Recombinant Bovine Transmembrane protein 215 (TMEM215)

What is the primary function of TMEM215 in endothelial cells?

TMEM215 plays a crucial role in protecting endothelial cells (ECs) from apoptosis during vessel pruning and regression. In developmental and pathological tissues, nascent vessel networks generated by angiogenesis require pruning to delete nonfunctional endothelial cells through apoptosis and migration .

TMEM215 forms a complex with BiP (binding immunoglobin protein) and BIK (BCL-2 interacting killer), a BH3-only proapoptotic protein. This interaction prevents BIK-triggered mitochondrial apoptosis mediated by calcium influx through mitochondria-associated ER membranes (MAMs) . Knockdown of TMEM215 in endothelial cells induces strong apoptotic cell death without affecting cell proliferation and migration, indicating that TMEM215 is specifically required for EC survival .

How is TMEM215 expression regulated in endothelial cells?

TMEM215 expression in endothelial cells is dynamically regulated by blood flow-derived shear stress. Specifically:

• Physiological high laminar shear stress (LSS >8 dyne/cm²) induces TMEM215 expression via downregulation of EZH2

• Quantitative real-time PCR and Western blotting show that TMEM215 expression is significantly upregulated at both mRNA and protein levels under laminar shear stress

• Oscillatory shear stress (OS), in contrast, downregulates TMEM215 in HUVECs compared to LSS

• TMEM215 expression is higher in ECs from descending thoracic aorta (with laminar blood flow) than those from aortic arch (with turbulent blood flow)

This shear stress-dependent expression pattern aligns with the physiological role of TMEM215 in protecting ECs from apoptosis in areas of high laminar flow while permitting apoptosis in low-flow zones during vessel pruning .

What is the molecular mechanism by which TMEM215 prevents endothelial cell apoptosis?

TMEM215 prevents endothelial cell apoptosis through a complex molecular mechanism involving the regulation of calcium signaling between the ER and mitochondria. The process involves:

• Complex formation with BiP and BIK: TMEM215 forms a complex with and facilitates the interaction of BiP with BIK. The C-terminus of TMEM215 associates with BiP within a protein complex .

• Regulation of mitochondria-associated ER membranes (MAMs): TMEM215 knockdown:

  • Increases the number of MAMs

  • Decreases the distance between outer mitochondrial membrane (OMM) and ER membrane

  • These alterations are rescued by simultaneous BIK knockdown

• Control of mitochondrial calcium influx: TMEM215 knockdown increases Ca²⁺ flux from ER to mitochondria. This effect is:

  • Dependent on BIK, as BIK knockdown rescues the increased calcium flux

  • Mediated through MAMs

  • Leads to mitochondrial apoptosis

• Prevention of apoptosis: Inhibiting mitochondrial calcium influx by blocking IP₃R (inositol 1,4,5-trisphosphate receptor) or MCU (mitochondrial calcium uniporter) abrogates TMEM215 knockdown-induced apoptosis .

This mechanism highlights TMEM215's role as a scaffold that assists the interaction of BIK and BiP to inhibit BIK-mediated apoptosis .

How does TMEM215 deficiency affect vascular development in animal models?

EC-specific Tmem215 knockout mice exhibit significant vascular development abnormalities:

• Retinal vasculature defects:

  • Reduced vessel density in the superficial plexus

  • Increased empty basement membrane sleeves (a marker of vessel regression)

  • Increased EC apoptosis

  • These effects are most pronounced during active angiogenesis

• Alterations in MAMs and calcium signaling:

  • Increased number of ER-M contacts

  • Decreased distance between OMM and ER membrane in retinal ECs

  • Decreased colocalization of BiP and BIK

• Organ-specific effects:

  • Disrupted ovary follicle development, consistent with impaired angiogenesis

  • Normal morphology in liver, kidney, spleen, and lung in adult mice

  • This suggests that TMEM215 deficiency primarily affects angiogenic ECs, while quiescent ECs in most adult organs do not depend on TMEM215 for survival

Importantly, intravitreous injection of TMEM215 siRNA results in delayed and abnormal development of retinal vasculature with poor perfusion, further confirming the importance of TMEM215 in proper vascular development .

