Recombinant Human Transmembrane protein 186 (TMEM186)

Basic Structural Characteristics of TMEM186

Q: What is the tissue-specific expression pattern of TMEM186 in humans, and how can researchers quantify its expression?

TMEM186 shows a broad expression pattern across human tissues, with notable presence in highly metabolically active tissues. The Human Protein Atlas data indicates expression in multiple tissues including brain regions, heart, liver, and kidney .

To quantify TMEM186 expression across tissues, researchers should consider the following methodological approaches:

• RT-qPCR analysis: Design primers specific to human TMEM186 mRNA for accurate quantification across tissue samples.

• Western blotting: When using commercial antibodies, validation is critical as cross-reactivity with other membrane proteins can occur. Prepare membrane fractions using differential centrifugation to enrich for membrane proteins.

• Immunohistochemistry: Use tissue microarrays for comparative analysis across multiple tissues simultaneously.

• RNA-seq analysis: For comprehensive transcriptomic profiling, analyze existing datasets from repositories such as GTEx or generate new data with appropriate tissue representation.

When analyzing expression data, researchers should normalize to appropriate housekeeping genes and consider that mitochondrial content varies significantly across tissue types, which can impact apparent expression levels of mitochondrial proteins.

Mitochondrial Function and ATP Synthase Assembly

Q: What role does TMEM186 play in mitochondrial function, particularly in ATP synthase assembly?

TMEM186 has been implicated in the assembly of the mitochondrial ATP synthase complex, particularly in the incorporation of specific subunits into the functional enzyme. Research indicates that TMEM186, along with COA1, is associated with the Mitochondrial Complex I Assembly (MCIA) complex in the inner mitochondrial membrane .

Specifically, TMEM186 appears to contribute to the incorporation of subunits of the ATP synthase. The assembly of mitochondrial ATP synthase is a complex process involving multiple intermediate modules representing the main structural elements of the enzyme, including the F1-catalytic domain, the peripheral stalk, and the c8-ring in the membrane part of the rotor .

To investigate TMEM186's role in ATP synthase assembly:

• Knockout/knockdown experiments: Generate TMEM186-deficient cell lines using CRISPR-Cas9 or RNAi approaches and analyze ATP synthase assembly using blue native PAGE followed by western blotting or in-gel activity assays.

• Complexome profiling: This state-of-the-art approach combines separation of native proteins by electrophoresis with mass spectrometry to identify protein complexes and their component subunits . This method can reveal how TMEM186 depletion affects the composition of mitochondrial complexes.

• Interaction studies: Co-immunoprecipitation followed by mass spectrometry can identify direct protein interactors of TMEM186 within the ATP synthase assembly pathway.

Experimental evidence suggests that deletion of TMEM186 affects but does not completely eliminate the assembly of ATP synthase, indicating functional redundancy or compensatory mechanisms .

Optimal Expression Systems for Recombinant TMEM186

Q: What expression systems are most effective for producing recombinant human TMEM186, and what optimization strategies should be considered?

Expression of membrane proteins like TMEM186 presents significant challenges due to their hydrophobic nature and specific folding requirements. Based on research with similar membrane proteins, the following systems and strategies should be considered:

Recommended Expression Systems:

Key Optimization Parameters:

• Clone design considerations:

  • Include a cleavable tag for purification (His6 tag has been successful)

  • Consider fusion partners to increase solubility

  • Codon optimization for the expression host

• Expression conditions:

  • Temperature reduction during induction (16-25°C) can improve folding

  • In prokaryotic systems, careful optimization of inducer concentration

• Co-expression strategies:

  • In prokaryotic systems, co-expression of molecular chaperones like DnaK/J has been shown to improve membrane protein folding

  • In eukaryotic systems, particularly for membrane proteins that interact with the ATP synthase, co-expression of interacting partners may stabilize the protein

Evidence from expression studies with transmembrane proteins indicates that E. coli expression is typically limited to proteins of 54 kDa and below, while S. cerevisiae performs better with smaller proteins (<60 kDa) . Given that TMEM186 is approximately 23 kDa, both systems could potentially be effective, but mammalian expression in HEK-293 cells has been documented to produce functional protein .

Purification Strategies and Functional Assessment

Q: What are the most effective purification approaches for TMEM186, and how can researchers assess its functional activity?

