Recombinant Human Transmembrane protein 87B (TMEM87B)

What is TMEM87B and what is currently known about its function?

TMEM87B is a highly conserved transmembrane protein whose function remains largely unknown . It is located within the critical region of the recurrent 2q13 microdeletion syndrome . Recent research suggests that TMEM87B plays a crucial role in cardiac development, as shown through morpholino-based knockdown experiments in zebrafish embryos that resulted in cardiac anomalies during development . The protein contains multiple transmembrane domains, with the Asn456 residue located at the end of the last transmembrane domain, a position that suggests functional importance .

What is the genomic location of TMEM87B and which syndromes is it associated with?

TMEM87B is located on chromosome 2, specifically within the 2q13 region (Chr 2:112856265 in hg19 reference genome) . It lies within the critical region of the recurrent 2q13 microdeletion syndrome . This syndrome is characterized by a variable phenotype that can include cardiac defects, intellectual disabilities, and craniofacial anomalies. The variability in the phenotypic presentation may be due to additional single-nucleotide variants on the non-deleted allele of genes within this interval, including TMEM87B .

What structural domains characterize TMEM87B protein?

TMEM87B contains multiple transmembrane domains typical of membrane proteins . The Asn456 residue, which is evolutionarily conserved across multiple species, is located at the end of the last transmembrane domain . This conservation suggests a critical role in protein function, and mutations at this position (such as p.Asn456Asp) have been associated with cardiac anomalies . The complete three-dimensional structure of TMEM87B has not been fully characterized in the provided search results, which is common for membrane proteins that present challenges for structural determination.

Which expression systems are most suitable for producing recombinant TMEM87B?

For transmembrane proteins like TMEM87B, the choice of expression system is critical and depends on research objectives. While the search results don't specifically address TMEM87B expression, general principles for membrane protein expression can be applied:

The optimal choice depends on specific research needs, whether focused on functional studies (where native folding is critical) or structural studies (where higher yields may be prioritized).

What are the advantages and limitations of different expression hosts for TMEM87B production?

Each expression system presents distinct advantages and limitations for membrane protein expression as outlined in the comparative table below:

For TMEM87B, mammalian expression systems would likely provide the most native environment for proper folding and function, though at higher cost and potentially lower yields than other systems .

How can expression of recombinant TMEM87B be optimized in E. coli systems?

While E. coli presents challenges for membrane protein expression, several strategies can optimize TMEM87B expression if this system is selected:

• Induction strategies: T7 RNA polymerase-based expression under control of the lac operon using IPTG induction is commonly employed . Auto-induction protocols, where protein production is induced by lactose in the medium after depletion of glucose, have shown better results for some membrane proteins .

• Strain selection: Engineered strains can address specific challenges like disulfide bond formation (SHuffle and Origami strains) and codon bias (Rosetta and CodonPlus RIL/RP strains) .

• Fusion tags: Addition of fusion partners that enhance solubility or membrane targeting can improve expression.

• Co-expression strategies: Co-expression of molecular chaperones and components of post-translational machinery can facilitate proper folding .

• Growth conditions: Lowering the temperature after induction can slow down protein synthesis, potentially allowing more time for proper folding and membrane insertion.

These approaches should be systematically tested to determine the optimal conditions for TMEM87B expression in E. coli systems.

What purification strategies are most effective for recombinant TMEM87B?

Purification of membrane proteins like TMEM87B requires specialized approaches:

• Membrane isolation: First, cellular membranes containing the recombinant TMEM87B must be isolated by differential centrifugation.

• Detergent selection: Critical for membrane protein extraction, detergent choice impacts protein stability and activity. For crystallization purposes, detergents forming relatively small micelles compared to the protein size are preferable, such as alkyl glycosides .

• Chromatography: After solubilization, purification typically involves:

  • Affinity chromatography using appropriate tags (His, GST, FLAG)

  • Size exclusion chromatography (SEC) for final purification and to confirm monodispersity

  • Ion exchange chromatography may be employed depending on protein properties

• Monodispersity assessment: Confirming homogeneity of purified/solubilized TMEM87B is crucial before structural studies. SEC, dynamic light scattering, analytical ultracentrifugation, or pulsed-field gradient diffusion NMR can verify sample integrity .

