positive neurons that express TRPA1 and/or TRPV1

  • Analyze co-expression in specific subpopulations (e.g., neurons innervating TMJ or masseter muscle)

• Sample preparation:

  • For neuronal studies: Fixation and processing of trigeminal ganglia

  • Retrograde tracer application to target tissues 3-7 days before tissue collection

How can researchers effectively evaluate TMEM100's functional impact on cellular processes?

To assess the functional consequences of TMEM100 modulation:

• Ca²⁺-imaging techniques:

  • Ex vivo preparations of GCaMP3-expressing TG neurons

  • Application of specific agonists (e.g., JT010 for TRPA1) with and without TMEM100 inhibitors

  • Quantification of the percentage of responsive neurons and signal intensity

• Cell growth and invasion assays:

  • MTT assay: Seed cells in 96-well plates, apply treatments, add MTT solution, and measure absorbance

  • Colony formation: Plate cells at low density, stain colonies after 7-14 days, and quantify

  • Scratch healing: Create a "wound" in a cell monolayer and measure migration rate

  • Transwell assay: Quantify cells that migrate through a membrane barrier

How might researchers address contradictions between TMEM100's roles in different biological contexts?

While TMEM100 serves as a tumor suppressor in colorectal cancer , it promotes pain signaling in sensory neurons . To reconcile these seemingly contradictory functions:

• Tissue-specific protein interaction mapping: Identify differential binding partners in various cell types using proteomics approaches.

• Comparative signaling pathway analysis: Investigate how TMEM100 modulates different signaling pathways (TGF-β vs. TRPA1-TRPV1) in a context-dependent manner.

• Domain-specific function analysis: Generate truncated or mutant TMEM100 proteins to determine which structural elements are responsible for different functions.

• Transcriptomic analysis: Compare gene expression profiles in response to TMEM100 manipulation across different cell types to identify context-specific downstream effects.

What are the most promising therapeutic applications of TMEM100 research?

Based on current findings, TMEM100 research has several potential therapeutic applications:

• Cancer therapy: Strategies to upregulate or restore TMEM100 expression in colorectal cancer could inhibit tumor growth and metastasis by suppressing the TGF-β pathway and EMT process .

• Pain management: TMEM100 inhibitors such as T100-Mut demonstrate efficacy in reducing temporomandibular disorder pain in animal models. Local administration into affected tissues provides targeted pain relief without systemic effects .

• Combined approaches: For cancer pain, dual targeting of TMEM100's tumor-promoting and pain-facilitating functions could address both the disease and its symptoms.

What are critical gaps in TMEM100 research that require further investigation?

Despite recent advances, several aspects of TMEM100 biology remain unexplored:

• Species-specific variations: More research is needed to characterize differences between bovine, murine, and human TMEM100 in structure and function.

• In vivo validation: The current research on TMEM100 in colorectal cancer lacks in vivo experimental validation and clinicopathological correlation analysis .

• Regulatory mechanisms: The factors controlling TMEM100 expression in different tissues and disease states remain poorly understood.

• Other pain conditions: Investigation is needed to determine if TMEM100 is involved in other types of trigeminal pain beyond TMD, such as dental pain, migraine, and trigeminal neuralgia .

• Structural biology: Detailed structural analysis of TMEM100's interaction with TRPA1-TRPV1 and TGF-β pathway components would provide insights for rational drug design.