Recombinant Human Transmembrane protein 240 (TMEM240)

What is the cellular localization of TMEM240 protein?

TMEM240 is expressed in various areas of the brain, with the highest levels in the hippocampus, isocortex, and cerebellum. Within the cerebellum, TMEM240 is detected in both deep nuclei and the cerebellar cortex, being expressed in all three layers of the cortex . At the subcellular level, TMEM240 is primarily localized to the post-synaptic side of synapses near the Purkinje-cell soma, as confirmed by co-immunostaining with synaptophysin, post-synaptic fractionation, and electron microscopy . This specific localization pattern suggests that TMEM240 may be involved in organizing cerebellar networks, particularly in synaptic inputs converging on Purkinje cells.

What are the known functions of TMEM240 in normal cellular physiology?

TMEM240 appears to have several key physiological functions, primarily related to cell cycle regulation and cellular migration. In colorectal cancer research, it has been demonstrated that TMEM240 can lead to G1 cell cycle arrest, effectively slowing down cell proliferation . When TMEM240 is overexpressed in cancer cell lines such as DLD-1, growth is repressed by approximately 66.6% compared to control groups . Conversely, knockdown of TMEM240 increases cell growth significantly, with some studies showing up to 15.9-fold increases in colon cancer cell growth . Additionally, transwell assays have revealed that TMEM240 overexpression suppresses the migratory ability of cancer cells by approximately 39.7% . In neurological contexts, TMEM240 appears to be involved in synaptic organization, particularly in Purkinje cells, suggesting a role in cerebellar functionality .

What genetic disorders are associated with TMEM240 mutations?

The primary genetic disorder associated with TMEM240 mutations is Spinocerebellar Ataxia 21 (SCA21). A variety of missense mutations and a stop mutation in the TMEM240 gene have been identified as causative mutations for this condition . SCA21 is characterized by progressive cerebellar ataxia, which aligns with the high expression of TMEM240 in the cerebellum, particularly in Purkinje cells and their synaptic connections . Testing for SCA21 via the TMEM240 gene is available for patients presenting with symptoms suggestive of this disorder .

How does TMEM240 expression influence cancer development and progression?

TMEM240 appears to function as a tumor suppressor in several cancers, particularly colorectal cancer. Research indicates that TMEM240 overexpression significantly inhibits cancer cell proliferation and migration. In DLD-1 colorectal cancer cells, TMEM240 overexpression reduced cell growth by 66.6% compared to vector controls . Mechanistically, this appears to occur through G1 cell cycle arrest, as flow cytometry data shows that the percentage of DLD-1 cells in the G1 phase increased by 4.28% with TMEM240 overexpression .

Hypermethylation of the TMEM240 promoter region is observed in 87.8% (480/547) of colorectal cancer tumors, as well as in benign tubular adenomas (55.6%) . This hypermethylation leads to decreased TMEM240 expression, potentially contributing to cancer progression by removing the protein's inhibitory effects on cell growth and migration. Similar patterns of hypermethylation have been observed in other cancer types, including esophageal cancer (80.0%), gastric cancer (100%), liver cancer (80.4%), and pancreatic cancer (44.4%) .

In breast cancer, circulating methylated TMEM240 in plasma has been investigated as a potential biomarker for monitoring treatment response and disease progression . This suggests that epigenetic regulation of TMEM240 may be a common mechanism across different cancer types.

What methodological challenges exist in studying TMEM240 protein-protein interactions?

Studying TMEM240 protein-protein interactions presents several challenges. As a transmembrane protein, TMEM240 contains extracellular and intracellular domains with distinct interaction partners . Standard co-immunoprecipitation techniques may be insufficient due to the hydrophobic nature of transmembrane regions.

For effective protein-protein interaction studies, researchers should consider:

• Using specialized detergents (such as CHAPS or n-dodecyl β-D-maltoside) that solubilize membrane proteins while preserving native interactions

• Employing split-ubiquitin yeast two-hybrid systems specifically designed for membrane proteins

• Utilizing proximity-dependent biotin identification (BioID) or APEX2 proximity labeling to identify interaction partners in living cells

• Applying crosslinking techniques prior to immunoprecipitation to capture transient interactions

Additionally, researchers should validate antibody specificity for TMEM240, as demonstrated in studies that confirmed specificity through both immunofluorescence and electron microscopy techniques .

How does the methylation status of TMEM240 correlate with clinical outcomes in cancer patients?