For structural biology applications, especially crystallization, maintaining a monodisperse preparation is essential for success .

How can the proper folding and stability of recombinant TMEM87B be assessed?

Assessing the proper folding and stability of recombinant TMEM87B is critical for functional and structural studies:

• Size exclusion chromatography (SEC): Monodisperse elution profiles suggest properly folded, non-aggregated protein .

• Dynamic light scattering (DLS): Provides information about the homogeneity and hydrodynamic radius of the protein-detergent complex .

• Circular dichroism (CD) spectroscopy: Can assess secondary structure content, particularly useful for α-helical transmembrane proteins.

• Thermal stability assays: Methods like differential scanning fluorimetry (DSF) can assess protein stability under various conditions.

• Functional assays: If known, functional assays specific to TMEM87B can indicate proper folding.

• Limited proteolysis: Properly folded proteins often show distinct, limited digestion patterns when compared to misfolded variants.

For membrane proteins like TMEM87B, stability in the chosen detergent or membrane mimetic is particularly important to monitor throughout the purification process.

What experimental approaches can be used to investigate TMEM87B's role in cardiac development?

Based on evidence suggesting TMEM87B's involvement in cardiac development, several experimental approaches can be employed:

• Animal models: Zebrafish models have already demonstrated that TMEM87B knockdown affects cardiac development . Similar approaches using CRISPR/Cas9 in zebrafish or mouse models could further characterize its role.

• Cardiac cell culture models: Human induced pluripotent stem cells (iPSCs) differentiated into cardiomyocytes could be used to study TMEM87B function through knockdown, overexpression, or mutation introduction.

• Transcriptomics: RNA-seq analysis of tissues with modified TMEM87B expression can identify affected pathways.

• Protein interaction studies: Techniques like BioID, proximity labeling, or co-immunoprecipitation can identify TMEM87B interaction partners in cardiac tissue.

• Functional recovery experiments: Rescue experiments in zebrafish or cellular models where wild-type TMEM87B is reintroduced after knockdown can confirm specificity of observed phenotypes.

• Structure-function analysis: Creating a series of TMEM87B mutants, particularly focusing on the conserved regions like Asn456, to determine which domains are essential for cardiac development.

These approaches would provide complementary insights into TMEM87B's specific role in cardiac development pathways.

How can researchers investigate the molecular mechanisms through which TMEM87B variants cause disease?

To investigate how TMEM87B variants like p.Asn456Asp lead to disease phenotypes:

• Site-directed mutagenesis: Introducing specific mutations (e.g., p.Asn456Asp) into expression constructs to study their effects on protein function .

• Cellular localization studies: Comparing localization patterns of wild-type and mutant TMEM87B using fluorescently tagged constructs.

• Patient-derived cells: iPSCs from patients with TMEM87B mutations can be differentiated into relevant cell types to study disease mechanisms.

• Molecular dynamics simulations: Computational approaches to predict how specific mutations affect protein structure and dynamics.

• Transgenic animal models: Generating animals expressing the human mutation to recapitulate and study disease phenotypes.

• Transcriptomic and proteomic profiling: Comparing expression profiles between wild-type and mutant conditions to identify dysregulated pathways.

• Functional assays: Developing assays specific to TMEM87B's function, once better understood, to quantitatively measure the impact of mutations.

For the p.Asn456Asp variant specifically, investigating how this mutation at the end of the last transmembrane domain affects protein stability, localization, or interaction with binding partners would be particularly informative .

How does the recurrent 2q13 microdeletion affect TMEM87B function, and how might this contribute to the variable phenotype?

The recurrent 2q13 microdeletion removes one copy of TMEM87B, creating haploinsufficiency . Research suggests the variability in phenotypic presentation may result from additional variants on the non-deleted allele of genes within this interval, including TMEM87B . This represents a "two-hit" model where:

• The first hit is the microdeletion removing one copy of TMEM87B.