What are the implications of TMEM215 targeting for anti-angiogenic cancer therapy?

TMEM215 represents a promising target for anti-angiogenic therapy, particularly for cancer treatment. Research indicates:

• Effects on tumor growth and vascularization:

  • EC-specific Tmem215 ablation inhibits tumor growth with disrupted vasculature

  • This is associated with reduced tumor blood vessels and increased EC apoptosis

• Impact on metastasis:

  • Tmem215 ablation in adult mice attenuates lung metastasis

  • This correlates with reduced expression of Vcam1, an adhesion molecule involved in endothelial inflammation and dysfunction

  • Vcam1 plays an important role in lung metastasis in several tumor models

• Therapeutic delivery approaches:

  • Nanoparticle-delivered Tmem215 siRNA inhibits tumor growth

  • Polyethyleneimine-polyethylene glycol functionalized with cyclic Arg-Gly Asp-D-Phe-Lys peptide (PEI-PEG-cRGD) can specifically deliver siRNA to activated ECs expressing integrin αVβ3

  • This approach showed no significant toxicity in preclinical studies

• Additional therapeutic applications:

  • Administration of nanoparticles carrying Tmem215 siRNA also inhibits choroidal neovascularization injury

  • This suggests potential applications for treating age-related macular degeneration

The differential effect of TMEM215 on angiogenic versus quiescent endothelial cells makes it an attractive target, as it primarily affects tumor vasculature while sparing normal blood vessels in most adult organs .

What expression systems are most effective for producing recombinant bovine TMEM215?

Several expression systems have been successfully used to produce recombinant TMEM215:

• E. coli expression system:

  • Suitable for producing full-length bovine TMEM215 (1-235 amino acids)

  • Can be fused with N-terminal His-tag for purification

  • Yields protein in lyophilized powder form

  • Purity greater than 90% as determined by SDS-PAGE

• Mammalian cell expression systems:

  • Used for producing mouse and human TMEM215

  • Allows for proper folding and potential post-translational modifications

  • May yield higher quantities of soluble protein compared to bacterial systems

• Insect cell lines (S2):

  • While not specifically documented for TMEM215, similar transmembrane proteins have been successfully expressed in stable S2 insect cell lines

  • Could be advantageous for obtaining properly folded protein with appropriate glycosylation patterns

When selecting an expression system, researchers should consider their specific experimental requirements, including the need for post-translational modifications, protein folding, yield, and downstream applications .

What are the recommended protocols for purification and storage of recombinant TMEM215?

For optimal purification and storage of recombinant bovine TMEM215:

Purification protocol:

• Express TMEM215 with an affinity tag (His-tag is commonly used)

• Lyse cells in appropriate buffer

• Purify using immobilized metal affinity chromatography (IMAC)

• Assess purity by SDS-PAGE (should be >90%)

• Consider additional purification steps if higher purity is required

Storage recommendations:

• Short-term storage: Store at 4°C for up to one week

• Long-term storage: Store at -20°C/-80°C

• Reconstitution: Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol (final concentration 5-50%, with 50% being typical) to prevent freeze-thaw damage

• Aliquot for long-term storage to avoid repeated freeze-thaw cycles

Storage buffer: Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 has been shown to maintain protein stability .

What functional assays can be used to assess the activity of recombinant TMEM215?