DNA hypermethylation of TMEM240 appears to be a frequent event in multiple cancer types and may serve as a prognostic marker. In colorectal cancer studies, 87.8% (480 of 547) of tumors exhibited hypermethylated TMEM240, with methylation levels significantly higher in tumor tissues compared to matched normal colorectal tissues . The median DNA methylation levels (normalized by ACTB) were 0.0098 in CRC tumors, 0.0015 in benign tumors, and 0.0001 in adjacent normal tissues .

In breast cancer, TMEM240 hypermethylation has been investigated as a predictor of poor hormone therapy response . Circulating methylated TMEM240 in plasma samples has been explored as a potential liquid biopsy marker for monitoring treatment response and disease progression. Quantitative methylation-specific PCR (QMSP) techniques are commonly used to assess methylation status, with TMEM240 considered hypermethylated when its methylation level relative to reference genes (such as ACTB) is at least two-fold higher in tumor tissue compared to paired normal tissue .

This correlation between TMEM240 methylation status and clinical outcomes suggests potential utility as both a diagnostic and prognostic biomarker, as well as a possible therapeutic target for epigenetic therapies.

What are the optimal methods for producing and purifying recombinant human TMEM240 protein?

For producing recombinant human TMEM240 protein, several expression systems can be employed, each with particular advantages depending on research needs:

• Mammalian cell expression systems: Given the success with human cell expression systems for related transmembrane proteins , HEK293 or CHO cells are recommended for TMEM240 expression to ensure proper folding and post-translational modifications. Transient transfection using lipofection or polyethylenimine (PEI) methods typically yields sufficient protein for most applications.

• Purification strategy:

  • Initial extraction using mild detergents (0.5-1% DDM or CHAPS)

  • Affinity chromatography using His-tag or Fc-tag systems (similar to approaches used for TNFRSF10B )

  • Size exclusion chromatography for final polishing

• Recommended tags and constructs:

  • C-terminal Fc-6His tag has shown good results for similar transmembrane proteins

  • Consider truncated constructs of the extracellular domain (for studies not requiring the transmembrane portion)

• Quality control metrics:

  • Purity assessment via SDS-PAGE (aim for >95% purity)

  • Endotoxin testing (<1.0 EU per μg by LAL method)

  • Functional validation through binding assays

For storage, lyophilized protein is generally stable for up to 12 months at -20 to -80°C, while reconstituted protein solution can be stored at 4-8°C for 2-7 days, with aliquots remaining stable at < -20°C for 3 months .

What are the most reliable techniques for measuring TMEM240 methylation status in clinical samples?

Based on current research, the most reliable techniques for measuring TMEM240 methylation include:

• Quantitative Methylation-Specific PCR (QMSP):

  • This technique has been successfully applied in multiple studies

  • QMSP should be performed using specific primers and methyl-TaqMan probes designed to bind to the junction between the promoter and exon 1 of TMEM240

  • Beta-actin (ACTB) gene is commonly used as a reference gene

  • QMSP conditions: preincubation at 95°C for 10 min followed by 50 cycles of amplification at 95°C for 10 s and 60°C for 10 s

  • Methylation threshold: TMEM240 is typically considered hypermethylated when its methylation level relative to ACTB is at least 2-5 fold higher in the tumor compared to normal tissue

• Illumina Methylation Arrays:

  • Both 450K and EPIC arrays have been used to identify differentially methylated CpG sites in TMEM240

  • Three key methylation sites (cg16601494, cg15487867, and cg16306898) in the promoter and exon regions of TMEM240 showed significant differences in colorectal cancer samples

  • Average beta value differences (ΔAvg_β) between paired tumor and normal tissues at these sites were 0.47, 0.45, and 0.41, respectively

• Bisulfite Sequencing:

  • Should be used to confirm the specificity of QMSP end products

  • Provides single-base resolution of methylation patterns

For clinical applications, it's recommended to validate findings using at least two complementary methods, such as QMSP followed by bisulfite sequencing confirmation.

What experimental designs are most effective for studying TMEM240's role in cell cycle regulation?