Purification Strategy:

Purification of TMEM186, like other transmembrane proteins, requires careful selection of detergents and chromatography methods to maintain protein stability and function:

• Membrane preparation:

  • Start with differential centrifugation to isolate membrane fractions

  • Wash membranes with high-salt buffer to remove peripheral proteins

• Solubilization optimization:

  • Screen multiple detergents (DDM, LMNG, digitonin) at various concentrations

  • For mitochondrial membrane proteins like TMEM186, mild detergents such as digitonin often preserve native interactions

  • Consider adding lipids during solubilization to stabilize the protein

• Affinity chromatography:

  • Use immobilized metal affinity chromatography (IMAC) for His-tagged TMEM186

  • Include detergent at concentrations above CMC in all buffers

  • Consider on-column detergent exchange to a more stabilizing detergent

• Size exclusion chromatography:

  • Final polishing step to separate monomeric from aggregated protein

  • Can also provide information about oligomeric state

Functional Assessment Methods:

• Binding assays:

  • If TMEM186 interacts with ATP synthase components, develop binding assays with purified interaction partners

  • Surface plasmon resonance (SPR) or microscale thermophoresis (MST) can quantify binding affinities

• Reconstitution into liposomes:

  • For functional studies, reconstitute purified TMEM186 into liposomes

  • Verify incorporation using proteoliposome flotation assays

• ATP synthase assembly assay:

  • Develop in vitro assays using isolated mitochondria from TMEM186-depleted cells

  • Add purified TMEM186 and assess rescue of ATP synthase assembly defects

• Structural characterization:

  • Negative-stain EM to verify protein homogeneity

  • For detailed structural information, cryo-EM is increasingly successful for membrane proteins

Researchers should monitor protein stability throughout purification using analytical size exclusion chromatography. Bis-Tris PAGE, anti-tag ELISA, Western blot, and analytical SEC (HPLC) have been used to achieve >90% purity for recombinant TMEM186 .

Protein-Protein Interactions and Complex Formation

Q: How does TMEM186 interact with other proteins in the mitochondrial membrane, and what methods are most effective for studying these interactions?

TMEM186 has been found to associate with the Mitochondrial Complex I Assembly (MCIA) complex in the inner mitochondrial membrane and appears to play a role in incorporating specific subunits into ATP synthase . Understanding these interactions is crucial for elucidating TMEM186's function.

Methodological Approaches:

• Complexome Profiling:
  This state-of-the-art approach combines blue native electrophoresis with mass spectrometry to identify protein complexes and assembly intermediates . The methodology involves:

  • Solubilization of mitochondrial membranes with mild detergents

  • Separation of protein complexes by blue native PAGE

  • Cutting the gel lane into slices and performing mass spectrometry on each slice

  • Computational analysis to determine protein migration profiles

• Co-immunoprecipitation (Co-IP) coupled with mass spectrometry:

  • Generate antibodies against TMEM186 or use epitope-tagged versions

  • Solubilize mitochondrial membranes with digitonin or other mild detergents

  • Perform immunoprecipitation with appropriate controls

  • Identify interacting proteins by mass spectrometry

• Proximity labeling approaches:

  • APEX2 or BioID fusions to TMEM186 can identify proximal proteins in living cells

  • These methods are particularly valuable for transient interactions

• Crosslinking mass spectrometry:

  • Apply chemical crosslinkers to stabilize interactions

  • Identify crosslinked peptides by specialized mass spectrometry approaches

  • This provides information about specific interaction interfaces

• Split reporter assays:

  • For validating specific interactions, techniques like split-GFP or BRET can be employed

  • These can be used in intact cells to confirm interactions in the native environment

Research Findings and Interaction Partners:

Based on the available literature, TMEM186 appears to interact with components of the ATP synthase assembly pathway. Research indicates it has a role similar to TMEM70, which interacts with subunit c of the ATP synthase c8-ring . The assembly of the c8-ring, which provides the membrane sector of the enzyme's rotor, is influenced by TMEM70 and potentially by TMEM186.

Knockout studies have shown that deletion of TMEM186 affects but does not completely eliminate ATP synthase assembly, suggesting functional redundancy or compensatory mechanisms .

Structural Studies of TMEM186

Q: What structural characterization methods are most appropriate for TMEM186, and what structural information is currently available?