Several functional assays can be employed to assess the activity and functional properties of recombinant TMEM215:

• Endothelial cell survival assays:

  • Measure cell viability using MTT, WST-1, or similar assays

  • Compare survival rates between control cells and cells treated with TMEM215 or TMEM215 knockdown constructs

  • Annexin V/PI staining combined with flow cytometry to quantify apoptotic cells

• Protein-protein interaction assays:

  • Co-immunoprecipitation to detect interactions between TMEM215, BiP, and BIK

  • Pull-down assays using recombinant TMEM215 as bait

  • Surface plasmon resonance (SPR) or bio-layer interferometry (BLI) to quantify binding kinetics

• Calcium flux measurement:

  • Fluorescent calcium indicators to measure calcium flux from ER to mitochondria

  • Use of Ca²⁺-sensitive probes targeted to specific organelles

  • Live-cell imaging to monitor calcium dynamics in real-time

• In vitro angiogenesis assays:

  • Tube formation assay using endothelial cells on Matrigel

  • Sprouting assay using endothelial cell spheroids

  • These assays can be performed after treatment with recombinant TMEM215 or after TMEM215 knockdown

• Mitochondria-associated ER membrane (MAM) analysis:

  • Transmission electron microscopy to visualize and quantify MAMs

  • Measure the distance between OMM and ER membrane

  • Super-resolution microscopy (SIM) to analyze colocalization of TMEM215, BiP, and BIK

These assays provide comprehensive methods to evaluate various aspects of TMEM215 function, from molecular interactions to cellular phenotypes.

How can researchers effectively use recombinant TMEM215 to study its role in endothelial cell apoptosis?

To effectively study the role of recombinant TMEM215 in endothelial cell apoptosis, researchers can employ several strategies:

• Loss-of-function approaches:

  • siRNA or shRNA knockdown of endogenous TMEM215 in endothelial cells (HUVECs are commonly used)

  • CRISPR-Cas9 genome editing to generate TMEM215 knockout cells

  • These approaches induce apoptotic cell death, allowing study of the mechanism

• Rescue experiments:

  • After TMEM215 knockdown, introduce recombinant TMEM215 to rescue the phenotype

  • Use site-directed mutagenesis to create TMEM215 variants with modifications in key domains

  • This approach can identify essential regions/residues for TMEM215 function

• Mechanistic studies:

  • Combine TMEM215 knockdown with knockdown of BIK to test rescue

  • Overexpress BCL-2 to test if it abrogates TMEM215 knockdown-induced apoptosis

  • Block IP₃R or MCU to inhibit mitochondrial calcium influx and test rescue of apoptosis

  • These strategies can dissect the molecular pathway of TMEM215 function

• Structural studies using recombinant protein:

  • Use purified recombinant TMEM215 for crystallography or cryo-EM

  • Focus on the C-terminal fragment (residues 60-235) for interaction studies

  • This can provide insights into how TMEM215 interacts with binding partners

• In vivo validation:

  • Deliver recombinant TMEM215 or TMEM215 siRNA in nanoparticles to animal models

  • Assess effects on angiogenesis in developmental or pathological contexts

  • This approach bridges in vitro findings with physiological relevance

By integrating these approaches, researchers can comprehensively characterize how TMEM215 prevents endothelial cell apoptosis and explore its potential as a therapeutic target.

How should researchers design experiments to study the differential effects of TMEM215 in angiogenic versus quiescent endothelial cells?

To effectively study the differential effects of TMEM215 in angiogenic versus quiescent endothelial cells, consider the following experimental design strategy:

• Cell model selection:

  • Angiogenic ECs: Use HUVECs stimulated with VEGF or bFGF to induce angiogenic phenotype

  • Quiescent ECs: Culture HUVECs in low serum conditions without growth factors, or use primary ECs isolated from adult tissues (lung, liver)

  • Comparison: Measure TMEM215 expression levels in both conditions using qRT-PCR and Western blotting

• Flow condition experiments:

  • Use ibidi apparatus to apply different shear stress conditions:

    • High laminar shear stress (>8 dyne/cm²) to mimic normal circulation

    • Low laminar shear stress (<4.5 dyne/cm²) to mimic conditions prone to vessel pruning

    • Oscillatory shear stress to mimic disturbed flow

  • Measure TMEM215 expression and EC apoptosis in each condition

• Tissue-specific analysis:

  • Compare TMEM215 expression and function in ECs from:

    • Actively angiogenic tissues (developing retina, ovary)

    • Quiescent vasculature (adult liver, lung, kidney)

  • Use laser capture microdissection to isolate ECs from different tissues for analysis