Based on published research, the following experimental design elements are effective for studying TMEM240's role in cell cycle regulation:

• Cell models and manipulation approaches:

  • Colorectal cancer cell lines (DLD-1, HCT116) have demonstrated clear TMEM240-dependent effects

  • Both overexpression (via plasmid transfection) and knockdown (using siRNA) approaches should be implemented

  • For knockdown studies, at least two different siRNAs (e.g., s50536 and s50534) should be used to control for off-target effects

  • Stable cell lines with inducible TMEM240 expression provide more consistent results for long-term studies

• Cell cycle analysis techniques:

  • Flow cytometry with propidium iodide staining has successfully demonstrated TMEM240's effect on G1 arrest

  • EdU incorporation assays to measure S-phase entry

  • Western blotting for cell cycle regulators (cyclins, CDKs) to determine mechanistic pathways

• Growth and viability assessments:

  • Sulforhodamine B (SRB) assays have effectively quantified TMEM240's impact on cell growth

  • Microscopic observations to corroborate growth inhibition findings

  • Real-time cell analysis systems for continuous monitoring of growth kinetics

• Migration and invasion studies:

  • Transwell assays have demonstrated TMEM240's effect on reducing cell migration by approximately 40%

  • Wound healing assays for complementary migration assessment

  • 3D spheroid invasion assays for more physiologically relevant models

A comprehensive experimental design would include time-course analyses of cell cycle progression following TMEM240 manipulation, coupled with gene expression profiling to identify downstream effectors mediating the observed G1 arrest.

How might TMEM240's role in synaptic organization inform therapeutic approaches for SCA21?

TMEM240's expression pattern and subcellular localization provide important insights for developing SCA21 therapeutic approaches. Immunofluorescence studies show TMEM240 is primarily expressed in the hippocampus, isocortex, and cerebellum, with particular enrichment in Purkinje cells . At the subcellular level, TMEM240 is localized to the post-synaptic side of synapses near Purkinje-cell soma, suggesting a critical role in synaptic organization and cerebellar network function .

Potential therapeutic strategies informed by this knowledge include:

• Gene therapy approaches:

  • Adeno-associated virus (AAV) vectors targeting Purkinje cells could deliver functional copies of TMEM240 in SCA21 patients

  • The relatively small size of TMEM240 makes it amenable to viral packaging constraints

• Synaptic modulation:

  • Compounds that enhance synaptic stability or compensate for TMEM240 dysfunction might ameliorate symptoms

  • Targeting post-synaptic protein complexes that interact with TMEM240

• Purkinje cell-specific interventions:

  • Neuroprotective strategies focused on preserving Purkinje cell function

  • Cerebellar stimulation techniques to modulate affected neural circuits

• Biomarker development:

  • TMEM240 expression analysis in accessible tissues or fluids might serve as surrogate markers for disease progression or treatment response

  • Cerebrospinal fluid analysis for TMEM240-related biomarkers

Future research should focus on identifying the exact molecular mechanisms through which TMEM240 mutations lead to Purkinje cell dysfunction and developing high-throughput screens for compounds that can compensate for mutant TMEM240 function.

What quality control measures should be implemented when working with recombinant TMEM240 protein?

To ensure reliable experimental results when working with recombinant TMEM240 protein, implement the following quality control measures:

• Purity assessment:

  • SDS-PAGE analysis with Coomassie or silver staining (aim for >95% purity)

  • Western blot confirmation using specific anti-TMEM240 antibodies

  • Mass spectrometry validation of intact protein mass and peptide coverage

• Functional validation:

  • Binding assays with known interaction partners

  • Circular dichroism to assess secondary structure integrity

  • Size exclusion chromatography to verify monomeric state or appropriate oligomerization

• Contaminant testing:

  • Endotoxin testing using LAL assay (<1.0 EU per μg is considered acceptable)

  • Host cell protein (HCP) ELISA to quantify residual expression system proteins

  • DNA contamination assay using PicoGreen or similar methods

• Stability assessment:

  • Thermal shift assays to determine protein stability

  • Time-course analysis at different storage conditions (4°C, -20°C, -80°C)

  • Freeze-thaw stability tests (limit to 1-2 cycles if possible)

• Batch consistency:

  • Establish reference standards for lot-to-lot comparison

  • Document critical quality attributes for each production batch

  • Implement consistent acceptance criteria across batches

Proper storage conditions are critical: store lyophilized protein at -20 to -80°C (stable for up to 12 months), reconstituted protein at 4-8°C (stable for 2-7 days), and aliquoted samples at < -20°C (stable for 3 months) .

How can researchers verify antibody specificity for TMEM240 in experimental applications?