Structural characterization of membrane proteins like TMEM186 presents unique challenges due to their hydrophobic nature and requirement for detergents or lipid environments. Here are the most appropriate approaches and current structural knowledge:

Structural Characterization Methods:

• Cryo-Electron Microscopy (Cryo-EM):

  • Currently the method of choice for membrane protein structures

  • Advantages: No need for crystallization; can capture different conformational states

  • Sample preparation considerations:

    • Protein concentration typically 2-5 mg/ml

    • Detergent selection critical (DDM, LMNG commonly used)

    • Consider reconstitution in nanodiscs or amphipols

• X-ray Crystallography:

  • Traditional approach for membrane protein structures

  • Challenges: Obtaining well-diffracting crystals

  • Strategies:

    • Lipidic cubic phase (LCP) crystallization

    • Use of fusion partners (T4 lysozyme, BRIL)

    • Antibody fragment co-crystallization to increase polar surface area

• NMR Spectroscopy:

  • Suitable for smaller membrane proteins or domains

  • Can provide dynamics information

  • Isotopic labeling required (15N, 13C)

• Integrative Structural Biology:

  • Combining low-resolution techniques:

    • Small-angle X-ray scattering (SAXS)

    • Hydrogen-deuterium exchange mass spectrometry (HDX-MS)

    • Crosslinking mass spectrometry

    • Molecular dynamics simulations

Current Structural Knowledge:

Currently, no high-resolution structure of human TMEM186 has been reported in the literature or structural databases. Structural predictions can be made based on:

• Homology modeling: Using structures of related transmembrane proteins

• Secondary structure predictions: Identifying transmembrane helices

• AI-based structure prediction: Tools like AlphaFold2 can provide structural models

Based on sequence analysis and prediction algorithms, TMEM186 likely contains multiple transmembrane helices. The protein sequence (213 amino acids) suggests a relatively compact structure with predominantly alpha-helical transmembrane segments .

For researchers pursuing structural studies of TMEM186, expression optimization and rigorous biochemical characterization should precede structural attempts. Stability assessment using techniques like differential scanning fluorimetry can help identify optimal buffer conditions.

TMEM186 in Disease and Pathological Conditions

Q: What is known about TMEM186 dysfunction in disease states, and how can researchers investigate its pathological roles?

While direct links between TMEM186 mutations and human diseases have not been extensively documented in the available literature, its role in mitochondrial function suggests potential implications in conditions associated with mitochondrial dysfunction.

Potential Disease Associations:

Given TMEM186's role in ATP synthase assembly , dysfunction could potentially contribute to:

• Mitochondrial disorders: Particularly those characterized by ATP synthase deficiency

• Neurodegenerative conditions: Due to the high energy demands of neural tissues

• Metabolic disorders: Reflecting disrupted energy homeostasis

Methodological Approaches for Investigating Pathological Roles:

• Patient cohort analysis:

  • Sequence TMEM186 in patients with unexplained mitochondrial disorders

  • Perform whole-exome sequencing in families with suspected mitochondrial diseases

  • Analyze TMEM186 expression in relevant pathological tissues

• Cell and tissue models:

  • Generate TMEM186 knockout cell lines using CRISPR-Cas9

  • Assess mitochondrial function parameters:

    • Oxygen consumption rate (Seahorse assay)

    • ATP production capacity

    • Mitochondrial membrane potential

    • Reactive oxygen species production

• Animal models:

  • Generate knockout or conditional knockout mice

  • Assess phenotype under normal and stress conditions

  • Perform tissue-specific analyses focusing on high-energy organs

• Functional complementation studies:

  • In patient-derived cells with TMEM186 mutations, assess rescue with wild-type TMEM186

  • Characterize function of patient-specific variants in knockout cell models

• Mitochondrial proteomics:

  • Compare the mitochondrial proteome in normal vs. TMEM186-deficient cells

  • Identify compensatory mechanisms or stress responses

Studies with TMEM186 knockout models could provide valuable insights into its physiological importance. Research with related proteins suggests that deletion of TMEM186 affects but does not completely eliminate ATP synthase assembly, which may indicate functional redundancy .

For clinical researchers, investigating TMEM186 expression and mutations in mitochondrial disease cohorts without known genetic causes could potentially identify new disease associations.

Advanced Experimental Design for TMEM186 Research

Q: What are the key considerations for designing comprehensive TMEM186 research projects, and what controls should be implemented?

Designing robust experiments for TMEM186 research requires careful consideration of controls, model systems, and analytical approaches. Here's a comprehensive guide for researchers:

Experimental Design Framework:

• Model System Selection:

  Model System	Advantages	Limitations	Best Applications
  HEK293/HeLa cells	Easy transfection; well-characterized	May not reflect tissue-specific interactions	Initial characterization; protein expression
  Primary cells	Physiologically relevant	Limited lifespan; donor variation	Validation of findings in relevant cell types
  Fibroblasts	Patient-derived options	May not reflect tissue-specific phenotypes	Disease modeling for mitochondrial disorders
  Mouse models	In vivo physiological context	Time and resource intensive	Systemic effects; tissue interactions

• Genetic Manipulation Approaches:

  • CRISPR-Cas9 knockout: Complete elimination of protein

  • CRISPR-Cas9 knock-in: Tag endogenous protein, introduce mutations

  • RNAi: Temporary knockdown for acute effects

  • Overexpression: Study gain-of-function effects

• Essential Controls:

  • Multiple independent clones for knockout lines

  • Rescue experiments with wild-type TMEM186

  • Isogenic control lines

  • Multiple siRNA/shRNA sequences for knockdown studies

  • Empty vector controls for overexpression

Assay-Specific Considerations:

• Mitochondrial Function Assays:

  • Oxygen consumption rate (OCR): Measure with and without inhibitors to assess specific complexes

  • ATP production: Both mitochondrial and total cellular ATP

  • Membrane potential: Multiple dyes (TMRM, JC-1) for cross-validation

  • Mitochondrial morphology: Confocal imaging with appropriate mitochondrial markers

• ATP Synthase Assembly Analysis:

  • Blue native PAGE: Assess complex formation

  • Complexome profiling: Identify assembly intermediates

  • In-gel activity assays: Functional assessment of assembled complexes

• Protein-Protein Interaction Studies:

  • Multiple complementary methods (Co-IP, proximity labeling, Y2H)

  • Appropriate negative controls (unrelated mitochondrial proteins)

  • Competition assays to confirm specificity

• Quantitative Considerations:

  • Biological replicates (minimum n=3)

  • Technical replicates to assess method variability

  • Blinding where applicable

  • Statistical power calculations for animal studies

Data Analysis and Interpretation:

• Mitochondrial Normalization:

  • Normalize to mitochondrial content (citrate synthase, VDAC)

  • Consider mitochondrial mass changes in knockouts

• Compensatory Mechanisms:

  • Assess expression of related proteins (e.g., TMEM70)

  • Consider adaptive responses in chronic knockouts vs. acute knockdowns

• Tissue/Cell Type Differences:

  • Compare findings across different cell types

  • Consider tissue-specific interaction partners

Incorporating these design elements will strengthen the validity and reproducibility of TMEM186 research and facilitate meaningful comparison with studies of related mitochondrial proteins.

Future Research Directions and Emerging Technologies

Q: What are the most promising future directions for TMEM186 research, and which emerging technologies might accelerate progress in this field?

As research on TMEM186 is still relatively limited compared to some other mitochondrial proteins, several promising avenues for future investigation exist. Emerging technologies will likely accelerate progress in understanding this protein's structure, function, and potential disease associations.

Priority Research Directions:

• Comprehensive Functional Characterization:

  • Define the precise role of TMEM186 in ATP synthase assembly

  • Investigate potential moonlighting functions outside mitochondria

  • Establish tissue-specific roles and expression patterns

• Structural Biology:

  • Determine high-resolution structure using cryo-EM or X-ray crystallography

  • Characterize conformational changes during function

  • Map interaction interfaces with binding partners

• Physiological Significance:

  • Develop tissue-specific knockout models to assess organ-specific effects

  • Investigate metabolic adaptation in TMEM186-deficient models

  • Explore potential roles in cellular stress responses

• Clinical Relevance:

  • Screen mitochondrial disease cohorts for TMEM186 mutations

  • Investigate expression changes in tissues from patients with mitochondrial disorders

  • Develop potential therapeutic approaches for TMEM186-related dysfunctions

Emerging Technologies with High Impact Potential:

• Single-Cell Omics:

  • Single-cell proteomics to identify cell type-specific expression patterns

  • Single-cell metabolomics to assess metabolic consequences of TMEM186 dysfunction

  • Integration of multi-omics data for systems-level understanding

• Advanced Imaging Techniques:

  • Super-resolution microscopy to visualize TMEM186 within mitochondrial substructures

  • Live-cell imaging with split fluorescent proteins to visualize protein-protein interactions

  • Correlative light and electron microscopy (CLEM) to link functional data with ultrastructural information

• Structural Biology Innovations:

  • Microcrystal electron diffraction (MicroED) for structure determination from tiny crystals

  • Cryo-electron tomography for visualizing TMEM186 in its native membrane environment

  • Integrative modeling approaches combining multiple experimental data sources

• Genome Engineering Advances:

  • Base editing and prime editing for precise mutation introduction

  • CRISPR interference/activation for temporal control of expression

  • Tissue-specific inducible systems for in vivo studies

• Mitochondrial Medicine Applications:

  • Mitochondria-targeted protein replacement strategies

  • Gene therapy approaches for mitochondrial disorders

  • Development of small molecule modulators of ATP synthase assembly

These future directions and emerging technologies promise to significantly advance our understanding of TMEM186's role in mitochondrial function and potentially reveal new therapeutic targets for mitochondrial disorders.