• Temporal regulation:

  • Study TMEM215 expression during developmental angiogenesis stages (e.g., in retinal vasculature from P1 to P28 in mice)

  • Correlate expression patterns with vessel pruning events and EC apoptosis markers

  • This approach can identify temporal windows when TMEM215 is most critical

• In vivo model systems:

  • Use inducible EC-specific Tmem215 knockout mice to deplete TMEM215 at different developmental stages

  • Compare effects on developmental angiogenesis (retina) versus adult vasculature

  • Analyze both physiological and pathological (tumor) angiogenesis in the same animal models

This comprehensive approach will help researchers understand why TMEM215 deficiency primarily affects angiogenic ECs while quiescent ECs in adult organs are less dependent on TMEM215 for survival.

What are the optimal conditions for studying TMEM215 interactions with BiP and BIK using recombinant proteins?

To optimize studies of TMEM215 interactions with BiP and BIK using recombinant proteins:

• Protein production considerations:

  • Express TMEM215-C (C-terminal fragment, residues 60-235) fused to GFP or another tag for enhanced solubility and detection

  • Express full-length BiP and BIK with compatible tags for co-immunoprecipitation

  • Consider using mammalian expression systems to ensure proper folding and post-translational modifications

• Interaction detection methods:

  • Co-immunoprecipitation (Co-IP): Optimal buffer conditions include:

    • HEPES or Tris buffer (pH 7.4-7.6)

    • 150 mM NaCl

    • 1% NP-40 or similar mild detergent

    • Protease inhibitor cocktail

  • Pull-down assays using recombinant proteins immobilized on beads

  • Surface plasmon resonance (SPR) or bio-layer interferometry (BLI) for quantitative binding kinetics

• Domain mapping experiments:

  • Create truncated variants of TMEM215 to identify minimal regions required for BiP binding

  • Focus on the C-terminal tail extending into the ER lumen, as this region has been identified as interacting with BiP

  • Perform alanine scanning mutagenesis of key residues to identify critical interaction sites

• Complex reconstitution:

  • Attempt to reconstitute the trimeric TMEM215-BiP-BIK complex in vitro

  • Use size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to determine complex stoichiometry

  • Consider cross-linking coupled with mass spectrometry to identify interaction interfaces

• Functional validation:

  • Test if recombinant TMEM215 can disrupt or enhance BiP-BIK interaction in cell lysates

  • Develop FRET-based assays using fluorescently labeled proteins to monitor interactions in real-time

  • Assess calcium flux in the presence of different protein combinations

These optimized conditions will facilitate detailed characterization of how TMEM215 forms a complex with BiP and BIK to regulate endothelial cell survival.

How can researchers effectively measure and quantify changes in mitochondria-associated ER membranes (MAMs) related to TMEM215 function?

To effectively measure and quantify changes in mitochondria-associated ER membranes (MAMs) related to TMEM215 function, researchers should employ the following methods:

• Transmission electron microscopy (TEM):

  • Sample preparation: Fix cells with glutaraldehyde and osmium tetroxide, dehydrate, and embed in resin

  • Analysis parameters:

    • Quantify the number of MAMs per mitochondrion

    • Measure the distance between outer mitochondrial membrane (OMM) and ER membrane at contact sites (optimal distance: 10-30 nm)

    • Calculate the length of MAM contact sites as a percentage of mitochondrial perimeter

  • Compare these parameters between control cells and cells with TMEM215 knockdown or overexpression

• Super-resolution microscopy:

  • Structured Illumination Microscopy (SIM) offers resolution beyond the diffraction limit (~100 nm)

  • Staining protocol:

    • Label ER with markers like calnexin or PDI

    • Label mitochondria with TOM20 or MitoTracker

    • Immunostain for TMEM215, BiP, and BIK

  • Quantify colocalization using Pearson's or Manders' correlation coefficients

  • Measure changes in colocalization after experimental manipulations of TMEM215 expression

• Proximity ligation assay (PLA):

  • Use antibodies against proteins located in ER (IP₃R) and mitochondria (VDAC)