Verifying antibody specificity for TMEM240 is crucial for reliable experimental results. A comprehensive approach includes:

• Positive and negative controls:

  • Positive: Tissues known to express TMEM240 (cerebellum, hippocampus, isocortex)

  • Negative: TMEM240 knockout cells/tissues or siRNA-treated samples

  • Competitive inhibition with purified recombinant TMEM240 protein

• Multiple antibody validation:

  • Compare results using antibodies targeting different epitopes of TMEM240

  • Verify concordance between monoclonal and polyclonal antibodies

  • Check for cross-reactivity with other TMEM family proteins

• Orthogonal techniques:

  • Complement immunohistochemistry/immunofluorescence with in situ hybridization for TMEM240 mRNA

  • Verify protein expression patterns with transcriptomic data

  • Confirm subcellular localization using fractionation followed by Western blotting

• Specific validation tests:

  • Western blotting should show bands of expected molecular weight

  • Peptide blocking experiments to demonstrate epitope specificity

  • Pre-absorption tests with the immunizing peptide

  • Testing in multiple species if cross-reactivity is claimed

• Application-specific validation:

  • For immunohistochemistry: Test different fixation methods and antigen retrieval protocols

  • For flow cytometry: Compare surface vs. permeabilized staining patterns

  • For immunoprecipitation: Validate pull-down efficiency and specificity

Previous studies have successfully validated TMEM240 antibodies using immunofluorescence labeling in mouse brain tissue, with confirmation through electron microscopy . Similar validation approaches in human tissue samples have shown consistent results, supporting antibody specificity across species.

TMEM240 Expression and Methylation Patterns Across Tissue Types

Effect of TMEM240 Manipulation on Cellular Functions

TMEM240 Methylation Quantification in Clinical Samples

What molecular mechanisms link TMEM240 dysfunction to cerebellar degeneration in SCA21?

Understanding the molecular mechanisms connecting TMEM240 dysfunction to cerebellar degeneration in SCA21 represents a critical research gap. Based on current knowledge of TMEM240's expression in Purkinje cells and its post-synaptic localization , several hypothetical mechanisms warrant investigation:

• Synaptic plasticity disruption: TMEM240 mutations may impair long-term depression or potentiation at parallel fiber-Purkinje cell synapses, compromising motor learning and coordination.

• Protein interaction network perturbation: As TMEM240 is localized near Purkinje cell soma at post-synaptic sites , mutations might disrupt critical protein complexes involved in post-synaptic signaling or structural maintenance.

• Calcium homeostasis dysregulation: Given the importance of calcium signaling in Purkinje cells, TMEM240 might participate in calcium channel regulation or downstream calcium-dependent processes.

• Protein misfolding and aggregation: Certain TMEM240 mutations might lead to protein misfolding, triggering endoplasmic reticulum stress and eventual Purkinje cell degeneration.

Future research should employ transgenic mouse models expressing SCA21-associated TMEM240 mutations to track cerebellar degeneration progression. Electrophysiological studies of Purkinje cells in these models could reveal functional deficits preceding morphological changes. Proteomics approaches, including proximity labeling techniques, could identify TMEM240 interaction partners disrupted by disease mutations, potentially revealing therapeutic targets.

How might TMEM240 be integrated into cancer pathway analysis for personalized medicine approaches?

TMEM240's demonstrated roles in cell cycle regulation, migration inhibition, and its frequent epigenetic silencing in multiple cancer types suggest several promising directions for integration into personalized medicine frameworks:

• Predictive biomarker development:

  • TMEM240 methylation status could potentially predict response to both conventional chemotherapies and targeted treatments

  • Circulating methylated TMEM240 has already shown promise as a monitoring biomarker in breast cancer and colorectal cancer

• Pathway integration:

  • RNA-seq and pathway analysis following TMEM240 manipulation could identify downstream effectors and signaling networks

  • These pathways might reveal synergistic drug combinations for tumors with TMEM240 silencing

• Therapeutic targeting strategy:

  • For tumors with hypermethylated TMEM240, combining demethylating agents with cell cycle inhibitors might provide synergistic benefits

  • TMEM240's role in G1 arrest suggests potential interaction with CDK4/6 inhibitor efficacy

• Patient stratification approach:

  • Multi-gene panels including TMEM240 methylation status could help classify tumors into molecular subtypes with distinct prognosis and treatment responses

  • The high frequency of TMEM240 hypermethylation across multiple cancer types (44.4-100%) suggests broad applicability