• The second hit could be a mutation on the remaining TMEM87B allele.

This was illustrated in a case where a patient inherited both a 2q13 microdeletion from the mother (removing one TMEM87B copy) and a hemizygous missense variant (c.1366A>G, p.Asn456Asp) in TMEM87B from the father . This combination resulted in effective loss of normal TMEM87B function and a severe cardiac phenotype .

Research strategies to investigate this further could include:

• Sequencing the remaining TMEM87B allele in patients with 2q13 microdeletion and varying phenotypes

• Creating cellular or animal models with different combinations of deletion and point mutations

• Quantitative assessment of TMEM87B dosage effects on cardiac development pathways

What experimental approaches can determine the three-dimensional structure of TMEM87B?

Determining the structure of membrane proteins like TMEM87B presents significant challenges. Based on general approaches for membrane protein structural biology , strategies include:

• X-ray crystallography: Requires production of well-diffracting crystals, which for membrane proteins often involves:

  • Screening multiple detergents to find optimal solubilization conditions

  • Using specialized crystallization screens designed for membrane proteins (MemStart, MemGold, MemSys)

  • Crystallizing protein-detergent complexes, bicelles, or lipid cubic phases (LCPs)

  • Ensuring monodispersity of the sample through SEC and other analytical methods

• Cryo-electron microscopy (cryo-EM): Increasingly popular for membrane proteins as it doesn't require crystallization:

  • May require incorporation into nanodiscs or amphipols for stability

  • Particularly useful for larger membrane protein complexes

  • Can resolve structures at near-atomic resolution

• NMR spectroscopy: Suitable for smaller membrane proteins or domains:

  • Requires isotope labeling (¹³C, ¹⁵N) of recombinant protein

  • Can provide dynamic information not available from static methods

  • May require specialized membrane mimetics like bicelles or nanodiscs

• Integrative structural biology: Combining multiple experimental approaches:

  • Homology modeling based on related proteins

  • Cross-linking mass spectrometry to constrain domains

  • Molecular dynamics simulations to predict conformational changes

Each method requires significant optimization of protein production, purification, and sample preparation conditions specific to TMEM87B.

How can researchers investigate potential TMEM87B interaction partners and signaling pathways?

Identifying TMEM87B interaction partners and signaling pathways is crucial for understanding its function in cardiac development. Advanced approaches include:

• Proximity labeling methods:

  • BioID or TurboID fusions with TMEM87B to biotinylate proximal proteins

  • APEX2 enzyme fusions for proximity-based biotinylation with temporal control

  • These approaches are particularly valuable for membrane proteins in their native environment

• Co-immunoprecipitation with mass spectrometry:

  • Requires development of specific antibodies or expression of tagged TMEM87B

  • Can be performed with crosslinking to capture transient interactions

  • Quantitative proteomics can identify enriched interactors

• Genetic interaction screens:

  • CRISPR screens in cardiac cell models to identify synthetic lethal or modifier genes

  • Genetic modifier screens in model organisms like zebrafish

• Phosphoproteomics and signaling pathway analysis:

  • Comparing phosphorylation patterns in wild-type versus TMEM87B-deficient cells

  • Pathway inhibitor studies to place TMEM87B in known signaling cascades

• Membrane yeast two-hybrid or split-ubiquitin assays:

  • Specialized for membrane protein interactions

  • Can screen libraries to identify novel interaction partners

• Live-cell imaging approaches:

  • FRET or BRET to detect protein-protein interactions in real-time

  • Can provide spatial and temporal information about interactions

These approaches would provide complementary data to build a comprehensive picture of TMEM87B's role in cellular signaling networks relevant to cardiac development.

How do the variants in TMEM87B correlate with specific clinical phenotypes?

The available research shows a correlation between TMEM87B variants and cardiac phenotypes, particularly when both copies of the gene are affected . Key findings include:

• The p.Asn456Asp variant (c.1366A>G) in TMEM87B, when present in combination with a deletion of the other allele, was associated with restrictive cardiomyopathy, atrial septal defect, craniofacial anomalies, and intellectual disabilities .