  • Quantify PLA signals as indicators of MAM formation

  • Compare signal intensity between control and TMEM215-manipulated samples

• Subcellular fractionation and biochemical analysis:

  • Isolate MAM fractions using differential centrifugation

  • Verify fraction purity with markers (MAM: FACL4; ER: calnexin; mitochondria: VDAC)

  • Quantify proteins of interest in each fraction using Western blotting

  • Compare MAM composition between control and TMEM215-manipulated cells

• Calcium flux measurement at MAMs:

  • Use genetically encoded calcium indicators targeted to ER-mitochondria interface

  • Analyze:

    • Basal calcium levels at MAMs

    • Kinetics of calcium transfer from ER to mitochondria upon stimulation

    • Mitochondrial calcium uptake capacity

  • Compare these parameters between control and TMEM215-manipulated cells

• Live-cell imaging of MAM dynamics:

  • Express fluorescently tagged ER and mitochondrial markers

  • Perform time-lapse imaging to measure:

    • MAM formation rate

    • MAM stability/persistence

    • Dynamics of ER-mitochondria contacts

  • Compare dynamics between control and TMEM215-manipulated cells

These methodologies provide complementary approaches to comprehensively characterize how TMEM215 influences MAM formation and function in endothelial cells.

What are the potential applications of TMEM215 research beyond cancer therapy?

Research on TMEM215 has applications extending beyond cancer therapy:

• Age-related macular degeneration (AMD) treatment:

  • Choroidal neovascularization is a hallmark of wet AMD

  • Nanoparticle-delivered Tmem215 siRNA inhibits choroidal neovascularization in animal models

  • This suggests potential for treating pathological ocular angiogenesis

• Diabetic retinopathy interventions:

  • Diabetic retinopathy involves abnormal retinal vessel formation and regression

  • TMEM215's role in retinal vascular development makes it relevant for diabetic retinopathy

  • Targeted modulation could help normalize retinal vasculature in diabetic patients

• Tissue engineering and regenerative medicine:

  • Controlled angiogenesis is crucial for engineered tissue survival

  • Manipulating TMEM215 expression could help regulate blood vessel formation in engineered tissues

  • Could improve vascularization of implanted tissues or artificial organs

• Atherosclerosis management:

  • Endothelial dysfunction is a key factor in atherosclerosis

  • TMEM215 is regulated by laminar shear stress, which is protective against atherosclerosis

  • Understanding TMEM215's role in endothelial health under different flow conditions could inform atherosclerosis prevention strategies

• Wound healing applications:

  • Angiogenesis is essential for proper wound healing

  • Modulating TMEM215 expression could enhance controlled angiogenesis in chronic wounds

  • Potential for developing TMEM215-based interventions for diabetic ulcers or other difficult-to-heal wounds

• Female reproductive health:

  • TMEM215 deficiency affects ovary follicle development

  • Further research could reveal roles in female reproductive disorders characterized by abnormal angiogenesis

  • Potential implications for treating conditions like polycystic ovary syndrome or endometriosis

These diverse applications highlight the broad significance of understanding TMEM215 function beyond its role in tumor angiogenesis.

How might gene editing technologies be applied to study or modulate TMEM215 function in vascular disorders?

Gene editing technologies offer powerful approaches to study and modulate TMEM215 function in vascular disorders:

• CRISPR-Cas9 for mechanistic studies:

  • Generate precise TMEM215 knockout or knockin cell lines and animal models

  • Create domain-specific mutations to understand structure-function relationships

  • Engineer conditional alleles to study temporal aspects of TMEM215 function

  • Advantages: Precise modification with minimal off-target effects compared to siRNA

• Base or prime editing for therapeutic applications:

  • Introduce single nucleotide variants to modulate TMEM215 expression or function

  • Target regulatory elements controlling TMEM215 expression

  • Modify EZH2 binding sites to influence TMEM215 regulation by laminar shear stress

  • Benefits: No double-strand breaks, reduced risk of unintended mutations

• RNA editing for reversible modulation:

  • Use ADAR (adenosine deaminase acting on RNA) systems to modify TMEM215 mRNA

  • Create temporary changes in TMEM215 expression or function

  • Ideal for situations requiring transient modulation of angiogenesis

  • Advantage: Reversible effects without permanent genomic alterations

• Epigenome editing approaches:

  • Target dCas9-DNMT (DNA methyltransferase) or dCas9-TET (ten-eleven translocation) to TMEM215 regulatory regions

  • Alter methylation status to modulate expression

  • Focus on regions influenced by EZH2, which is known to regulate TMEM215 expression

  • Benefit: Modulates expression without altering DNA sequence

• Organ-specific delivery systems:

  • Use adeno-associated virus (AAV) vectors with endothelial-specific promoters

  • Target gene editing components to specific vascular beds (retina, tumor)

  • Deliver via nanoparticles functionalized with endothelial-targeting moieties like cRGD

  • Advantage: Reduces systemic effects, concentrates therapeutic impact

• Diagnostic applications:

  • CRISPR-based detection systems to quantify TMEM215 expression in patient samples

  • Create reporter systems to monitor TMEM215 activity in real-time

  • Develop high-throughput screening platforms to identify modulators of TMEM215 function

  • Benefit: Personalized approach to diagnosing and treating vascular disorders

These gene editing applications represent cutting-edge approaches to translate basic TMEM215 research into clinical interventions for vascular disorders.

What emerging technologies could enhance our understanding of TMEM215's role in calcium signaling and mitochondrial function?

Several emerging technologies could significantly advance our understanding of TMEM215's role in calcium signaling and mitochondrial function:

• Genetically encoded calcium sensors with subcellular targeting:

  • GCaMP variants specifically targeted to MAMs, ER, or mitochondrial membranes

  • Dual-wavelength sensors for simultaneous monitoring of calcium in multiple compartments

  • These tools enable real-time visualization of calcium dynamics at ER-mitochondria contact sites in living cells

  • Would reveal how TMEM215 influences calcium transfer between organelles

• Cryo-electron tomography (cryo-ET):

  • Visualize native MAM structures at near-atomic resolution

  • Map the 3D organization of protein complexes at ER-mitochondria interfaces

  • Locate TMEM215, BiP, and BIK within these complexes

  • Would provide structural insights into how TMEM215 organizes protein complexes at MAMs

• Proximity proteomics in live cells:

  • BioID or APEX2 tagging of TMEM215 to identify proximal interacting proteins

  • TurboID for rapid labeling of the TMEM215 interactome under different conditions

  • Split-BioID to specifically identify proteins at MAMs

  • Would comprehensively map TMEM215's interaction network in its native environment

• Optogenetic tools for manipulating calcium signals:

  • Light-activated calcium channels or pumps targeted to ER or mitochondria

  • Optogenetic control of IP₃R or MCU activity

  • These tools allow precise temporal control of calcium flux between organelles

  • Would help dissect the causal relationships between calcium signaling and TMEM215-mediated protection against apoptosis

• Single-cell multi-omics approaches:

  • Integrated single-cell transcriptomics, proteomics, and metabolomics

  • Spatial transcriptomics to map TMEM215 expression in tissue context

  • These approaches reveal cell-to-cell variability in TMEM215 expression and function

  • Would identify cell subpopulations with differential sensitivity to TMEM215 manipulation

• Mitochondrial membrane potential and bioenergetics analysis:

  • High-throughput Seahorse analysis of mitochondrial respiration

  • Live-cell imaging of mitochondrial membrane potential using voltage-sensitive dyes

  • Real-time measurements of ATP production

  • Would reveal how TMEM215-mediated calcium signaling affects mitochondrial function and energy production

• Microfluidic devices with controlled shear stress:

  • Systems allowing precise control of flow patterns and shear stress magnitudes

  • Integration with live-cell imaging of calcium dynamics and apoptosis markers

  • Would establish direct links between flow conditions, TMEM215 expression, calcium signaling, and EC survival