• This variant is extremely rare and has very high conservation scores across several algorithms, indicating likely pathogenicity according to multiple mutation prediction algorithms .

• The patient's genotype represented a compound effect of a maternally inherited 2q13 microdeletion and a paternally inherited hemizygous missense variant, effectively eliminating normal TMEM87B function .

The table below summarizes variant characteristics from the research:

Further research with larger patient cohorts would help establish more detailed genotype-phenotype correlations for TMEM87B variants.

What experimental models best recapitulate human TMEM87B-associated disorders?

Based on current knowledge, several experimental models could effectively recapitulate human TMEM87B-associated disorders:

• Zebrafish models: Already demonstrated utility in TMEM87B research, with morpholino-based knockdown showing cardiac anomalies that parallel human phenotypes . CRISPR/Cas9-engineered zebrafish with specific human mutations could further refine these models.

• Mouse models: Engineering mice with the specific p.Asn456Asp mutation or other TMEM87B variants could provide mammalian models of the condition. Conditional knockout approaches could also help determine tissue-specific effects.

• Patient-derived iPSCs: Cells from patients with TMEM87B mutations differentiated into cardiomyocytes would maintain the exact genetic background of affected individuals, providing highly relevant in vitro models.

• Engineered heart tissues (EHTs): Three-dimensional cardiac tissues derived from patient iPSCs could model functional cardiac defects at the tissue level.

• CRISPR-engineered cell lines: Introduction of specific TMEM87B variants into cardiac cell lines could isolate the effects of individual mutations.

The choice between these models depends on specific research questions, with complementary approaches likely yielding the most comprehensive understanding of TMEM87B's role in cardiac disorders.

What are the most promising therapeutic approaches based on TMEM87B research findings?

Though current knowledge about TMEM87B is still developing, several therapeutic approaches could be explored based on existing findings:

• Gene therapy approaches: For loss-of-function mechanisms, delivery of functional TMEM87B copies to affected tissues could restore normal function.

• Small molecule screening: Once TMEM87B's function is better characterized, high-throughput screens could identify compounds that enhance residual activity of mutant proteins or modulate downstream pathways.

• Antisense oligonucleotides: For mutations affecting splicing, antisense approaches could potentially correct aberrant splicing patterns.

• CRISPR-based therapeutic strategies: Precise correction of pathogenic variants like p.Asn456Asp could be explored, particularly in combination with advances in cardiac-targeted delivery systems.

• Pathway-based interventions: As signaling pathways affected by TMEM87B dysfunction are identified, existing drugs targeting these pathways could be repurposed.

Development of these therapeutic approaches requires further fundamental research to better understand TMEM87B's normal function and the molecular mechanisms through which mutations lead to disease.

How might high-throughput functional genomics approaches advance our understanding of TMEM87B?

High-throughput functional genomics approaches offer powerful tools to accelerate TMEM87B research:

• Massively parallel reporter assays (MPRAs): To assess the impact of hundreds of TMEM87B variants simultaneously on gene expression and splicing regulation.

• Saturation genome editing: Systematic CRISPR editing to create libraries of TMEM87B variants, particularly focusing on conserved regions like the transmembrane domains.

• Single-cell transcriptomics: Analysis of gene expression changes in individual cells with TMEM87B perturbations could reveal cell type-specific effects and identify affected pathways.

• Proteomics approaches: Global protein expression and modification analysis in models with altered TMEM87B function could identify downstream effectors.

• CRISPR screens: Genome-wide screens for synthetic lethal interactions or genetic modifiers could place TMEM87B in broader biological pathways.

• High-content imaging screens: Automated microscopy combined with machine learning analysis to identify cellular phenotypes associated with TMEM87B variants.

These approaches would generate comprehensive datasets to build a systems-level understanding of TMEM87B function and pathogenic mechanisms, potentially identifying novel therapeutic targets.